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‘l
1
i
a

FUNCTK

FUNCTIONAL ANALYSIS OF ATVTI1, A FAMILY OF SNARE

PROTEINS IN ARABIDOPSIS

BY

Haiyan Zheng

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Genetics Program

2001

FUNCTIONAL A

Most Va“:

The ones carry
secretory proié
coated transo
vacuolar com
targeting of th
To und

he PVC in m.
typical SNAF
Arabldopsis F
63d AIVAM3

QiOUp SNARE

ABSTRACT

FUNCTIONAL ANALYSIS OF ATVTI1, A FAMILY OF SNARE PROTEINS IN
ARABIDOPSIS

By
Haiyan Zheng

Most vacuolar proteins carry short peptidal sequence as targeting signals.
The ones carrying an N-temiinal pro-peptide (NTPP) are sorted away from other
secretory proteins by NTPP receptors at the TGN and packaged into clathrin-
coated transport vesicles. These vesicles are further transported to the pre-
vacuolar compartment (PVC) before finally reach the vacuole. The correct
targeting of these vesicles to the PVC most likely involves SNARE proteins.

To understand the vacuolar protein trafficking step between the TGN and
the PVC in more detail, I have isolated several genes from Arabidopsis encoding
typical SNARE proteins. First, I identified a SNARE protein called AtPLP,
Arabidopsis PEP12 Like Protein. Due to its high similarity with AtPEP12 (SYP21)
and AtVAM3 (SYP22), it is grouped into SYP2 and renamed as SYP23. SYP2
group SNAREs are located on the PVC membrane. They might be the t-SNAREs
for accepting incoming vesicles.

I have also isolated AtVTI11 and AtVTI12, two related SNARE genes from
Arabidopsis genome. AtVTI11 is localized on the domain of TGN where AtELP,
an NTPP cargo receptor and sporamin, an NTPP cargo reside. It also colocalizes

with SYP2 on the PVC. AtVTI11 is found in SNARE complexes with SYP2 and

SYPS group SN»?
in directing NTPF
Although

33. level). trey
and AtVTllZ a
g'adéent experii
and SYP6 inst
imoived in d‘.
between AtV‘
£31172. althoi
observable p
a CQITTDIEX w
Taker
SNARE prc
between it

ove‘fl‘aDl-‘iinc

SYP5 group SNAREs. All these data suggest that AtVTI11 is a SNARE involved
in directing NTPP cargo containing vesicles towards the PVC.

Although AtVTI11 and 12 share high sequence similarity (65% identical at
a.a. level), they complement different yeast vti1 mutants. In Arabidopsis, AtVTI11
and AtVTI12 are localized on different membranes based on an Accudenz
gradient experiment. The majority of AtVTI12 form SNARE complexes with SYP4
and SYP6 instead of with SYP2 and SYP5. It is likely that AtVTI11 and 12 are
involved in different vesicle targeting steps in vivo. However, the duty division
between AtVTI11 and 12 might not be exclusive. In a T-DNA insertion line of
Atvti12, although the homozygous mutant plant lacks AtVTI12 protein, it has no
observable phenotype because AtVTI11 takes the position of AtVTI12 and forms
a complex with SYP4 and SYP6.

Taken together, I have identified and characterized AtVTl1 family of
SNARE proteins. AtVTI11 is shown to be involved in NTPP cargo transport
between the TGN and the PVC. The closely related AtVTI12 might have

overlapping as well as different functions.

lwould ha‘.
PhD study the r
feel so much aio'
daft. In her lab.
wood also like ti
Dis. Ken Keegs
to thank Drs T
collaboration 0
DIS. Scott Pe
patence in he
g'aleful Iowarc
the nEllie of c.
My the
like to thank .
and my art re
omthe Immu
pmQIESS 30 .
lily Ie$eamh

like“ the lhir

ACKNOWLEDGEMENTS

I would have to thank Dr. Natasha Raikhel for making these five years of
PhD. study the most significant period of my life. She encouraged me when I
feel so much alone. She pointed out the sun to me when I felt completely in the
dark. In her lab, I learned how to be a good scientist and to be a better person. I
would also like to acknowledge the guidance offered by my committee members:
Drs. Ken Keegstra, John Wang, Greg Howe, and Michael Garavito. I would like
to thank Drs. Tom Stevens and Gabrille Fischer von Mollard for their generous
collaboration on yeast complementation of AtVTI1 proteins. Many thanks go to
Drs. Scott Peck for introducing me to the new world of proteomics and his
patience in helping me with 2-D gel and mass spectrometry. I am especially
grateful towards Scott and Antje for their hospitality when I intruded their home in
the name of collaboration.

My thanks go to all the past and present numbers of Raikhel lab. I would
like to thank Dr. Enrique Rojo for his unselfish help on my science, my English
and my art related problems. Dr. Valya Kovaleva for her collaboration in carrying
out the immunocytochemistry studies. Without her, my research can not possibly
progress so fast. Drs. Diane Bassham and Tony Sanderfoot for their advice on
my research and my writing. Dr. Jan Zouhar, for being the only one who always
liked the things I wrote. Sharif Ahmed, for being a valuable information source.
Thanks to the PRL staff for their help that made my study so much easier. In
particular, Kurt and Marlene for their expert on photographic service, and Jim for

his help on growth chamber problems.

iv

l woe eve?
Tao my most WO
lam mad. Thai".
hem.

Maxi the
angel sent by l
assignments as
she thinks It i5
writing proces:
abreak,

Finally

will and to a»

I woe everything to my parents who love me without condition. Thanks to
Tao, my most wonderful sister, who has the power to make me laugh even when
I am mad. Thanks to all my true friends, I feel privileged to have so many of
them.

Maxi, the cat who has made an apartment a sweet home, is no doubt an
angel sent by God at a difficult time of my life. She has never forgotten her
assignments as to stop me whenever I want a snack and to wake me up when
she thinks it is time. Besides occasional editing, she slept through the whole
writing process on my laps so that l was forced to remain sit even when I wanted
a break.

Finally, I believe in God's miracles. My life is a process to understand his

will and to appreciate his boundless wisdom.

LlST OF TABLES
LlST OF FlGUREE

LIST OF ABBREV

CHAPTER I

introduction an 0

l. The End(
llSNARE
Ill. Vacuolg
IV. Vacuol.

IV-a

th
Sys

V. CODCIUE

Vl- Thesis

TABLE OF CONTENTS

LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xii
CHAPTER I
Introduction: an Overview of the Plant Vacuolar Protein Transport Pathways
l. The Endomembrane System in Eucaryotic Cells 2
II. SNARE Hypothesis 4
III. Vacuolar Protein Trafficking in Mammalian and Yeast Cells 6
IV. Vacuolar Protein Transport in Plants 9
lV-a. Traffic Routes for Plant Vacuoles 9
IV-b. Components of the Plant Vacuolar Protein Delivery
System 13
V. Conclusion 21
VI. Thesis Scheme 24
References 26

CHAPTER II

The Syntaxin Family of Proteins in Arabidopsis: a New Syntaxin Homologue
Shows Polymorphism Between Two Ecotypes 35

Abstract 36

vi

Invoduchor
Materials 3
Resots

DlSCUSSiO.’

Reference

CHAPTER III

The Part v-SN.
the PVC

Abstract
InUOduc
Iyflatefla'
Resuhs
DISCUSS

CHAPTER Iv

ITSGl‘IlOn M Ufa

Abstra:
Introdu
Maleri;
RESUME

Introduction

Materials and Methods
Results

Discussion

References

CHAPTER III

37

43

45

58

62

The Plant v-SNARE AtVTl1a Likely Mediates Vesicle Transport from the TGN to

the PVC
Abstract
Introduction
Materials and Methods
Results
Discussion

References

CHAPTER IV

66
67
68
71
78
109

114

Comparison of Two AtVTI1 Proteins and Characterization of Atvti12, a T-DNA

Insertion Mutant of AtVTI12
Abstract
Introduction
Materials and Methods
Results

Discussion

vii

119

120

121

124

129

147

 

Referen:

CHAPTER V

Characterizati:
Columns

Abstract
IanOdUCII

Materiais

 

Resots
Discusslt

REIETEnC

CHAPTER Vl

CORC USIOHS an

REIEIEW

References 1 55

CHAPTER V

Characterization of AtVTI1-Containing Vesicles Purified by Immune-affinity

Columns 1 58
Abstract 159
Introduction 1 60
Materials and Methods 162
Results 165
Discussion 174
References 1 80

CHAPTER VI

Conclusions and Future Directions 182

References 1 94

viii

Table M. T a

Table 3-1. R

Table 5-1. 6

compartmer -

>C

 

 

 

LIST OF TABLES

Table 1-1. The name changes for SNARE proteins used in this dissertation.
23

Table 3-1. Relative sequence identity between Vti1 protein homologues
82

Table 5-1. Relative distribution of T7-AtVTl11 and sporamin in intracellular
compartments of transgenic Arabidopsis roots. 174

ix

 

 

Figure H. T?“-

Figure 1-2. T“-
ith and the P

   

Figure 2-1. Arr
mammalian sy.

Figure 2-2. T‘
eco'tyoes

Figure 2-3. Db

Figure 2-4. A
ecotyoe Col-an

Figure 2-5. lr
ALDLP USIrtg a

Figure 2-6. lr

FiSure 3~1. 5
Of the Tamil\
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figure 3.3
Sam to W
gigs-Inmate
FIQUre 3.4
FIQUre3_5

FIQUre 3‘6

LIST OF FIGURES

Figure 1-1. The routes for protein transport to the vacuole in plants.

1 1
Figure 1-2. The likely players involved in the NTPP cargo transport between the
TGN and the PVC. 18

Figure 2-1. Amino acid sequence alignment of AtPLP and other plant, yeast and
mammalian syntaxins. ‘ 47

Figure 2-2. The AtPLP amino acid sequence differs between Arabidopsis

ecotypes. 48
Figure 2-3. DNA gel blot analysis of the AtPLP gene. 51
Figure 2-4. AtPLP mRNA distrubution pattern among tissues of Arabidopsis
ecotype Columbia and RLD. 53
Figure 2-5. Immunoprecipitation of in vitro translation products of AtPEP12 and
AtPLP using antiserum against AtPEP12p. 56
Figure 2-6. In vitro membrane assay for AtPLP : TMD. 57

Figure 3-1. Sequence comparison of AtVTI1a and AtVTI1b with other members
of the family including thi1p (Saccharomyces cerevisiae, accession no.
2497184), thi1p (Homo sapiens, accession no. 268740), thi1a (Mus
musculus, accession no. 3213227), thi1b (Mus musculus, accession no.
3213229). 80

Figure 3-2. Expression of either AtVTHa or AtVTI1b allows yeast cells to grow in
the absence of Vti1p, but only AtVTl1a functions in TGN-to-PVC traffic. 85

Figure 3-3. AtVTI1b but not AtVTl1a could replace yeast Vti1p in ALP and API
traffic to the vacuole, which are transported to the vacuole via two different

biosynthetic pathways. 88
Figure 34. Northern blot analyses of AtVTI1a and AtVTI1b. 91
Figure3-5. AtVTl1a is an integral membrane protein. 94

Figure 3-6. Subcellular fractionation of AtVTl1a by step sucrose gradient. 97

Figure 37 TI
iraesgenio pla'

Figure 3-8. If
Arabidopss ro .
localzed on tr.-

 

 

 

Figure 3-9. TTI

Arabrdopsrs ro
Figure 3-10. A

Figure 41. Ch

 

 

FiQure 4,2. SL
Accudenz grad

FiQure t3. Ar.
Figure 4-4 At
Figure 4-5. Pt

Figure 4~6. lr
plants.

Figme 4-7, 8

F‘QUre 5.1. A

FIQUre 5.2 C

. ~3,
ANTI” and 3

om Arabldo,

Figure 5
i“ -5. F

Figum 5‘1, c

Figure 3-7. T7-tag does not affect AtVT|1a function and is expressed in
transgenic plants. 100

Figure 3-8. In situ localization of T7-AtVTl1a and AtELP on ultrathin sections of
Arabidopsis roots from T7-AtVTl1a transgenic plants. T7-AtVTl1a and AtELP are
localized on the TGN and on dense vesicles. 104

Figure 3-9. T7-AtVTI1a and AtPEP12p colocalize on the PVC in cryosections of

Arabidopsis roots from T7-AtVTI1a transgenic plants. 107
Figure 3-10. AtVTl1a associates with AtPEP12p 110
Figure 4-1. Characterization of the specificity of anti-AtVTI11 and anti-AtVTI12.
Figure 4-2. Subcellular fractionation of AtVTI11 and AtVTI12 by discontinuous
Accudenz gradient. 136
Figure 4-3. Atvti12 is a null mutant for AtVTI12. 139
Figure 4-4. Aleurain processing in Atvti12 cells. 143
Figure 4-5. Phenotype of Atvti12. 144
Figure 4-6. Immunoprecipitation of AtVTI11 from wild type and Atvti12 mutant
plants. 146
Figure 4-7. Summary of results. 148
Figure 5-1. AtVTI1 vesicles were enriched though a step sucrose gradient.166
Figure 5-2. Characterization of affinity-purified AtVTI1 vesicles. 168

Figure 5-3. The plants crossed between transgeneic plant expressing T7-
AtVTI11 and sporamin. 170

Figure 5-4. Immuno-Iocalization of T7-AtVTl11 and sporamin on thin sections
from Arabidopsis roots. 171

Figure 5-5. Representative 2-D gels showing protein profiles of affinity-purified
AtVTI1 vesicles. 175

Figure 6-1. Schematic model for the AtVTI1 protein functions in plant cells.
186

xi

a a.
AAP
ALP
Ans-l
AP
API
ARF
AtPLP
CCV
CPY
CTPP
CIVSS
CHI
EM
ER
ESl
EST

GOA
GFP
CST
lCAT
LV

IleLDl-TOF

Map
Meet;
ht
NTPP
NSF
ORF
PBS
RSV
DSVSS
PVC

SNAP25

SRP
SYP
SSVSS

 

 

a.a.
AAP
ALP
Ams-1
AP
AP1
ARF
AtPLP
CCV
CPY
CTPP
ctVSS
Cvt
EM
ER
ESI
EST
FT
GGA
GFP
GST
ICAT
LV

MALDl-TOF

MS
M-6-P
M-6—PR
nt
NTPP
NSF
ORF
PBS
PSV
psVSS
PVC
SNAP
SNAP-25
SNARE
SD
SRP
SYP
ssVSS

LIST OF ABBREVIATIONS

= amino acid

= Abridged Anchor Primer (

= Alkaline phosphatase

=or-manosidase

= adaptor protein

= aminopeptidase 1

= ADP-ribosylation factor

= Arabidopsis thaliana Pep12 like protein
=clathrin-coated vesicle

= carboxy-peptidase Y

= C-terminal pro-peptide

= C-terminal sorting signals

= =cytoplasm-to-vacuole

= Electron microscopy

= Endoplasmic [eticulum

= electrospray ionization

=expressed sequence tag

= flow through

=Golgi-localized, y-ear-containing, ARFs-binding proteins
= green fluorescence protein
Glutathione-S-transperase
isotope-coded affinity tags

=lytic vacuole

= matrix-assisted laser desorption ionization- time of flight
= mass spectrometry
Mannose—6-phosphate
Mannose-6-phosphate receptor

= nucleotide

= N-terrninal pro-peptide
N-ethylmaleimide-sensitive factor

open reading frame

= phosphate-buffered saline

=protein storage vacuole

= physical structure signals

Pre-vacuolar compartment

Soluble NSF attachment protein

= 25 kD synaptosomal associated protein
= SNAP receptors

= standard medium

= signal recognition particle

= syntaxin protein

= sequence-specific vacuolar sorting signals

xii

TBS
TGN
TIP
TlrlD
t-SNARE
y-SANE
WE
VSD
\ISR
V88
VTl
w l.
YEPD

 

II II

I

II II II II H II

 

 

TBS = Tris buffered saline

TGN = trans-Golgi network

TIP =tonoplast intrinsic protein
TMD = Transmembrane domain
t-SNARE = target-SNARE

v-SANE = vesicle SNARE

VPE = vacuolar processing enzyme
VSD = vacuolar sorting determinant
VSR = vacuolar sorting receptor
VSS = Vacuolar sorting signal

VTI = Vps10 interacting factor

w.t. = wild type

YEPD = yeast extract peptone dextose medium

xiii

Chapter I

Introduction

An Overview of Plant Vacuolar Protein Transport Pathways

 

I. The Endoml
The er

eukaryotic ce

membrane. tr

enclosed org;

 

 

membrane sy
biochemical a
Golgi. endoso
targeted to thi
cell biology. I
Signal peptic;
translational»:
folding and II
the ER. The
vesicles and
1‘994; Bar-P.
aLiberatus by

G059] SIaCkE

UDOn aTl’lVa
domains 0‘
destinatbns

I. The Endomembrane System in Eucaryotic Cells

The endomembrane system is an important feature that distinguishes the
eukaryotic cell from the prokaryotic cell. Originated as invaginations of the plasma
membrane, the endomembrane system has evolved into a series of membrane
enclosed organelles in modern eukaryotic cells. Owing to this sophisticated
membrane system, cells are organized into compartments featuring different
biochemical and biophysical conditions, such as the endoplasmic reticulum (ER),
Golgi, endosomes, lysosomes (vacuoles) and plasma membrane. How proteins are
targeted to the correct compartment becomes a fundamental question of eukaryotic
cell biology. Most lumenal and many membrane proteins have specific N-terminal
signal peptides that are recognized by signal recognition particle (SRP) and co-
translationally imported into the endoplasmic reticulum (ER) (Rappoport, 1990). The
folding and initial modification of newly synthesized protein molecules take place in
the ER. The protein molecules ready to leave the ER are sorted into coated
vesicles and transported to the next compartment, the Golgi complex (Rothman
1994; Bar-Peled et al., 1996). These cargo proteins further travel through the Golgi
apparatus by vesicle transport or maturation (review see Pelham 1998). Along the
Golgi stacks, proteins are extensively modified by the resident Golgi enzymes.
Upon arrival at the trans-Golgi network (TGN), proteins are sorted into different
domains of the TGN based on specific sorting signals that indicate their
destinations, before eventually packaged into distinct vesicles. Via these vesicles,

cargoes are transported to their final destination, such as the extracellular matrix,

endosomes 0

system. Con.
extracellular r‘
tar-sported to

lonyard and r:

 

time allowing t
Homecstas
era'omembrar
selection me:
destinatson. I
and these p
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endosomes or lysosomes. This fonNard trafficking system is termed the secretory
system. Conversely, proteins localized on the plasma membrane and in the
extracellular matrix can be taken back by the clathrin coated vesicles (CCVs) and
transported to the endosomes or lysosomes through the endocytic pathway. These
forward and retrograde pathways maintain the membrane in balance at the same
time allowing transport of proteins between organelles.

Homeostasis of each compartment is important for its proper function. The
endomembrane system developed multiple ways to achieve this. One is the active
selection mechanism. Many proteins carry peptide signals as the address for their
destination. These signals are recognized by membrane anchored signal receptors
and these proteins are selectively packaged into coated transport vesicles and
delivered to the correct compartment: i.e. many Iysosomal proteins in mammalian
cells carry a mannose-6 phosphate (M6P) signal that is recognized by M6P
receptors at the TGN. These receptors recruit the proteins into CCV that transport
them to the Iysosome. Some transport pathways or steps are less selective. In that
case, proteins are transported forward by bulk flow and selection takes place
downstream. The resident proteins belonging to the upstream compartment are
delivered back based on the retrieval signal they carry. Many ER resident proteins
are retained in the ER by an efficient retrieval system. Some ER proteins can reach
the plasma membrane before being delivered back to the ER. It is now believed
that in most compartments, the active selection in forward transport and bulk flow
plus selective retrieval are combined to maintain the stable composition of the

organelle. For example, in the maturation theory, most resident Golgi enzymes are

 

restricted at c:

 

Golgi apparaf.

see Allan and

  
  

ll. SNARE Hy
Both a:
seéected prcte

set of compai

 

Based on the.
ll993‘i first p:
Protein comp
attachment

Secondary s,
are Type II m
terminus of ‘
that m SOme
SNARES (R
SNARES‘U;
that is impc
Specific,ty 0
Either Q or I
a COnSeWeg
of proteins

group Of D Fr

restricted at certain stacks by retrieval from more advanced stacks. Proteins in the
Golgi apparatus are matured into the next stack by receiving these vesicles (review
see Allan and Balch, 1999).
II. SNARE Hypothesis

Both active selection and selective retrieval require vesicles to deliver the
selected proteins. The correct targeting of vesicles is thought to be fulfilled by a
set of compartment specific membrane proteins and common soluble factors.
Based on their research on neuronal synaptic vesicle secretion, Sbllner et at,

(1993) first proposed the SNARE hypothesis that specified these membrane

protein components to be soluble NSF (N-ethylmaleimide-sensitive factor)

attachment protein receptors or SNAREs. SNARE proteins share similar

secondary structures. Most SNAREs, with the exception of the SNAP 25 family,
are type II membrane proteins with a stretch of hydrophobic residues at the C-
terminus of the protein that form a transmembrane domain. It has been shown
that in some cases, the transmembrane domain determines the location of the
SNAREs (Rayner and Pelham, 1997; Watson and Pessin, 2001). For typical
SNAREs, upstream of the transmembrane domain, there is a coiled-coil domain
that is important for interacting with other SNAREs and may contribute to the
specificity of SNARE interactions (Sutton et al. 1998). SNAREs are classified as
either Q or R SNAREs based on the presence of either a glutamate or arginine at
a conserved position, respectively (Fasshauer et al., 1998a). The SNAP25 family
of proteins are a special group of SNAREs with unique structural features. This

group of proteins lack transmembrane domain. Instead, they are anchored on the

 

 

   
  

membrane by
typical SNAR.
have two "0" ‘.

Based

arbitrarily; v-E

 

membrane of
are on the ta'
MmsaQ
SDBC‘iTICale II
this comple
(Antonin et.
ll“fernbrane
IEESI. ii he
membrane
stable, F

snaptObre

membrane by a post-translationally added lipid group (Oyler et al., 1989). Unlike
typical SNAREs that contain one coiled coil domain, SNARE 25 type SNAREs
have two "Q" type coiled-coil domains.

Based on their locations, SNAREs can also be divided into two groups
arbitrarily: v-SNAREs (a lot of them are also R SNAREs) are localized on the
membrane of transport vesicles and t-SNAREs (Most of them are Q SNAREs)
are on the target membranes. Typically, 3 t-SNAREs (or 2 t-SNAREs if one of
them is a SNAP25 type SNARE) form a cis-SNARE complex. The v-SNARE
specifically recognizes this cis-SNARE complex and align its coil-coil region with
this complex to form a structure called 4-helices bundle or the SNAREpin
(Antonin at al., 2000). The formation of this structure brings the vesicle and target
membrane together for fusion. In the case of syntaxin-SNAP 25 and VAMP2 at
least, it has been shown 3 SNARE complexes are needed to fulfill the task of
membrane fusion (Hua and Scheller, 2001). The SNARE complex is extremely
stable. For instance, the SNAREpin formed by Snytaxin-1, SNAP25 and
snaptobrevin does not unfold until heated up to 95°C (Fasshauer et al., 1998b).
To dissociate the SNAREpin, soluble factors, N-ethylamleimide sensitive factor
(NSF) and soluble NSF attachment protein (or-SNAP) are recruited afterwards to
form a 205 complex with the SNAREs. Energy is used by NSF, an AAA type
ATPase, to dissociate the SNAREpin. (Whiteheart et al., 1994; Tolar and
Pallanck, 1998). The freed v-SNAREs are then recycled back to the original

compartment.

 

 

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see Hay and e
eridence ind

require other :

iSandertoot e:
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The SNARE hypothesis is supported by large number of studies (review
see Hay and Scheller, 1997; Chen and Scheller, 2001). However, a large body of
evidence indicate that in vivo, the specificity of vesicle docking and fusion may
require other protein and lipid factors besides SNAREs (review see Pfeffer 1999).

There are 55 genes identified as SNAREs in Arabidopsis genome so far
(Sanderfoot et al., 2000). Several SNAREs have been found to play important
roles in development and physiology of plants. For example, KNOLLE is found to
be involved in cell plate formation (Lukowitz et al., 1996; Lauber et al., 1997).
SYR1, a SNARE in tobacco is identified by its involvement in plasma membrane
K-channels regulation in guard cells (Leyman et al., 1999). SYP21, SYP22 and
AtVTI11 might be involved in vacuolar protein transport (Bassham et al., 1995;
Sato, et al., 1997; Zheng et al., 1999a). In plants, the basic machinery of vesicle
trafficking is most likely to be conserved. However, those trafficking steps are
important for the cell and the whole plant's development in unique ways different
from Its mammalian and yeast counterpart. This made functional study of SNARE
proteins in plant more intriguing.

III. Vacuolar Protein Trafficking in Mammalian and Yeast Cells

Plant vacuoles are thought to be equivalent organelles to mammalian
lysosomes and yeast vacuoles. In mammals and yeast, there is one kind of
vacuole, although in mammals, differentiated cells sometimes develop
specialized lysosomes like melanosomes and platelet dense granules. For most
lysosomes and vacuoles, the basic function is to digest expired proteins and

recycle cellular components or sequester toxic secondary metabolite. They are

 

 

normally ma
biosynthetic C
maiority ot the
he third one
In maz'l‘
(At-5p) I0 N’g

lysosome (G?

 

catalyzes the
membrane b:
sorts them a-.
l.l6PR46 and
diieucine mo
motif are effic
'r-ear-contair
recrtlit's the c
to form clath
SNAREs tr
Cori‘PaTllher
Vacuoles of

the IYSOSOrr

toss),

recognizes a

normally maintained at low pH and contain mostly hydrolyases. For the
biosynthetic pathway, there are three routes to the vacuole. One is taken by
majority of the soluble protein. Another is mostly by vacuolar membrane proteins.
The third one is a direct cytoplasm-to-vacuole (Cvt) transport pathway.

In mammalian cells, a post-translational addition of mannose-6-phosphate
(M-6-P) to N-glycans acts as a signal to target the majority of hydrolyases to the
Iysosome (Griffiths et al., 1988). The enzyme GlcNAc phosphotransferase
recognizes a certain patch of the amino acid sequence in the protein and
catalyzes the modification of N—linked oligosaccharide. At the trans-Golgi, a
membrane bound M-6-P receptor recognizes the M6P group on the cargo and
sorts them away from the bulk flow (Albert et al., 1994). M-6-P receptors like
M6PR46 and M6PR300 contain a signal comprised of an acidic stretch and a
dileucine motif at their C-terminus which faces cytosol (Chen et al., 1997). This
motif are efficiently recognized by a cytosolic protein called GGA (Golgi-localized,
y—ear—containing, ARFs-binding proteins, Puertollano et al., 2001). GGA then
recruits the clathrin, a type of cytosolic coat protein, and other accessory factors
to form clathrin-coated vesicles (CCV) at the TGN. These CCVs probably carry v-
SNAREs that promote fusion with the late endosome, an intermediate
compartment on the way to the Iysosome (an equivalent compartment to
vacuoles of yeast and plant cells). There is also a M-6-P independent pathway to
the Iysosome which is utilized by certain cells like pepatocytes (Lodish et al.,

1995).

 

 

Yeast '

report indicate

l
the sorting of

 

oi this protein
nechanisms t
In Sac
most other so.
to the M-B-P c
a sugar more
teminus of ti
the Precursor
9181., 1994,
IOaded With
Dtevacuolar
targeting Ste
Win, a mul
IBEChETer e
for pr de l
Meir
SNARE Va
b‘YDaSSES 1
Componem

OI GGA, u.

Yeast vacuolar proteins are not modified with M-6-P. However, a recent
report indicates that a yeast homologue of mammalian M6PR also plays role in
the sorting of vacuolar hydrolases (Whyte and Munro, 2001). Although the ligand
of this protein has not been identified, it is possible yeast also utilize some similar
mechanisms to deliver a subset of vacuolar proteins.

In Saccharomyces cerevisiae, hydrolase caboxypeptidase Y (CPY) and
most other soluble proteins are transported to the vacuole through a route similar
to the M-6-P pathway. One difference, however, is that instead of decorated with
a sugar moiety, the cargo protein carries a peptide targeting signal at the N-
terminus of the protein (Johnson et al., 1987). At the TGN, this sorting signal in
the precursor CPY is recognized by a membrane receptor, Vps10p (Marcusson
et al., 1994). GGA recognizes Vps10p and leads to the formation of vesicles
loaded with CPY and other vacuolar proteins, which are then targeted to the
prevacuolar compartment (PVC)/late endosome (Black et al., 2000). The
targeting step of these vesicles to the PVC was recently found to be facilitated by
Vti1p, a multifunctional Q-SNARE and Pep12p, a t-SNARE localized on the PVC
(Becherer et al., 1996; Fischer von Mollard et al., 1997). The subsequent steps
for CPY delivery from the PVC to the vacuole are not well characterized.

Membrane proteins like alkaline phosphatase (ALP) and the vacuolar t-
SNARE Vam3p in yeast (Darsow et al., 1998), use an alternative pathway that
bypasses the PVC. Thus their transport is not affected by some mutations in
components of the CPY pathway (Cowles et al., 1997; Piper et al., 1997). Instead

of GGA, this pathway requires another adaptor called AP3 (Adaptor Protein 3;

 

l.

Cowies et al

the TGN. Ir-

 

 

 

proteins taro:
isea. DeliAr
nammalan

membrane p'

 

In yeas
u-nanosrdasi
vacuole vra tr
mechanism 0
involved in (i
wtn the Sec‘
transDon (A:
loutes for p
aUIPPhagy.
PTOlelns‘ IDS

VaCUOIQ

W' vaCuQIa
‘V~a.

The

to study It

fanctlbns n

Cowles et al., 1997; Stepp et al., 1997; Darsow et al., 1998) to form vesicles from
the TGN. In mammalian cells, the proper delivery of lysosomal membrane
proteins lamp-2, limp-2 and CD63 (DellAngelica et al., 1999; Le Borgne et al.,
1998; DellAngelica et al., 2000) has also been reported to require AP3. Thus,
mammalian cells also need different pathways for transport soluble and
membrane proteins.

In yeast, some vacuolar resident proteins like aminopeptidase 1 (AP1) or
a—manosidase (Ams1p) are synthesized in the cytosol and transported to the
vacuole via the Cvt pathway (Klionsky 1998; Hutchins et al., 2001). The transport
mechanism of Cvt pathway is just beginning to be unraveled. Membrane fusion is
involved in this process, since SNAREs TIgZp, Vam3p, th7 and Vti1p, along
with the sec1 homologue Vps45p that interacts with Tngp are required for AP1
transport (Abeliovich et al., 1999). Besides biosynthetic pathways, there are other
routes for protein delivery to the vacuole, such as through endocytosis and
autophagy. However, proteins transported by those routes are not resident
proteins, instead, most of them are delivered as substrates for digestion in the

vacuole.

IV. Vacuolar Protein Transport in Plants

IV-a. Traffic Routes for Plant Vacuoles

The plant vacuolar system is much more complex and much more difficult
to study than its yeast and mammalian counterpart. Vacuolar content and

functions not only vary depending on the cell types and developmental stages,

 

 

but differenl -

 

preteilis to 1"
plant vacuole
munlefpari ll“

indicate their

 

 

orting sigoa‘
signais lssVS
signals (psVE
l§99l In the
ssVSS are r
e‘rentualty c1
1991; Matsul
Conserved rr
their functior
precursor of
active A Cha
IS CIeaVed i

but different vacuoles containing different soluble and membrane proteins may
co-exist in the same cell (Paris at al., 1996; Jauh et al., 1999; Di Sansebastiano
et al., 2001). It is likely that distinct transport pathways are used to transport
proteins to these different types of vacuoles. (Robinson and Hinz, 1997). The
plant vacuolar protein transport systems are illustrated as Figure 1-1. Like their
counterpart in mammals or yeast, plant vacuolar proteins carry specific signals to
indicate their destination. For soluble cargo proteins, three types of vacuolar
sorting signals (VSS) have been identified: sequence-specific vacuolar sorting
signals (ssVSS), C-terminal sorting signals (ctVSS), and physical structure
signals (psVSS) (review see Vitale and Raikhel, 1999; Matsuoka and Neuhaus,
1999). In the cases of prosporamin and the cysteine protease proaleurain,
ssVSS are propeptides at the N-terminus of the protein, (NTPP) which are
eventually cleaved to release the mature protein. (Matsuoka and Nakamura,
1991; Matsuoka et al., 1995). The NTPPs from aleurain and sporamin contain a
conserved motif (NPIR). The N-terminal location of the signal is not required for
their function as shown for sporamin A (Koide et al., 1997). For instance, the
precursor of ricin carries an NPIR signal in a 12aa linker joining the catalytically
active A chain and the sugar-binding B chain. On reaching the vacuole, this linker
is cleaved and mature proteins are released (Frigerio et al., 2001a). Many
proteins carrying ssVSS are acidic hydrolyases. They are transported to the lytic
vacuoles by a route reminiscent to the yeast CPY pathway and the mammalian

Mannose-6-phosphate pathway. The VSS is recognized at the TGN by vacuolar

10

ER Golgi

   
  
  
 
 
  
  
 

TG
CCV.
.03...
Central
NTPP pathway
00 PSV

mp? 093“”

 

Figure 1-1. The routes for protein transport to the vacuole in plants. In plants, the
lytic vacuole (LV) and the protein storage vacuole (PSV) may co-exist in the
same cell. Proteins take different routes to these two vacuoles. Most vacuolar
proteins are transported by endomembrane system. They all carry a N-terminal
signal peptide that direct them to be co-translationally transported to the
endoplasmic reticulum (ER). Proteins that belong to the lytic vacuole normally
carry a vacuolar sorting signal at the N-terminus called N-treminal pro-peptide
(NTPP). The route they travel is called NTPP pathway. From ER, these proteins
are transported to the Golgi by vesicle transport. At the TGN, they are sorted
away from other proteins by the NTPP signal and are transported from the TGN
to the PVC via clathrin coated vesicles (CCV). The NTPP cargo eventually reach
the lytic vacuole possibly are mediated by fusion of the PVC to the lytic vacuole.
The proteins targeted to the PSV are transported either through the Golgi-
apparatus or follow a Golgi-independent pathway. depending on whether they
form aggregates at the ER or later. Some of these proteins carry the sorting
signals at the C-terminal of the protein and the pathway they use is called the C-
terminal propeptide (CTPP) pathway. In most mature cells, the lytic and the
protein storage vacuoles may fuse to form the central vacuole.

 

 

sorting recet'

unknown ”‘9

 

Putative SOFT

described in '
indicate that i
the pre-vacu:

The lower pH

 

after dissociat
d‘1rectiy wrth ti

Most s
piopeptides I
lack consens
that CTPP rT
al., 1999). M
(PSV). Little
transpon DI
Phaseolin, I!
lhe involver

attempts to

sorting receptors (VSRs) and sequestered to certain areas of the TGN by some
unknown mechanism. The VSRs then recruit cytosolic factors to form CCVs.
Putative sorting receptors have been identified by several groups and will be
described in more detail below. Instead of directly to the lytic vacuole, most data
indicate that CCV transports its cargo proteins to an intermediate compartment or
the pre-vacuolar compartment (PVC, Paris at al., 1997; Conceicao et al., 1997).
The lower pH in the PVC lumen allows the VSRs to be recycled back to the TGN
after dissociated with and the cargo. The PVC eventually fuse with each other or
directly with the vacuole, delivering the cargo to its final destination.

Most storage proteins and certain hydrolyases carry ct-VSS, or C-terminal
propeptides (CTPP) that is cleaved upon arrival to the vacuole. These signals
lack consensus sequence, but the C-terminal location is critical. It is suggested
that CTPP might share similar secondary structures (Matsuoka and Neuhaus et
al., 1999). Most CTPP- type cargoes are transported to protein storage vacuoles
(PSV). Little is known about how these proteins are selected from the bulk of
transport proteins and how they are transported. Although in the case of
phaseolin, the transport has been shown to be a saturable process, suggesting
the involvement of protein factors (Frigerio at al, 1998). However, numerous
attempts to identify CTPP receptors have failed. It is conceiVable that CTPP
cargo is not selected by particular protein receptors. Rather, by the help of some
protein factors, the hydrophobic CTPP signals promote the formation of
aggregates that exit from ER as precursor accumulating vesicles or dense

vesicles from Golgi (Hara-Nishimura et al., 1998; Toyooka et al., 2000 Frigerio et

12

 

al.. 2001b). ‘

 

the storage
is cleaved 0
different ma:
concentratior

not affect the

 

Yet an
barley phyte
vacuolar sor
proteins are
Schaewen a
°-' the route

The
indePendEr
protein STOI

(TIP). The“

al., 2001b). These vesicles either fuse with the existing PSV or are taken up by
the storage vacuoles by autophagy at the vacuole (Levanony et al., 1992). CTPP
is cleaved once the protein arrives at the PSV. CTPP pathway apparently use
different machinery from NTPP pathway since CTPP pathway is sensitive to a
concentration of wortmannin (a phosphatidyl-inositol 3-Kinase inhibitor) that does
not affect the NTPP protein transport (Matsuoka et al., 1995).

Yet another group of cargo proteins such as bean phytohemagglutinin and
barley phytepsin, use an internal sequence of the mature protein as their
vacuolar sorting signal (Kervinen et al., 1999). In most cases examined, these
proteins are targeted to the PSV (Tague et al., 1990; Saalbach et al., 1991; von
Schaewen and Chrispeels, 1993). However, little is known about either the signal
or the route they pass.

The delivery of vacuolar membrane proteins has been shown to be
independent of soluble proteins (Gomez and Chrispeels, 1993). Since lytic and
protein storage vacuoles carry different isoforms of tonoplast intrinsic proteins
(TIP). Their delivery routes might also be different (Jauh et al., 1998; Jauh et al.,
1999)

IV-2. Components of the Plant Vacuolar Protein Delivery System

Several potential components of the vacuolar protein delivery machinery
have been identified in plants. Most of them appear to be involved in the NTPP
pathway. BP80 is a protein purified from CCV enriched fraction of pea developing
cotyledon with apparent molecular weight of 80 kDa. Its ability to specifically bind

to NTPP signals at an optimal pH of 6.0 and dissociate at pH5.5 (Kirsch et al.,

13

 

 

   
  

l994, Pans ._
AtELP. first If
homologue c I
in CCV prep
al.,1997; Sa
sgnals wrth .'
peptides (A
homologues
plants and

expression

Suggesting

may also h:
VSRs is L:
identzfied I
1999). Ho
SEQUEHCQE

binding six

1994; Paris at al., 1997) makes it a good candidate for NTPP cargo receptor.
AtELP, first identified by its structural similarity to mammalian EGF receptor, is a
homologue of BP80 in Arabidopsis (Ahmed et al., 1997). AtELP is also enriched
in CCV preparations and has been localized to the TGN and the PVC (Ahmed et
al., 1997; Sanderfoot et al., 1998). Like BP80, it has the similar affinity to NTPP
signals with a pH sensitive manner but fails to bind to CTPP or mutated NTPP
peptides (Ahmed et al., 2000). In the Arabidopsis genome, there are 6
homologues of AtELP, VSR1-6. Preliminary results from individual knock-out
plants and RT-PCR show that these 6 genes might have different spatial
expression patterns (Avila-Teeguarden E and Raikhel NV, unpublished data),
suggesting that these proteins might function in different developing stages. They
may also have different affinity to different NTPPs. The ligand specificity of these
VSRs is under investigation. Homologues of AtELP or BP80 have also been
identified in pumpkin (Shimada et al., 1997) and Nicotiana alata (Miller et al.,
1999). However, in both cases, these VSRs recognize internal or C-terminal
sequences that has no NPIR motif. BP80 may also has two separate ligand
binding site that recognize different VSS (Cao et al., 2000).

SNAREs are important components for vesicle transport machinery in all
eukaryotic cells, so they are likely to play a role in vacuolar protein transport in
plants. The completion of Arabidopsis genome allows us to examine the
composition of SNARE genes at genome range. Based on sequence similarities
and secondary structural analysis, Sanderfoot et al. (2000) reported 55 SNARE

genes in Arabidopsis. The largest group is syntaxins. Syntaxins all share similar

14

 

 

secondary 5'

 

Tnere are 2~
and named
extensively
trafficking, E
functzonaily Q

complex of S

 

 

 

and an NSF I
{Bassham a:
SYP21 IS lo
previously c
structure ml
and recycle
Sanderfoot .
incoming A
SYP2 homc
One of they
CPY in Yea
lQCIallzed O
relateu to t
iOCaI‘lzed tc
have fOUn C

(Sandem,

secondary structures. They are always found as a part of the t-SNARE complex.
There are 24 syntaxins in Arabidopsis. They can be grouped to 10 subgroups
and named SYP (syntaxin protein) 1 to 10. One group, SYP2, has been
extensively studied due to its likelihood to be involved in vacuolar protein
trafficking. SYP21, a homologue of yeast Pep12p, was found by its ability to
functionally complement a yeast pep12 mutant (Bassham et al., 1995). A 208
complex of SYP21 has been partially characterized. It contains SYP21, oc-SNAP
and an NSF homologue. This complex readily dissociates when ATP is present
(Bassham and Raikhel, 1999). lmmuno-electron microscopy studies show that
SYP21 is localized to an electron dense tubular structure that had not been
previously characterized. Since AtELP is also found in this compartment, this
structure might be the plant PVC where AtELP dissociates with the NTPP cargo
and recycles back to the TGN. (Bassham et al., 1995; Conceicao et al., 1997;
Sanderfoot et al., 1998). Thus, SYP21 may be a SNARE involved in receiving the
incoming AtELP-containing CCV derived from the TGN. There are two other
SYP2 homologues in Arabidopsis. They show around 65-70% identity to SYP21.
One of them, SYP22 was first identified by its ability to restore the transport of
CPY in yeast vam3 mutant (Sato et al., 1997). In yeast, Vam3p is the t-SNARE
localized on the vacuole that is responsible for all membrane fusion events
related to the vacuole (Darsow et al., 1997). However, in Arabidopsis, SYP22 is
localized to different places in different tissues by different labs. In root tips, we
have found SYP21 and SYP22 are both localized on the same PVC membrane

(Sanderfoot et al., 1999). In shoot meristem, however, SYP22 has been

15

 

observed or

and SYP22

 

SYP21 or S

I
most each ;
and probab }.
was first ides

SYP21 and ;

lacking a he

 

lanother eco

observed on the tonoplast of small vacuoles (Sato et al., 1997). Although SYP21
and SYP22 interact with identical set of other SNAREs, knockouts of either
SYP21 or SYP22 result in a pollen-lethal phenotype. SYP21 and SYP22 thus
must each perform critical and nonredundant functions in pollen development
and probably in later stages of development (Sanderfoot et al., 2000b). SYP23
was first identified from Arabidopsis EST collections due to its high similarity with
SYP21 and 22. Surprisingly, in Columbia ecotype, this gene encodes a protein
lacking a transmembrane domain due to a frame shift. The same gene from RLD
(another ecotype) background, on the other hand, encodes a full length SYP23
with a transmembrane domain similar to the other two SYP23 (Zheng et al.,
1999b). Previous reports show that some syntaxins lacking a transmembrane
domain can be localized correctly by interacting with other SNAREs. That may
explain why SYP23 without transmembrane domain has no dominant negative
effect on membrane fusion. Alternatively, SYP23 are not abundantly expressed
(Zheng et al., 1999b) and may have redundant functions with other SYP25. Thus
this mutation in recent evolution is tolerated and preserved with no adverse
effects.

SYP4 group of syntaxins are most closely related to TIgZp in yeast and
Syntaxin 16 in mammals. ln yeast and mammals, this syntaxin is found on the
Golgi and the TGN (Holthuis et al., 1998; Simonsen et al., 1998). Among other
functions, TIgZp is involved delivering vacuolar proteins via the Cvt pathway
(Abeliovich et al., 1999). In Arabidopsis, there are 3 genes in this group. SYP41

and 42 are both localized on the TGN but on separate domains (Bassham et al.,

16

zoo-OI. Like

 

SYP42 lead

roles in pie”
The \

syntaxrn Tlg

IS only one E

 

membranes

both SYP41

other (Sande

NTPP cargo

 

Vllip
associate w
DTOVen an 8
been show
trafficking. 5
Cause the r
5119995th

V01) MOHard

Vam3p an:

GOCking an;

SNARES II
that are ret

Interaetion

2000). Like SYP2 group syntaxins, single knock-out mutation of SYP41 and
SYP42 lead to pollen lethal phenotype, suggesting that they play non-redundant
roles in plant development.

The SYP5 and SYP6 group of syntaxins share similarity with yeast
syntaxin Tlg1p. The SYP5 gene family is composed of 2 members, where there
is only one SYP6. Both SYP5 and 6 are localized on both the TGN and the PVC
membranes. SYP5 proteins associate with SYP2 at the PVC, while SYP6 with
both SYP41 and SYP42 on the TGN. SYP5 and SYP6 also interact with each
other (Sanderfoot et al., 2001). Figure 1-2 shows the likely players involved in the
NTPP cargo transport between the TGN and the PVC.

Vti1p is a v-SNARE first identified in yeast because of its ability to
associate with Vps10p, the cargo receptor for CPY. This interaction was later
proven an artifact (Fischer von Mollard and Stevens, 1997). However, Vti1 has
been shown to be an important SNARE with multiple functions in vesicle
trafficking. Some temperature sensitive mutant alleles of this v-SNARE in yeast
cause the mistargeting of CPY to the extracelluar space as a precursor form,
suggesting that the CPY transport be blocked at the step after the TGN (Fischer
von Mollard and Stevens 1997). More over, VTl1p forms a SNARE complex with
Vam3p and Vam7p on the vacuolar membrane that is necessary for vesicle
docking and fusion to the vacuole (Fischer von Mollard and Stevens 1997). Five
SNAREs, including Vti1p, Vam3p, Vam7p, th6p, Nyv1p form SNARE complex
that are required for vacuolar homotypic fusion (Ungermann et al., 1999). The

interaction between Vam3p and Vti1p contributes to all membrane docking and

17

 

 

GOLC

 

 

 

Figure 1.
TGN and
lemons-re
fOmla‘Jtm
ThESe v-5
Pchand
°Und 0n .

PVC

 

TGN
GOLGI
AtELP
AtELP
AtSYP6 ”SYP2
AtSYP4 AtSYP5
AtELP

l ’
. “é t-SNARE m:- v-SNARE

D: NTPP cargo receptor
0 NTPP cargo

 

 

 

Figure 1-2. The likely players involved in the NTPP cargo transport between the
TGN and the PVC. NTPP signals are recognized by AtELP at the TGN. This
recognition leads to the sorting of NTPP cargo away from other proteins and the
formation of CCV. On the CCV membrane, besides AtELP, there are v-SNAREs.
These v-SNAREs interact with SYP2 and SYP5, the t-SNAREs localized on the
PVC, and leads to membrane fusion. Because the pH is lower in the PVC, AtELP
dissociates NTPP cargo and is recycled back. SYP4 and SYP6 are t-SNAREs
found on the TGN membrane. The functions of these SNAREs are not clear.

 

fusion event

 

vacuole (Fis

routes to the

  
  
 

titp also i"

and Tng. the.
in multiple 5
acheeve spe
needed for s

Vtilp
1997. Fischi
M0 Vlll QSr

bel‘fleen m C

36% Slmlla

organlsms 3
tasks. 1n m,

Cell lXu eta

In A
seqUEHCE
peptide 86

DTESenCe ‘

80d AtVTI

fusion events with the vacuolar membrane and thus affect all traffic routes to the
vacuole (Fischer von Mollard et al., 1999). Thus, Vti1p plays important roles in all
routes to the vacuole in yeast. Besides the roles in delivering vacuolar proteins,
Vti1p also interacts with Sed5p, the t-SNARE on the cis-Golgi (Lupashin et al.,
1997; Fischer von Mollard et al., 1997), probably as part of the retrograde
transport system. Vti1p also interacts with Tlg1p, a syntaxin on early endosomes
and TIgZ, the syntaxin on the TGN (Holthuis et al., 1998). Vti1p is thus involved
in multiple steps of the endomembrane transport system in yeast. The way to
achieve specific targeting is not clear. It is possible that other SNAREs are
needed for specific SNARE complex formation and membrane docking.

Vti1p homologues are also found in mouse and human (Lupashin et al.,
1997, Fischer von Mollard and Stevens 1998). Interestingly, in higher organisms,
two Vti1 genes are found although there is only one gene in yeast. The homology
between mouse and human Vti1a is 98% while mouse Vti1a and Vti1b only share
30% similarity. The notion is that the endomembrane systems in higher
organisms are more complex. Two Vti1 genes thus evolved to share the multiple
tasks. In mouse, Vti1a and Vti1b are localized on different membranes within the
cell (Xu et al., 1998). Presumably, they perform different functions.

In Arabidopsis, several EST cDNA clones were found to share significant
sequence similarity with Vti1p of other organisms (about 25-34% identity at
peptide sequence level). A search of the Arabidopsis genome reveals the
presence of three VTI1 gene homologues. We named them AtVTI11, AtVTI12

and AtVTl13. The comparison of their deduced amino acid sequences show that

19

 

may are be’.

identical wr.

 

proteins ha
long. They

sequence c
SNAREs bel
AtVTI13 is c
at high leve'
genes are

Sequence. ,
AtVTlll is a
to the PVC
transoon of:
p{01910) and

1999a). In A.

the TGN are
SYP21 (Zhe
AtVTI12 is ft
(8

 

andenbot
been Shown
mutant has
Drotein eXlr;

comparing tc

they are between 65% (AtVTI11-12, AtVTI11-AtVTI13) to 75% (AtVTI12-AtVTI13)
identical with each other. Like other Vti1 proteins, these three Arabidopsis VTI1
proteins have a C-terminal hydrophobic domain around 17 to 20 amino acids
long. They also contain a coiled-coil region that shows highest amino acids
sequence conservation. Like other Vti1 proteins, they are classified as "Q"
SNAREs because of a conserved glutamate residue at the 0 layer of the helices.
AtVT|13 is only expressed at very low level. AtVTI11 and AtVTI12 are expressed
at high levels. Thus, we characterized AtVTI11 and AtVTI12 in more detail. Both
genes are expressed in all tissues of the plant. Although very similar in
sequence, AtVTI11 and AtVTI12 complement different vti1 mutant in yeast.
AtVTI11 is able to substitute for yeast Vti1 p in the transport of CPY from the TGN
to the PVC. AtVTI12, on the other hand, is more efficient at facilitating the
transport of the vacuolar membrane protein alkaline phosphatase (the membrane
protein) and aminopeptidase 1 (a marker of the Cvt pathway, Zheng et al.,
1999a). In Arabidopsis root tips, AtVTI11 is found to be located with AtELP on
the TGN area and with SYP21 on the PVC where it physically interacts with
SYP21 (Zheng et al., 1999a) and SYP51 (Sanderfoot et al. 2001). In contrast,
AtVTI12 is found in the complex with SYP4 (Bassham et al., 2000) and SYP6
(Sanderfoot et al., 2001). A T-DNA insertion mutant of AtVTI12 (Atvti12) has
been shown to lack AtVTI12 gene expression and the protein. However, this
mutant has no apparent phenotypes. Immunoprecipitation of AtVTI11 from
protein extract of this mutant co-precipitated much more SYP4 and SYP6

comparing to that from the wild type plant. Thus, it is likely that AtVTI11 takes

20

 

place of rr

Recently. A

 

deletron mq
indicating a
function. To

between the

mutant of Al

 

oiAtvnianJ

BESlC
charactenzg
sec1 homc
presumably
the interact
There 18 m
Protein is ;

and SYP4

SandEiffoc
V' Conch

0L
been dral

place of missing AtVTI12 to maintain the normal functioning of the plant.
Recently, A shoot meristem gravitropic mutant zig1 (sgr4) is found to have a
deletion mutation in AtVTI11 gene (Morita et al., 2001; Kato et al., 2001),
indicating at least some level of non-overlap between AtVTI11 and AtVTI12
function. This mutant might give us more information about the relationship
between the endomembrane system and gravity sensing in plants. The double
mutant of Atvti12 and zig-1 will also be helpful for us to understand the function
of AtVTl family proteins.

Besides SNAREs, a homologue of the yeast Vps45 protein has been
characterized in Arabidopsis (Bassham and Raikhel, 1998). Vps45 protein is a
sec1 homologue that associates with Pep12p and Tngp. This association
presumably helps the syntaxin to adopt a functional conformation or to facilitate
the interactions between syntaxins and other proteins (Bryant and James, 2001).
There is no transmembrane domain on AtVPS45. However, in Arabidopsis, this
protein is associated peripherally with membrane and co-Iocalizes with AtELP
and SYP4 in subcellular fractionation studies and immune-electron microscopy.
Moreover, AtVPS45 physically interacts with SYP4 (Bassham et al., 2000;
Sanderfoot et al., 2001). Thus, AtVPS45 might be involved in regulating the
functions of SYP4.

V. Conclusion

Our understanding about how protein is transported to the vacuole has

been dramatically advanced by the knowledge of the Arabidopsis genome and by

tools available such as reverse genetics in Arabidopsis. Our approach of

21

 

 

characteriz
between the
trafficking s:
the basic rr
lansoon nah
other Systey»
involved in r
The

 

isoforms of s
’Wundancy
have gaffe“;
Secreiow DE
Cope With '
Chanefiges

machine”
ot the plant

AIQ‘~
of the SN;
induded t

0396. T0

are apple;

characterizing the molecular components of vesicle trafficking machinery
between the TGN and the PVC has already offered a lot of information of this
trafficking step and the vacuolar protein transport. We have shown that although
the basic machinery is conserved in all eukaryotic cells, plant vacuolar protein
transport has unique features that can not be inferred from studies conducted in
other systems. In particular, mutant studies have shown that vesicle trafficking is
involved in many specific aspects of plant development and physiology.

The presence of small families of related proteins are common
phenomena for secretory machinery components in plants. These multiple similar
isoforms of secretory machinery components probably indicate some functional
redundancy. However, the few cases studied thus far, indicate these proteins
have different duties to meet the unique and complex requirement for plant
secretory pathway. Alternatively, they could be required in different tissues or to
cope with different environment conditions. These gene families are great
challenges in addressing functional questions of the various components of the
machinery, but they also provide us opportunities to examine the unique features
of the plant cell machinery in the context of the whole organism

Along the rapid advance in our knowledge about plant genome, the names
of the SNAREs have been changed during past several years. This dissertation
included two chapters that are published papers where historical names were
used. To clarify the confusion, Table 1-1 indicates all the names changed that

are appeared in this dissertation.

22

 

 

Table M. T
current nan

AtVTI1 1
ANTI 12
SYP21
SYP22
SYP23
SYP41

SYP42

l

 

 

Table 1-1. The name changes for SNARE proteins used in this dissertation.

 

 

current name old name

AtVTI1 1 AtVTI1 a

AtVTI12 AtVTI1 b

SYP21 AtPEP12p, atPEP12p
SYP22 AtVAM3p. atVAM3
SYP23 AtPLP

SYP41 AtTLGZa

SYP42 AtTLGZb

 

23

 

VI. Thesis 8*
Wher

been identil

PEP12 and .

plants that r

 

identified a

database. I

 

Columbia a
surprising.)
transmemiC
gone ll‘tTQu
sequence
To
partners
ldemrfiec
“NEE-ed
System I:
SpeCUla:

ATVTH

‘OCaiize

VI. Thesis Scheme

When ljoined the lab, SYP21 (AtPEP12p) and SYP22 (AtVAM3p) had just
been identified by their ability to complement the yeast mutation in genes of
PEP12 and VAM3 respectively. These were the first cases of syntaxins found in
plants that might be involved in vacuolar protein transport. Subsequently, I
identified a partial cDNA for AtPLP or SYP23, from an Arabidopsis EST
database. I cloned full-length cDNAs from Arabidopsis thaliana ecotypes
Columbia and RLD. The RLD cDNA sequence encodes a full length protein but
surprisingly, the cDNA from Columbia encoded a protein that lacked the
transmembarne domain. Careful examination revealed that this gene has also
gone through evolutionary changes in its DNA sequence recently that caused the
sequence difference among different ecotypes.

To further characterize the SYP2 gene family, I searched the potential
partners of this group syntaxins. At the time, the yeast Vti1p had just been
identified and shown that it interacts with yeast Pep12p. A database search
revealed three Vti1 type genes in Arabidopsis. Since plant endomembrane
system is more complicated and advanced than that of yeast, it is reasonable to
speculate that these three Arabidopsis proteins might have functional difference.
Since AtVT|13 is only expressed in very low level, I extensively studied the
ATVTI11 (AtVTI1a) and AtVTI12 (AtVTI1b). I was able to show that AtVTI11 is
localized on the TGN and the PVC. It also interacts with SYP21 (Chapter III).

AtVTI12, although very similar to AtVTI11, may have different functions. The first

24

 

 

 

evidence to
of yeast vti1
III). In p'
immunoore:
AtVTI12 inte'
In an AtVT I
SYP4 and E

dsfi'erent for

will take ove

 

 

evidence to support this notion is that these two proteins rescue different alleles
of yeast vti1 mutant that block different trafficking routes to the vacuole (Chapter
III). In plants, their functional difference has been indicated in
immunoprecipitation assays. AtVTI11 interacts with SYP2 and 5, whereas
AtVTI12 interacts preferentially with SYP4 and SYP6 groups syntaxins. However,
In an AtVTI12 knock-out mutant (Atvti12), AtVTI11 is found to associate with
SYP4 and SYP6 (Chapter IV). This indicates that, although AtVTI11 and 12 have
different functions, in the absence of one family member, the remaining member

will take over its function.

25

 

 

Referencq

 
 
   
  

Abeliovich
of aminoce:
\lbsASp. Ell."

Ahmed SU.
location ot a
protein-sort:

Ahmed 80,
K, and Raik
in the trans
Arabidopsis

Albert 8, E
biology of ti

Allan BB, 5
COmpartmg

Al'ltonin I.
00338.”qu

BaT‘Pele
eukaWOtt.

Basshar
S‘thaxin
mutant, ,

Bassha
[mmltati

References

Abeliovich H, Darsow T, and Emr SD (1999) Cytoplasm to vacuole trafficking
of aminopeptidase 1 requires a t—SNARE-Sec1p complex composed of Tngp and
Vps45p. EMBO 18: 6005-6016.

Ahmed SU, Bar-Peled M, and Raikhel NV (1997). Cloning and subcellular
location of an Arabidopsis receptor-like protein that shares common features with
protein-sorting receptors of eukaryotic cells. Plant Physiol. 1 14: 325-336.

Ahmed SU, Rojo E, Kovaleva V, Venkatarmam S, Dumbrowski JE, Matsuoka
K, and Raikhel NV (2000) The plant vacuolar sorting receptor AtELP is involved

in the transport of NH2-terminal propeptides-containing vacuolar proteins in
Arabidopsis thaliana. J. Cell Biol. 149: 1335-1344.

Albert B, Bray D, Lewis J, Raff M, Roers K, and Watson J (1994). Molecular
biology of the cell. 3'd ed. Pp613-618.

Allan BB, and Balch WE (1999). Protein sorting by directed maturation of Golgi
compartments. Science 265: 63-66.

Antonin W, Holroyd C, Fasshauer D, Pabst SV, Mollard, GF, and Jahn R
(2000). A SNARE complex mediating fusion of late endosomes defines
conserved properties of SNARE structure and function. EMBO J. 19: 6453-6464.

Bar-Peled M, Bassham DC, and Raikhel NV (1996). Transport of proteins in
eukaryotic cells: More questions ahead. Plant Mol. Biol. 32: 223-249.

Bassham DC, Gal 8, Conceicao AS, and Raikhel NV (1995). An Arabidopsis
syntaxin homologue isolated by functional complementation of a yeast pep12
mutant. Proc. Natl. Acad. Sci. USA 92: 7262-7266.

Bassham DC, and Raikhel NV (1998) An Arabidopsis VPS45p homolog
implicated in protein transport to the vacuole. Plant Physiol. 117: 407-415

Bassham DC, and Raikhel NV (1999). The pre-vacuolar t-SNARE AtPEP12p
forms a 208 complex that dissociates in the presence of ATP. Plant J. 19: 509-
603.

Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, and Raikhel NV (2000).
AtVPS45 complex formation at the trans-Golgi network. Mol. Biol. Cell 11: 2251-
2265.

Becherer KA, Rieder SE, Emr SD and Jones EW (1996). Novel syntaxin
homologue, Pep12p, required for the sorting of lumenal hydrolases to the
Iysosome-like vacuole in yeast. Mol. Biol. Cell 7: 579-594.

26

 

 

Black M. 3“
endosomes

  
  

Bryant NJ...
Tngp and I-
3388.

030 X, Ro
requiremer
Plant Cell 1

Chen H,I Yl
CBTIOTT-Iflde;
Cyloplasmic
function in l'

CONCBIan
Rillkhel N\
60191 com;

Cowles C

Complex is
09 1 18

DarSow n

DaISOw 1

depenc
vac“Olar 1

Dell
(199
he

Ang
9) A
tQ m

Dew An.
BODifac
:Emans

Disa n1

Black M, and Pelham H (2000). A selective transport route from Golgi to late
endosomes that requires the yeast GGA proteins. J Cell Biol. 151: 587-600.

Bryant NJ, and James DE (2001). Vps45p stabilizes the syntaxin homologue
TIgZp and positively regulates SNARE complex formation. EMBO J. 20: 3380-
3388.

Cao X, Rogers SW, Bulter J, Beevers L, and Rogers JC (2000). Structural

requirements for ligand binding by a probable plant vacuolar sorting receptor.
Plant Cell 12: 493-506.

Chen YA, and Scheller RH (2001). SNARE-mediated membrane fusion. Nat.
Rev. Mol. Cell Biol. 2: 98-106.

Chen H, Yuan K, and Lobel P (1997). Systematic mutational analysis of the
cation-independent mannose 6-phosphate/insulin-like growth factor II receptor
cytoplasmic domain. An acidic cluster containing a key aspartate Is important for
function in Iysosomal enzyme sorting. J. Biol. Chem. 14: 7003-7012. '

Conceicao AS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, and
Raikhel NV (1997). The syntaxin homologue AtPEP12p resides on a late post-
Golgi compartment in plant. Plant Cell 9: 571-582.

Cowles CR, Snyder WB, Burd CG and Emr SD (1997). The AP-3 adaptor

complex is essential for cargo-selective transport to the yeast vacuole. Cell 91:
109-1 18.

Darsow T, Rieder SE, and Emr SD (1997). A multispecificity syntaxin

homologue, Vam3p, essential for autophagic and biosynthetic protein transport to
the vacuole. J. Cell Biol. 138: 517-529.

Darsow T, Burd CG and Emr SD (1998). Acidic dileucine motif essential for AP-
3 dependent sorting and restriction of the functional specificity of the Vam3p
vacuolar t-SNARE. J Cell Biol. 142: 913-922.

Dell‘Angelica EC, Shotelersuk V, Aguilar RC, Gahl WA, and Bonifacino JS
(1999) Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome
due to mutations in the 63A subunit of the AP-3 adaptor. Mol Cell 3: 11-21.

Dell‘AngeIica EC, Aguilar RC, Wolins N, Haxelwood S, Gahl WA, and
Bonifacino JS (2000). Molecular characterization of the protein encoded by the
Hemansky—Pudlak syndrome type 1 gene. J. Biochem. 275: 1300-1306.

Di Sansebastiano GP, Paris N, Marc-Martin S, and Neuhaus JM (2001)
Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be

27

 

visualized b
Physiol. 126

 

Fasshauer
structural 16
as 0- and F.

 
    
  

Fasshauer
minimal core
and dlSBSSE'

the t-SNARE

Fisher von
leplace the j
273: 2624-2

Fisher von
V-SNARE \
vacuole. Mi

F(”Mario L
Dhaseolin 1
Plant C911 1

Frigerio L,
Carton: A
prchlSor .
Physloi. 12

FriSerio L
fate 0f tn”
[Cute t0 Va

visualized by green fluorescent proteins targeted to either type of vacuoles. Plant
Physiol. 126: 78-86.

Fasshauer D, Sutton RB, Brunger AT, and Jahn R (1998a). Conserved
structural features of the synaptic fusion complex: SNARE proteins reclassified
as Q- and R-SNAREs. Proc. Natl. Acad. Sci. U S A. 95: 15781-15786.

Fasshauer D, Eliason W, Brunger AT, and Jahn R (1998b). Identification of a
minimal core of the synaptic SNARE complex sufficient for reversible assembly
and disassembly. Biochemistry 37: 10354-10362.

Fischer von Mollard G, Northwehr SF, and Stevens TH (1997). The yeast v-
SNARE Vti1p mediates two vesicle transport pathways through interactions with
the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137: 1511-1524.

Fisher von Mollard and Stevens TH (1998). A human homolog can functionally
replace the yeast v-SNARE Vti1 p in two vesicle transport pathways J. Biol Chem.
273: 2624-2630.

Fisher von Mollard G, and Stevens, TH (1999). The Sacchromyces cerevisiae
v-SNARE Vti1p is required for multiple membrane transport pathways to the
vacuole. Mol. Biol. Cell 1 0: 1719-1732.

Frigerio L, de Virgilio M, Prada A, Faoro F, Vitale A (1998) Sorting of
phaseolin to the vacuole is saturable and requires a short C-terminal peptide.
Plant Cell 10: 1031-1042.

Frigerio L, Jolliffe NA, Di Cola A, Felipe DH, Paris N, Neuhaus JM, Lord JM,
Ceriotti A, and Roberts LM (2001). The internal propeptide of the ricin

precursor carries a sequence-specific determinant for vacuolar sorting. Plant
Physiol. 126: 167-175.

Frigerio L, Pastres A, Prada A, and Vitale A (2001). Influence of KDEL on the
fate of trimeric or assembly-defective phaseolin: selective use of an alternative
route to vacuoles. Plant Cell 13: 1109-1126.

Gomez L and Chrispeels MJ (1993). Tonoplast and soluble vacuolar proteins
are targeted by different mechanisms. Plant Cell 5: 1113-1124.

Griffiths G, Holflack B, Simons K, Mellman I, and Kornfeld S (1988). The
manose-6-phospate receptor and the biogenesis of lysosomes. Cell 52: 329-341.

Hara--Nishimura I, Shimada l, Hatono Y, Takeuchi Y, and Nishimura M

(1998). Transport of storage proteins to protein storage vacuoles is mediated by
large precursor accumulating vesicles. Plant Cell 10: 825-836.

28

 

 

'Hay JC an

fusion. Curr

 

Holthuis J
syntaxm hc
126.

Hua Y, and.
membrane

Hutchins

mannosida
pathway co
23498.

Jauh GY,
t0”(mast ir
Acad. Sci. L

Jauh (3y,
lSOfOl’ms as

GEtermma,
prOTEa$e\ (

Kato T! M1

Kerwinen
and VaCuc

KHOnS
Chem. 27

and inltia
Nat ACac

. Hay JC and Scheller RH (1997). SNAREs and NSF in targeted membrane
fusion. Curr. Opin. Cell Biol. 9: 505-512.

Holthuis JCM, Nichols BJ, Dhruvakumar S, and Pelham HRB (1998). Two
syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 17: 113-
126.

Hua Y, and Scheller RH (2001). Three SNARE complexes cooperate to mediate
membrane fusion. Proc. Nat. Acad. Sci. USA 98: 8065-8070.

Hutchins MU, and Klionsky DJ (2001). Vacuolar localization of oligomeric or-

mannosidase requires the cytoplasm to vacuole targeting and autophagy
pathway components in Saccharomyces cerevisiae. J. Biol. Chem. 276: 20491-
20498.

Jauh GY, Fischer AM, Frimes HD, Ryan CA, and Rogers JC (1998). 8-

tonoplast intrinsic protein defines unique plant vacuole functions. Proc. Nat].
Acad. Sci. USA 95:-12995-12999.

Jauh GY, Philips TE, and Rogers JC (1999). Tonoplast intrinsic protein
isoforms as markers of vacuolar functions. Plant Cell 11: 1867-1882.

Johnson LM, Bankaitis VA and Emr SD (1987). Distinct sequence
determinants direct intracellular soting and modification of a yeast vacuolar
protease. Cell 48: 875-885.

Kato T, Morita MI, Fukaki H, Yoshiro Y, Uehara M, Nihama M, and Tasaka M
(2001). SGR2, a phospholipase-Iike protein, and ZIG/SGR4, a SNARE, are
involved in the shoot gravitropism of Arabidopsis (Submitted).

Kervinen J, Tobin GJ, Costa J, Waugh DS, Wlodaver A, and Zdanov A
(1999). Crystal structure of plant aspartic proteinase prophytepsin: inactivation
and vacuolar targeting. EMBO J. 18: 3947-3955.

Klionsky DK (1998). Nonclassical protein shorting to the yeast vacuole. J. Biol.
Chem. 273: 10807-10810.

Kirsch T, Paris N, Butler JM, Beevers L, and Rogers JC (1994). Purification
and initial characterization of a potential plant vacuolar targeting receptor. Proc.
Nat. Acad. Sci. USA 91: 3403-3407.

Koide Y, Hirano H, Matsuoka K and Nakamura K (1997). The N-terminal
propeptide of the precursor to sporamin acts as a vacuole-targeting signal even
at the C-terminus of the mature part in tobacco cells. Plant Physiol. 114: 863-
870.

29

 

 

Lauber Mr
Lukowitz \
cytokinesis-

Le Borgne
3 adaptor
membrane

Levanony l
route of Wl‘i

Leyman B.“
WMamm.

Lodish H,
(7995). MC

741
ii_

LUpashin
ChaTaCteriz
traffic Mo].

 

LUkOWItz v
Embryo anC

MarcuSSOn
SD (1994)

matsUOka
34-838.

MatSUOka
senSltlvity ,
dlStant SO!

913'“ Vacu

Miller

Chara Cter
1013 0t Ce].

Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I,
Lukowitz W, and Jiirgens G (1997). The Arabidopsis KNOLLE protein is a
cytokinesis-specific syntaxin. J. Cell Biol. 139: 1485-1493.

Le Borgne R, Alconda A, Bauer U, and Hoflack B (1998). The mammalian AP-
3 adaptor-like complex mediates the intracellular transport of lysosomal
membrane glycoproteins. J. Biol. Chem. 273: 29451-29461.

Levanony HR, Rubin Y, Altschuler Y and Galili G (1992). Evidence for a novel
route of wheat storage proteins to vacuoles. J. Cell Biol. 1 19: 1117-1128.

Leyman B, Geelen D, Quintero FJ, and Blatt MR (1999). A tobacco syntaxin
with a role in hormonal control of guard cell ion channels. Science 283: 537-540.

Lodish H, Baltimore D, Berk A, Zipursky L, Matsudaira P, and Darnell J
(1995). Molecular Cell Biology 3rd ed. Scientific American Books, Inc., pp709-
711.

Lupashin VV, Pokrovskaya ID, McNew JA, and Waters MG (1997)
Characterization of a novel yeast SNARE protein implicated in Golgi retrograde
traffic. Mo]. Biol. Cell 8:1379-1388.

Lukowitz W, Mayer U, and Jilrgens G (1996). Cytokinesis in the Arabidopsis
embryo involves the syntaxin-related KNOLLE gene product. Cell 84: 61-71.

Marcusson EG, Horazdovsky BF, Ceregjino JL, Gharakhanian E and Emr
SD (1994). The sorting receptor for yeast vacuolar carboxypeptidase Y is
encoded by the VPS10 gene. Cell 77: 579-586.

Matsuoka K, and Nakamura K (1991). Propeptide of a precursor to a plant
vacuolar protein required for vacuolar targeting. Proc. Nat]. Acad. Sci USA 88:
834-838.

Matsuoka K, Bassham DC, Raikhel NV, Nakamura K (1995). Different
sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of
distinct sorting machineries in tobacco cells. J. Cell Bio. 130: 1307-1318.

Matsuoka K, and Neuhaus JM (1999). Cis-elements of protein transport to the
plant vacuoles. J. Exp Bot. 50: 165-174.

Miller EA, Lee MCS, and Anderson MA (1999). Identification and

characterization of a prevacuolar compartment in stigmas of Nicotiana alata.
Plant Cell 1 1: 1499-1508.

30

 

 

 

Morita MT,
(2001). min
C'avrtrOpisn

Oyler GA,
Wilson Ml
protein, Sh
Bio. 109: 321

Paris N, St
mnctionally

 

Paris N, R:
(17997), M,

\

V3Cuolar so

PfEffer SR

PElham HF

49.

PuertOIIan
Sorting 01 r

Rap°Pon
BIOChem- “

Rayner J(

the plant
WSOSOme

Sital!)acf
'fiETent
595708.

Sander-f

Morita MT, Kato T, Nagafusa K, Saito C, Ueda T, Nakano A and Tasaka M
(2001). Involvement of the vacuoles of the endodermis in early process of shoot
gravitropism in Arabidopsis (Submitted).

Oyler GA, Higgins GA, Hart RA, Battenberg E, Billingsley M, Bloom FE, and
Wilson MC (1989). The identification of a novel synaptosomal-associated
protein, SNAP-25, differentially expressed by neuronal subpopulations. J. Cell
Bio. 109: 3039-3052.

Paris N, Stanley CM, Jones, RL, and Rogers JC (1996). Plant cells contain two
functionally distinct vacuolar compartments. Cell 85: 563-572.

Paris N, Rogers SW, Jiang L, Kirsch T, Beevers L, Philips TE, Rogers JC
(1997). Molecular cloning and further characterization of a probable plant
vacuolar sorting receptor. Plant Physiol. 115: 29-39

Pfeffer SR (1999). Transport-vesicle targeting: tethers before SNAREs. Nature
Cell Biol. 1: E17-E22.

Pelham HR (1998). Getting through the Golgi complex. Trends Cell Biol. 8: 45-
49.

Puertollano R, Aguilar R, Gorshkova I, Crouch R, and Bonifacino J (2001).

Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science 292:
1712-1716.

Rapoport TA (1990). Protein transport across the ER membrane. Trends.
Biochem. Sci. 15:355-358.

Rayner JC, and Pelham HR (1997). Transmembrane domain-dependent sorting
of proteins to the ER and plasma membarne in yeast. EMBO J. 16: 1832-1841.

Rothman JE (1994). Mechanisms of intracellular protein transport. Nature 372:
55-63.

Robinson D6, and Hinz G (1997). Vacuole biogenesis and proteins transport to
the plant vacuole: a comparison with the yeast vacuole and the mammalian
Iysosome. Protoplasma 197: 1-25.

Saalbach G, Jung R, Kunzy G, Saalbach I, Adler K, and Muntz K (1991).
Different Iegumin protein domains act as vacuolar targeting signals. Plant Cell 3:
695-708.

Sanderfoot AA, Ahmed SU, Marty Mazars D, Rapopot I, Kirchhausen I,
Marty F, and Raikhel NV (1998). A putative vacuolar cargo receptor partially

31

 

 

l“

 

colocalizes
Proc. Natl.

Sanderfoo
AtVAMBp r:
Plant Phys

  
 

Sanderfoo
An abund

Sato MH, |
NIShlI’Tlura
molecule ii
Chem. 272

Shimada '
pumpkin 1
characteris

Simmlser
Syntaxm

S5nller 1
(1993‘). A
Sequentia
4T8

Stepp JI

ComDIEX‘

the aftErn

colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis Roots.
Proc. Nat]. Acad. Sci. USA 95: 9920-9925.

Sanderfoot AA, Kovaleva V, Zheng H, and Raikhel NV (1999). The t-SNARE
AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells.
Plant Physiol. 1 21: 929-938.

Sanderfoot AA, Assaad, FF, and Raikhel NV (2000), The Arabidopsis genome.
An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein
receptors. Plant Physiol. 1 24: 1558-1 569.

Sanderfoot AA, Kovaleva V, Bassham DC, Raikhel NV (2001). Interactions
between syntaxins identify at least five SNARE complexes within the
Golgi/prevacuolar system of the Arabidopsis. Mol. Biol. Cell (submitted)

Sato MH, Nakamura N, Ohsumi Y, Kouchi H, Kondo M, Hara-Nishimura I,
Nishimura K, and Wada Y (1997). The atVAM3 encodes a syntaxin-related
molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J Biol.
Chem. 272: 24530-24535.

Shimada T, Kuroyanagi M, Nishimura M, and Hara-Nishimura I (1997). A
pumpkin 72 kDa membrane protein of precursor-accumulating vesicles has
characteristics of a vacuolar sorting receptor. Plant Cell Physio]. 38: 1414-1420.

Simonsen A, Bremnes B, Ronning E, Aasland and Stenmark H‘(1998).
Syntaxin-16, a putative Golgi t-SNARE. Eur. J. Cell Biol. 75: 223-231.

Sbnller T, Bennett MK, Whiteheart SW, Scheller RH, and Rothman JE
(1993). A protein assembly-disassembly pathway in vitro that may correspond to

sequential steps of synaptic vesicle docking, activation and fusion. Cell 75: 409-
418.

Stepp JD, Huang K, and Lemmon SK (1997). The yeast adaptor protein
complex, AP-3, is essential for the efficient delivery of alkaline phosphatase by
the alternate pathway to the vacuole. J. Cell Biol. 139: 1761-1764.

Sutton RB, Fasshauer D, Jahn R, and Brunger AT (1998). Crystal sturcture of

a SNARE complex involved in synaptic excocytosis at 2.4 A resolution. Nature
395: 347-353.

Tague BW, Dickinson CD, and Chrispeels, MJ (1990). A short domain of the

plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole.
Plant Cell 2: 533-546.

32

 

Tolar LA
involves I"
vesicle doc

 

  
 

endoplasn
germinatin ;

vacuoles?

Ungennanl
Wickner l]l
Dentamerir
homotypic

Von Scha
ll'lfOflTlBtiOl
44'. 339-31

Watson F
intracellm;
Phl’s/o]. C

Tolar LA and Pallanck L (1998). NSF functions in neurotransmitter release
involves in rearrangement of the SNARE complex downstream of synaptic
vesicle docking. J. Neuronsic. 18: 10250-10256.

Toyooka K, Okamoto I, and Minamikawa T (2000). Mass transport of pro-form
of a KDEL tailed cysteine protease (SH-EP) to protein storage vacuoles by
endoplasmic reticulum-derived vesicles is involved in protein mobilization in
germinating seeds. J. Cell Biol. 148: 453-463.

Vitale A and Raikhel NV (1999). What do proteins need to reach different
vacuoles? Trends Plant Sci. 4. 149- 155.

Ungermann C, van Mollard GF, Jensen ON, Margolis N, Stevens TH,
WicknerW (1999). Three v-SNAREs and two t-SNAREs, present in a
pentameric cis-SNARE complex on isolated vacuoles, are essential for
homotypic fusion. J. Cell Biol. 145: 1435-1442.

Von Schaewen A, and Chrispeels MJ (1993). Identification of vacuolar sorting
information in phytohemagglutinin, an unprocessed vacuolar protein. J. Exp. Bot.
44: 339-342.

Watson RT, and Pessin JE (2001). Transmembarne domain length determines
intracellular membarne compartment localization of syntaxins 3, 4, and 5. Am. J.
Physiol. Cell Physiol. 28: 0215-0223.

Whiteheart SW, Rossnagel K., Buhrow SA, Brunner M, Jaenicke R, and
Rothman JE (1994). N—ethylmaleimide-sensitive fusion protein: a trimeric

ATPase whose hydrolysis of ATP 3 required for membrane fusion. J. Cell Biol.
126: 945-954.

Whyte JRC, and Munro 8 (2001). A yeast homolog of the mammalian mannose
6-phosphate receptors contributes to the sorting of vacuolar hydrolases. Cur.
Bio]. 11: 1074-1078.

Xu Y, Wong SH, Tang BL, Subramaniam VN, Xhang T, and Hong W (1998). A
29-kilodalton Golgi soluble N-ethylmaeimide-sensitive factor attachment protein
receptor (Vti1-rp2) implicated in protein trafficking in the secretory pathway. J.
Biol. Chem. 273: 21783-21789.

Zheng H, van Mollard GF, Kovaleva V, Stevens TH, and Raikhel NV (1999a).
The plant vesicle associated SNARE AtVT|1a likely mediates vesicle transport

from the trans-Golgi network to the prevacuolar compartment. Mol. Biol. Cell 10:
2251-2264.

33

Zheng H.
family of
polymorp“

 

 

 

Zheng H, Bassham DC, Conceicao AS, and Raikhel NV (1999). The syntaxin
family of proteins in Arabidopsis: a new syntaxin homologue shows a
polymorphism between two ecotypes. J. Exp Bot. 50: 915-924.

34

 

 

This I

Harya
V Re

Z.H.l

Chapter II

The Syntaxin Family of Proteins in Arabidopsis: a New Syntaxin

Homologue Shows Polymorphism Between Two Ecotypes

This chapter was published in:
Haiyan Zheng, Diane C. Bassham, Alexandre de Silva Conceicao and Natasha
V. Raikhel (1999) Journal of Experimental botany 50: 915-924.

Z.H. contributed to all the figures.

35

 

,d
Iut

Pr

‘—
MC

Ar

Abstract

The syntaxin family of proteins are involved in vesicle sorting, docking and
fusion in the secretory pathway. The discovery of several syntaxin homologues in
plants (AtPEP12p, KNOLLE, AtVAM3p) suggested that the general mechanisms
of protein trafficking through the secretory pathway also apply to plant cells. The
identification of another syntaxin homologues, the AtPLP (AtPEP12p-Like
Protein) gene, in Arabidopsis thaliana, which is not present in yeast, is reported
here. The putative amino acid sequence of AtPLP shows high similarity to
AtPEP12p and AtVAM3p, and AtPEP12p antibodies cross-react with in vitro
translated AtPLP. The AtPLP gene shows polymorphism between two
Arabidopsis ecotypes: In ecotype Columbia, it encodes a syntaxin homologue
lacking the C-terminal transmembrane domain (TMD), whereas in ecotype RLD,
due to a frame shift in the genomic sequence, it results in a typical syntaxin like
protein with a C-terminal TMD. The distribution pattern of AtPLP mRNA among
various tissues is different from that of AtPEP12, AtVAM3 and KNOLLE mRNA,

indicating that the protein may play a novel role in vesicle trafficking in plant cells.

36

Introduction

The plant secretory system is a series of organelles including the
endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, and vacuole.
Soluble proteins containing an N-terminal signal sequence are co-translationally
inserted into the ER lumen and from here are transported through the secretory
system in transport vesicles which bud from one compartment and fuse with the
next. From the ER, proteins are transported through the Golgi, cisternae to the
trans-Golgi network, where a major sorting event occurs. Proteins are either
secreted to the outside of the cell (the default pathway) or they are targeted to
the vacuole via a prevacuolar compartment (PVC), a route that is believed to be
receptor-mediated.

Protein Sorting to the Vacuole

For a soluble protein to reach the vacuole, it must contain a specific
vacuolar sorting signal. Three types of vacuolar sorting signal have been
identified. Some proteins contain a propeptide which comprises the sorting signal
and which is removed from the protein upon deposition in the vacuole. Several
proteins are known to contain a propeptide at their N-terminus, including sweet
potato sporamin (Matsuoka and Nakamura, 1991) and barley aleurain (Holwerda
et al., 1992). Deletion and mutagenesis studies have shown that the N-terminal
propeptide is required for transport to the vacuole, and have defined a conserved
motif within the propeptides which is essential for proper sorting and thus is
thought to be recognized by the sorting machinery (Holwerda et al., 1992;

Nakamura et al., 1993).

37

The second type of propeptide signal is found at the C-terminus of several
vacuolar proteins, including barley lectin (Bednarek et al., 1990) and tobacco {
chitinase (Neuhaus et al., 1991). No common motif can be identified between
these sortig signals, and while the propeptides have been shown to be both
necessary and sufficient for vacuolar transport, extensive mutagenesis studies
have determined that there is no sequence motif that is required for their function
(Dombrowski et al., 1993; Neuhaus et al., 1994). It is still not known whether a
receptor protein is present at the trans-Golgi network that recognizes this type of
signal.

Finally, a region of the mature protein can also serve as a sorting signal.
Bean phytohaemagglutinin contains this type of signal (von Schaewen and
Chrispeels, 1993), although the nature of the signal means that it is very difficult
to identify precisely the residues responsible for targeting information. Legumin
also appears to contain vacuolar sorting information within the mature protein,
and the entire Iegumin or chain is required for efficient sorting (Saalbach et al.,
1991). The mechanism of which these signals work is not known.

Studies using the fungal metabolite wortmannin, an inhibitor of
phospholipid metabolism in tobacco cells, have shown that proteins containing
an N-terminal signal are transported to the vacuole by a different mechanism to
proteins containing a C-terrninal propeptide signal (Matsuoka et al., 1995). In the
presence of wortmanin, proteins containing the barley lectin C-terminal
propeptide were secreted, whereas those containing the sporamin N-terminal

signal were correctly sorted to the vacuole. In addition, membrane proteins are

38

transported by a different pathway compared to soluble proteins (Gomez ad
Chrispeels, 1993). Therefore, it appears that multiple pathways to the vacuole
exist within a single plant cell. In fact, some specialized cell types have been
shown to contain two distinct types of vacuoles, which have their won discrete
complement of proteins (Paris et al., 1996; Di Samsebastiano et al., 1998). In
mature vegetative cells, these vacuoles are thought to fuse to form a single large
central vacuole upon which all the vacuolar targeting pathways converge
(Schroeder et al., 1993).

By affinity chromatography using the aleurain N-terminal propeptides, a
protein was isolated from pea clathrin coated vesicles which is thought to
represent a sorting receptor for this signal (Kirsch et al., 1994). This protein (BP-
80) is an integral membrane protein which can also bind to other N-terminal
signals, but not to the barley lectin C-terrninal propeptide (Kirsch et al., 1996). An
Arabidopsis homologue of BP-80 (named AtELP) was identified by searching the
DNA sequence database using motifs common to sorting receptors from various
eukaryotic species (Ahmed et al., 1997). Both the pea and Arabidopsis versions
have been localized by electron microscopy to the Golgi apparatus (Paris et al.,
1997; Sanderfoot et al., 1998) and in addition, AtELP was found at the
prevacuolar compartment (Sanderfoot et al., 1998). These receptor-like proteins
are proposed to bind to the cargos (Proteins containing an N-terminal vacuolar
sorting signal). At the trans-Golgi network and package them into clathrin coated

vesicles for transport to the prevavuolar compartment.

39

 

 

Ilechanis
Ah
lanctSc
baton
whth at
vesdei
hson 2

stages r

mEmbr
al., (9
memh
SNAP
assoc

assor

Mechanisms of Vesicle Docking and Fusion

After the formation of vesicles containing vacuolar cargo proteins from the
trans-Golgi network, the vesicles have to be directed to the PVC, and fuse with
that organelle. A number of proteins have been identified from various organisms
which are required for the correct targeting, docking and fusion of a transport
vesicle with its target membrane. As some components for vesicle targeting and
fusion appear to be similar between many different organisms and different
stages of the secretory pathway, the basic mechanisms for these processes are
probably conserved. Information from the better studied yeast and mammalian
systems can therefore, be very useful in the study of vesicle trafficking in plants.

Syntaxin 1 was originally isolated as a protein on the synaptic plasma
membrane of neurons, where it is involved in neurotransmitter release (Bennet et
al., 1992). It has been shown to form a complex with several soluble and
membrane-bound proteins such as NSF (N-ethylmaleimide-sensitive factor), or-
SNAP (soluble NSF attachment protein), SNAP-25 (25 kDa synaptosomal
associated protein), and synaptorbrevin (also known as VAMP, vesicle-
associated membrane protein; Sbllner et al., 1993). These proteins are believed
to function in vesicle trafficking according to the SNARE (SNAP-receptor)
hypothesis (Bennett, 1995). Syntaxin 1 and SNAP-25 (t-SNAREs) on the target
membrane (the pre-synaptic plasma membrane) are able to interact. with
synaptobrevin (a v-SNARE on the synaptic vesicle). or-SNAP and NSF can bind
to this complex, and fusion of the vesicle with the target membrane occurs by a

mechanism which is yet unclear. A number of syntaxin homologues have now

40

 

been id
Scheller
predzcze
similar i
dilieren

5803310

memi
the C
tells
lag;
hon
lnv

Cor

been identified in non-nerve cells and in various organisms (Bennett and
Scheller, 1993; Ferro—Novick and Jahn 1994). They have similar sequences and
predicted secondary structures to syntaxin 1 and therefore, may function in a
similar way in a variety of vesicle fusion events. It has been hypothesized that a
different syntaxin isoform may be required for each transport step of the
secretory pathway (Rothman, 1996).

Typical syntaxin homologues are type II transmembrane proteins. Most of
the protein is hydrophilic and faces the cytosol, except for a hydrophobic domain
about 10-30 amino acids long at the C-terminus of the protein. They hydrophilic
part is thought to interact with other factors involved in vesicle trafficking,
whereas the hydrophobic domain is responsible for anchoring the protein in the
membrane (Aalto et al., 1993; Jantti et al., 1994). Interestingly, syntaxins without
the C-terminal transmembrane domain have also been found in mammalian
cells, although their functions remain unknown (Hirai, 1993; lbaraki et al., 1995;
Jagadish et al., 1997; Simonsen et al., 1998; Tang et al., 1998). Several syntaxin
homologues have now been identified in plants which presumably also are
involved in transport through the secertory pathway. By functional
complementation of the yeast (Saccharomyces cerevisiae) secretory pathway
mutant pep12, Bassham eta]. (1995) have isolated a cDNA clone (AtPEP12)
from Arabidopsis thaliana ecotype Columbia that encodes a protein homologous
to yeast Pep12p and other members of the syntaxin family. The yeast pep12
mutant is defective in transport of some soluble proteins to the vacuole (Becherer

et al., 1996) and is thought to function in transport between the trans-Golgi

41

 

ndvular

 

mtnnspo
mtmscop
Gugineb
finnxnli
htheva:
Anbdoc
hasbeer
etal l
KNOLLE
embyo

7997i:

them it

Prote
ANA
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item

network and the prevacuolar compartment. AtPEP12p may therefore play a role
in transport to the vacuole in plants. AtPEP12p has been localized by electron
microscopy to a PVC in Arabidopsis roots, and may act as a receptor for trans-
Golgi network derived transport vesicles en route to the vacuole. A second
syntaxin-like protein, Vam3p, is thought to be involved in transport from the PVC
to the vacuole in yeast (Darsow et al., 1997; Wada et al., 1997). AtVAM3p is an
Arabidopsis Vam3p homologue that can complement the yeast vam3 mutant and
has been localized to the tonoplast in Arabidopsis shoot apical meristems (Sato
et al., 1997). By genetic analysis, another Arabidopsis syntaxin homologue,
KNOLLE (Lukowitz et al., 1996), is presumably involved in cytokinesis in the
embryo, and is located at the forming cell plate during cell division (Lauber et al.,
1997). Several syntaxin homologues therefore exist in Arabidopsis and each of
them may be involved in separated steps of the secretory system or function at
different developmental stages, or in different tissues.

Another syntaxin homologue in Arabidopsis, AtPLP (AtPEP12-Like
Protein), which encodes a protein showing high similarity to AtPEP12p and
AtVAM3p, is reported here. The original cDNA clone isolated from the Columbia
ecotype encodes a protein that has no transmembrane domain. Interestingly,
when the AtPLP sequence of another ecotype, RLD, was examined. It was found
that a typical syntaxin syntaxin transmembrane domain was regenerated by a
frame shift at the 3' end of this gene. Although the protein sequences of AtPLP
and AtPEP12p are very similar, and AtPEP12p antibody cross-react with AtPLP,

the AtPLP mRNA has a distinct distribution pattern from that of AtPEP12. This

42

indicates that they may have different functions. The function of AtPLP in plants
remains to be determined. But due to its high similarity to other proteins thought

to be involved in vacuolar protein transport, it may also function in this process.

Materials and Methods

DNA and RNA Preparation

One gram of Arabidopsis rosette leaves was used for genomic DNA
preparation according to Murry and Thompson (1980). Total RNA extraction from
plant tissues was performed based on Bar-Peled et al., (1995), except that the
RNA was further purified by phenol:chloroform: sioamyl alcohol (25: 24: 1, by
vol.) and chlorofrom sioamyl alcohol (24:1 v/v) extraction followed by ethanol
precipitation. The pellets were resuspended in 200 pl DEPC-treated water. The
concentrations of DNA or RNA in the extracts were determined by the 00250
value.
Reverse Transcription of mRNA and PCR

Reverse transcription reactions are performed using AMV (avian
myeloblastosis virus) reverse transcriptase (Boehringer Mannheim) following the
manufacturer's protocol. 0.5-2 ug of total Arabidopsis RNA were added as
template and 10 pmol of oligonucleotide (Primer 2: AAC ATA TAC TCA TTG
ATG CTT) as the antisense primer. The reaction was incubated at 42°C for 1
houn
For PCR reactions, 10 pl of reverse transcribed product or 0.1 ug of genomic

DNA were amplified by using Taq polymerase (Gibco BRL) with primers

43

designed for AtPLP (Primer 1: C'IT CCC TCA AGC CTA CAC ATC CAG and
primer 2 as shown above) under the general conditions offered by Gibco BRL.
To construct a cDNA encoding AtPLP containing the transmembrane domain,
reverse transcrition of RNA from roots of ecotype RLD was performed using oligo
d(T) as a primer. Primer 1 (see above) and oligo d(T) were used to further
amplify a DNA fragment from the reverse transcription product. This fragment
was cloned into the EcoRV site of Bluescript SK (-) (Stratagene). The full length
cDNA was reconstructed from this PCR fragment and the AtPLP EST cDNA
(from Columbia) using a Bglll site located within the PCR-amplified region.
DNA sequence analysis

The AtPLP cDNA was isolated as an Expressed Sequence Tag (EST
clone F3C11T7, GeneBank accession number N95926) and inserted at the
EcoRI site of Bluescript SK(-). The complete sequence was determined by the
DNA sequencing facility at the WM Keck Foundation of Yale University and
deposited in the GenBank database (Accession number U85036). For PCR and
RT-PCR products, blunt ends were generated using Klenow fragment and
inserted into the EcoRV site of Bluescript KS (-). Sequencing was performed by
using a Sequenase 2.0 kit (United States Biochemical). Three independent
clones of each product were analyzed to determine a final sequence.
Blotting Procedure

For northern analysis, 20 pg of Arabidopsis RNA were applied to each
lane of a formaldehyde denaturing agarose gel which was run as described by

Sambrook et a]. (1989). For Southern analysis, 5 ug of genomic DNA were sued

44

for each restriction endonulcease reaction and digested DNA fragments were
separated on a 0.7% agarose gel. Separated RNA or DNA samples were
transferred to Hybound-N (Amersham) nylon membrane. Alternatively, various
amounts of in vitro-transcribed mRNA were applied to a membrane for dot blot
analysis. Blots were hybridized with either RNA or DNA probes. Antisense RNA
probes were labeled using 32P or-UTP by in vitro transcription of linearized
plasmid. DNA probes were generated by random priming using 32P or-dATP.
In vitro translation followed by immunoprecipitation

In vitro transcription, translation and immunoprecipitation were performed
as described by Conceicao et a]. (1997). Antiserum used for immunoprecipitation
was generated against AtPEP12p (Conceicao, et al., 1997).
In vitro Membrane Association Assay

Two pl of canine pancreas microsomes (Promega) were added to 25 pl of
in vitro translation product described above. The reaction was incubated at room
temperature for 30 min. One ml of buffer A (50 mM trimethanolamine, pH 7.5, 0.4
M sucrose) was added to the reaction at the end of the incubation. Microsomes
were pelleted by ultracentrifugation at 140, 000 g for 10 min at 4°C. The pellets
were washed with 1 ml of buffer A and resuspended in 25 pl of SNS sample
buffer. Pellets and supernatant were separated on a SDS-PAGE gel and

analyzed by autoradiography.
Results

AtPLP Encodes a Putative Syntaxin Homologue in Arabidopsis

45

Pep12p is a yeast syntaxin homologue required for the sorting of certain lumenal
proteins to the Iysosome-like vacuole (Becherer, et al., 1996). An Arabidopsis
homologue of PEP12 (AtPEP12) was isolated by complementation of the yeast
pep12 mutant (Bassham et al., 1995) and may be involved in protein transport to .
the vacuole in Arabidopsis (Bassham et al., 1995; Conceicao et al., 1997). In
order to identify other proteins potentially involved in transport to the plant
vacuole, the AtPEP12 nucleotide sequence was used to search the Arabidopsis
EST database using the BLAST algorithm (Altschul et al., 1990). A sequence
was identified that showed similarity with AtPEP12. This 1263bp cDNA (From the
PRL2 cDNA library, Newman et al., 1994) was named AtPLP (Arabidopsis
thaliana PEP12p-Like Protein) and sequence analysis indicated that it contains
an open reading frame of 256 amino acids. This putative protein is 55% identical
to AtPEP12p (Figure 2-1) and 74% identical to another syntaxin homologue
found recently in Arabidopsis, AtVAM3p (Sato et al., 1997). It displays a much
lower sequence similarity to yeast Pep12p (Becherer et al., 1996; 20% identity)
and a mammalian Pep12p homologue synatxin 7 (Wang et al., 1997; 26%
identity), and no equivalent gene to AtPLP can be identified in the yeast genome.
However, the AtPLP sequence lacks one feature contained within most members
of the syntaxin family, including AtPEP12p and AtVAM3p: a C-terminal
transmembrane domain (TMD), important for anchoring the protein in the target

membrane and therefore for its function as a t-SNARE.

46

 

181
183
172
161

 

220

222

21.1

201

253 AtPLP

262 AtPEP12p
251 AtVAM3p
241 I ’ syntaxin?

 

Figure 2-1. Amino acid sequence alignment of AtPLP and other plant, yeast and
mammalian syntaxins. The sequence alignment was generated using the
MegAlign program (DNASTAR lnc., Madison, WI, USA). The AtPLP sequence is
based on Arabidopsis ecotype Columbia. Consensus amino acid residues are
shaded.

47

 

Figure 2-2. The AtPLP amino acid sequence differs between Arabidopsis
ecotypes. A) Nucleotide and amino acid sequences of the AtPLP cDNA from
Arabidopsis thaliana ecotype Columbia. The sequence difference from ecotype
RLD is shown in bold. Primers used for RT-PCR and PCR are marked here as
primer 1 (sense) and primer 2 (antisense). B) Partial nucleotide sequence and
derived amino acid sequences of AtPLP cDNA from Arabidopsis ecotype RLD
based on RT-PCR products. The sequence difference from Columbia is shown in
bold.

48

79

157

235

313

781

859

937

1015

1093

1171

1249

547

625

703

781

859

GTT

TTC

ATC

ACT

TTA

AAT

CGT

GAG

CCC

CTT

GTT

GTT

CAC

TCT

ATC

GAG

ATC

CTC

AAG

TGT

CTG

CTT

ATA

GAC

ATC

TCT

GCG

GAC

GGT

CAT

ATT

AAG

GCT

GAT

AAT

GTG

GAT

TGA

CTC

TCC

AAG

GCC GCG AGA

TGT TCG

GAG AGA

GGT GGT AGT

CGT CTT GTC

Q
GGA CAG TTA

AAG AAG ATT

GCT GAA AGA

GTG AAT GGA

GAG ATT GCG

CAC GAG ATC

AAC TCT TAC
GCT

ATA

TAC AAT TTA
GGC TTG TGT

CGG CCG CG

A E
GCT GAA

R
AGA

V N
GTG AAT GGA
E I
GAG ATT

H E
CAC GAG

N S
AAC TCT

T C
ACG TGC

GAG

AGA

AAT

AGA

AGA

AAT

GTG

GTG

GAT

GAA ACC

GAT AAG

TTC

AAT

TTC

GCT

TCT

TGG ACA

GTC TGT

TTG CTG

AAC

GTT

AGT

GAC

CTT

GAT

GTA

CAT

GAG

GAC

ACT

TGA

AGT

AGT

TCA

ACG

GGT

ACA

AAG

TAT

CCA

GCT

TTG

GCC

GCT

ACT

Y
TAT

CCA

CCG GGG AAT CTT GAA ATC ACC ATC ACA TCC TCT CTT CCC TCG
GCT TTG TTT TCT TGG GCG AAT AAA AGA GGA TAA TTT AGT GGC
Q D L E A G R G R S L A S S
CAA GAT TTA GAG GCG GGA AGA GGA AGA TCA TTA GCT TCT TCA
T Q D A S G I P Q I N T 3
ACT CAA GAT GTT GCT TCT GGT ATA TTT CAG ATC AAT ACA AGT
T P K D T P E L R E K L H K
ACT CCT AAA GAT ACG CCT GAG CTC AGA GAG AAG CTG CAT AAG
S A K L K E A S E T D H Q R
TCA GCT AAA CTT AAA GAA GCT AGT GAA ACT GAT CAT CAA AGA
L A K D P Q A V L K E F Q K
CTT GCA AAG GAC TTT CAA GCT GTG TTG AAA GAG TTT CAA AAG
A P L V H K P S L P S S Y T

GCT CCT CTT GTC CAC AAG CCA TCT
primerl
E Q R A L L V E S K R Q E L
GAG CAG CGT GCC CTT CTT GTG GAA TCA AAA AGA CAA GAA CTT
V I E E R E Q G I Q E I Q Q
GTT ATT GAG GAA AGA GAG CAA GGG ATA CAA GAA ATT CAG CAG
A V L V H D Q G N M I D D I
GCA GTG TTG GTG CAC GAT CAA GGA AAC ATG ATA GAT GAT ATT
Q G K S H L V R H Q R H K D
CAA GGA AAA TCC CAT CTC GTA AGO CAT CAA AGA CAC AAA GAT
TTG GTA TCG TGC TCA TGA TTG TTA TTA TAG TGC TCG CAG TTT
TCA TCA TCA ATC AAG AAG_QAI_CBA_IGB_§IA_IAI_QII_TGT CAC
primerz
ATT GGT CTC TTG TTT CTT GTT ATC GAG TAT ATA GAG TAG TTC
GGT TCT GAA GGA GAG AGT GTT ACT GCT TTG TCT CTG TAA TAT
A P L V H K P S L P S S Y T
GCT CCT CTT GTC CAC AAG CCA TCT CTT CCC TCA AGC TAC ACA
E Q R A L L V E S K R Q E L
GAG CAG CGT GCC CTT CTT GTG GAA TCA AAA AGA CAA GAA CTT
V I E E R E Q G I Q E I Q Q
GTT ATT GAG GAA AGA GAG CAA GGG ATA CAA GAA ATT CAG CAG
A V L V H D Q G N M I D D I
GCA GTG TTG GTG CAC GAT CAA GGA AAC ATG ATA GAT GAT ATT
Q G K S H L V K A S K T Q R
CAA GGA AAA TCC CAT CTC GTA AAA GCA TCA AAG ACA CAA AGA
F G I V L M I V I I V L A V
TTT GGT ATC GTG CTC ATG ATT GTT ATT ATA GTG CTC GCA GTT

49

ATC TCC

GTT

AGG AAC

GTT TCC

ACA AGA

GGT GTA

GCT CAG

S S

V L
GTA CTG

I
ATT

Q
CAA

G
GGT

T
ACT

I
ATT

Q
CAA

GAT TTC

GAC TCT

ACT

AAC TTG

S S
TCC AGT

V L
GTA CTG

Q I
CAA ATT

G T
GGT ACT

S N
TCA AAT

* F
TGA TTT

The AtPLP Gene Displays an Ecotype Difference Between RLD and
Columbia

From sequence analysis of the cDNA, AtPLP appeared to be a syntaxin
homologue that lacked a transmembrane domain. However, the C-terminal
transmemberane domain is thought to be critical for the membrane targeting of t-
SNAREs (Jantti et al., 1994; Banfield et al., 1994). In addition, membrane
association is thought to be important for the function of syntaxin homologues in
vesicle docking and fusion steps. To establish that this truncation was not due to
an artifact during the PRL2 cDNA library generation, RT-PCR was performed
using a pair of primers that would amplify the 3' end of the AtPLP mRNA. The
same set of primers was also used for PCR from genomic DNA. Two ecotypes of
Arabidopsis were used: Columbia, the ecotype from which the cDNA library was
derived, and RLD, the ecotype used for most of the work on AtPEP12 gene in
this laboratory. The sequences derived from RT-PCR and genomic DNA PCR
generated from Arabidopsis ecotype Columbia confirmed the cDNA sequence
and therefore, that the protein truncation was genuine. However, the PCR
sequences from RLD, unlike those from Columbia, turned out not to be in
agreement with the original EST clone (Figure 2-2). Compared to the EST clone
or Columbia sequence, the sequence from RT-PCR of RLD (and the genomic
sequence at the corresponding region) had an additional adenosine that
generated a frame shift as well as an a G-to-A transition (905-TCT CGT AAA

AGO-916 instead of 905-TCT CGT AAG GC-915). Such a changed sequence

50

Columbia RLD
E H X E H X

 

 

2.3kb- ,
2.0kb—

0.5kb- 5

Figure 2-3. DNA gel blot analysis of the AtPLP gene. 5 pg of Arabidopsis DNA
digested with EcoRI (E), Hind/II (H) or Xbal (X) was hybridized with a 32P-Iabeled
AtPLP probe generated by random priming from the complete cDNA and
visualized by autoradiography. Molecular size markers in kilobases (kb) are
shown on the left

51

results in a longer open reading frame with an extended C-terminus that shows a
higher similarity to the C-terminal transmembrane domain of AtPEP12p and
AtVAM3p.

Southern blot analysis (Figure 2-3) suggested that this gene is a single
copy gene in both ecotypes, which indicates that the sequences from RT—PCR
products of the two ecotypes probably represent the same gene. Each ecotype
may therefore contain a different version of the AtPLP protein. AtPLP in ecotype
RLD would be a typical syntaxin-like protein, anchored in the membrane by a C-
terminal transmembrane region, whereas in Columbia the protein would lack the
transmembrane domain, and therefore is predicted to be soluble. Interestingly,
direct genomic sequencing of two different lines of the Columbia ecotype also
revealed a polymorphism at this site (Dr. C Schueller, MIPS Arabidopsis genomic
sequencing project, Max-Planck-lnstitut FIJr Biochemie Martinsried, Germany,
personal communication).

Expression Pattern of AtPLP

To determine in which tissues AtPLP is expressed, RNA hybridization
analysis was performed. Total RNA of flowers, leaves, roots and stems was
extracted from Arabidopsis ecotypes Columbia and RLD. Equal amounts of RNA
from the different tissues were separated on an agarose/formaldehyde gel and
probed using a 3' gene-specific RNA probe of AtPLP including the 3' part of the
translated region and the whole untranslated region. This probe specifically
recognizes AtPLP (Figure 2-4A), but not AtPEP12. Figure 2-4B shows that

AtPLP is expressed in both ecotypes and the expression patterns are similar to

52

 

Figure 2-4. AtPLP mRNA distribution pattern among tissues of Arabidopsis
ecotype Columbia and RLD. A) Dot blot to demonstrate the specificity of the
AtPLP probe. In vitro-transcribed mRNA of AtPLP and AtPEP12 (1 ng or 0.1 r129)
were applied to the membrane and hybridized with an in vitro transcribed a3 P
UTP-Iabeled RNA probe. B) RNA gel blot analysis of AtPLP expression in
different tissues of Arabidopsis. 20 pg of total RNA extracted from flowers (F) ,
leafs (L), roots (R) and stems (S) at the comparable stages of ecotypes Columbia
and RLD were separated by gel eletrophoresis, transferred to nylon membrane
and hybridized with the AtPLP-specific probe described in A. C) RNA gel blot
analysis of AtPEP12 expression in different tissues of Arabidopsis. Identical to B

except hybridized with a 32P-iobied AtPEP12 probe derived by random priming of
the whole cDNA.

53

 

1ng 0.1ng
PLP PEP12 PLP PEP12

 

     

Columbia RLD
F L R S F L R S

 

 

54

each other. Unlike AtPEP12, which is highly expressed in roots but low in leaves
(Figure 2-40), AtPLP was expressed at higher levels in leaves, flowers, and
stems than in roots.
AtPEP12p Antibodies Cross-react with AtPLP

The molecular weight of the in vitro-synthesized product of the AtPLP
cDNA is about 33 kDa, smaller than in vitro synthesized AtPEP12p (about 35
kDa, Figure 2-5). This is consistent with AtPLP from the Columbia ecotype
lacking the 17 amino acids at the carboxy-terminus that are present on
AtPEP12p. Antiserum generated against AtPEP12p (Conceicao et al., 1997) was
used to immunoprecipitate in vitro translation products of the AtPEP12 and
AtPLP cDNAs. The antibodies were able to efficiently immunoprecipitate the
AtPLP product in addition to AtPEP12p (Figure 5). This result indicates that the
antibody against AtPEP12p cross-reacts with AtPLP in vitro.
AtPLP Requires a Transmembrane Domain to Associate with Membranes in
vitro

The transmembrane domain is important for the function of syntaxin
homologues. There are two versions of AtPLP in different ecotypes of
Arabidopsis. One has a TMD and the other one does not. To determine whether
the TMD is essential for anchoring AtPLP to the membrane, an in vitro
membrane association assay was performed. AtPLP was synthesized in vitro
with or without the TMD and mixed post-translationally with canine pancreatic
microsomes to allow membrane insertion. Figure 2-6 shows that AtPLP + TMD

was associated with the microsomal fraction, but AtPLP-TMD was not. Thus, the

55

AtPEP12 AtPLP
T P I T P I

    

Figure 2-5. Immunoprecipitation of in vitro translation products of AtPEP12 and
AtPLP using antiserum against AtPEP12p. T: 1/25 of total In vitro translation
products. P: 1/10 of immunoprecipitated pellets using pre-immune serum. I: 1/10
of immunoprecipitated pellets using AtPEP12p antiserum. Molecular mass
markers in kilodaltons (kDa) are given on the left.

56

 

Figur92-6. In vitro membrane association assay for AtPLP i TMD. The mRNA
encoding AtPLP-TMD or AtPLP+TMD was translated in vitro and incubated with
canine pancreatic microsomes. Microsomes were pelleted and equal amounts of
the total reaction (T) or pellet (P) were separated by SDS-PAGE and visualized
by fluorography. Molecular weight markers in kilodaltons (KDa) are given on the
left.

57

C-terminal hydrophobic domain is likely to be critical for membrane association of
syntaxin homologues. AtPLP lacking this domain, unlike other syntaxin
homologues, probably would not associate with the membrane. If this were the
case in vivo, AtPLP in Columbia (i.e. without the TMD) would be cytosolic. To
test this, two ecotypes of Arabidopsis (RLD and Columbia) were examined using
the antiserum against AtPLP12p that cross-reacted with AtPLP in vitro. However,
no signal was detected in cytosolic fractions of either ecotype (Conceicao et al.,
1997, data not shown). This indicates that AtPLP is either not expressed at a

detectable level or it is associated with membranes by some other means.
Discussion

By searching the Arabidopsis EST database, an AtPEP12 homologue,
AtPLP was identified. The RNA distribution pattern of AtPLP was distinct from
any of the know Arabidopsis syntaxin homologues, including AtPEP12 (Bassham
et al., 1995), AtVAM3 (Sato et al., 1997) and KNOLLE (Lukowitz et al., 1996).
KNOLLE mRNA accumulates chiefly in flowers and siliques, and the protein is
probably involved in cell cytokinesis (Lukowitz et al., 1996), The AtPEP12 and
AtVAM3 mRNA level is high in roots but very low in leaves. In contrast, AtPLP
mRNA accumulated more in leaves than in roots. If similar mechanisms of
vesicle sorting, docking and fusion found in mammalian cells and yeast operate
in plant cells, there are likely to be several different syntaxin homologues located
on the surface of different syntaxin homologues located on the surface of
different organelles. AtPLP may encode a syntaxin homologue responsible for a

step in the secretory pathway different from AtPLP12p or AtVAM3p, or it may

58

perform its function in a separated developmental stage or tissue. For example,
there are known to be several pathways for the transport of soluble and
membrane proteins to the vacuole in plants (Gomez and Chrispeels, 1993,
Matsuoka et al., 1995, Hohl eta]., 1996). It is possible that AtPEP12p functions in
one pathway and the APLP in another.

Each syntaxin homologue is thought to be associated with a specific
membrane by its C-terminal hydrophobic domain. The cytosolic part of the
protein interacts with other factors of the secretory pathway. The lack of the
transmembrane domain in the Columbia from of AtPLP is of great interest. There
are some examples of syntaxin-like proteins without the transmembrane domain
in mammalian cells (Bennett et al., 1993; Hirai 1993; lbaraki et al., 1995;
Jagadish et al., 1997; Simonsen et al., 1998; Tang et al., 1998). Possibly the
result of alternative splicing. In our case, however, a frame shift probably
transforms this protein from an integral membrane protein to a soluble protein.
Based on in vitro experiments done here and with yeast Ss02p (Jantti et al.,
1994), syntaxin homologues without the C-terminal hydrophobic domain cannot
insert into the membrane. It is possible that they are localized in the cytosol and
function by binding to other factors, such as synaptobrevin, NSF etc. that
normally interact with membrane-associated syntaxin homologues. These
soluble forms of syntaxin homologues apparently occur in both mammalian cells
and plant. Thus, they may play some specialized role. These proteins may
regulate vesicle sorting, clocking and fusion by binding to factors interacting with

membrane-bound syntaxins.

59

In Arabidopsis ecotype Columbia, AtPLP without the transmembrane
domain is expressed at least at the mRNA level, and antiserum raised against
AtPEP12p cross-reacts with the in vitro translation product of AtPLP. Using this
antiserum, it would be expected that AtPEP12p and AtPLP are localized both to
the membrane (AtPEP12p) and cytosolic (AtPLP) fractions of plant protein
extracts. However, no signal has ever been detected in the soluble fraction (data
not shown) using these antibodies. The AtPLP protein level may not be high
enough to be detected by the antibody. It is also possible that this protein still
associates with the membrane by a post-translational modification or through
protein-protein interactions. In that case, AtPLP could perform a normal function
as a syntaxin homologue in plants. The authors had some difficulty in generating
a specific antibody against this protein that dos not cross-react with AtPEP12p.
Therefore, in order to determine the subcelluar location of this protein, transgenic
plants containing epitope-tagged AtPLP are being generated. Introducing the
untagged AtPLP gene (Without the TMD) into plant will also be useful to
investigate possible dominant negative or regulatory effects of this syntaxin
homologue in plants.

Even before the genomic sequencing of Arabidopsis has been completed,
genes encoding putative syntaxin-like proteins have been identified which do not
exist in yeast. This is not surprising, given the complexity of different tissues and
developmental stages in plants. It is expected that the complete sequence of the
Arabidopsis genome will reveal that the total number of syntaxin-like proteins in

plants is greater than that in yeast. The challenge for the future will be to discern

60

the function of all of these homologues and their role in vesicle trafficking

between different organelles of the secretory pathway and in different cell types.

61

References

Aalto MK, Ronne H, and Keranen S (1993). Yeast syntaxins 8301p and Ss02p
belong to a family of related membrane proteins that function in vesicular
tranport. EMBO J. 1 2: 4095-4104

Ahmed SU, Bar-Peled M, and Raikhel NV (1997). Cloning and subcellular
localization of an Arabidopsis receptor-like protein that Shares common features
with protein-sorting receptors of eukaryotic cells. Plant Phys. 114: 325-336.

Altschul SF, Gish V, Miller W, Myers EW, and Lipman DJ (1990). Basic local
alignment search tool. J. Mol. Biol. 215: 403-410.

Banfield DK, Lewis MJ, Rabouille C, Warren G, and Pelham HRB (1994).
Localization of Sed5, a putative vesicle targeting molecule to the cis-Golgi

network, involves both its transmembrane and cytoplasmic domains. J. Cell Biol
127: 357-371.

Bar-Peled M, Conceicao AS, Frigerio L, and Raikhel NV (1995). Expression
and regulation of aERDZ, a gene encoding the KDEL receptor homologue in

plants, and other genes encoding proteins involved in ER-Golgi vesicular
trafficking. Plant Cell 7: 667-676.

Bassham DC, Gal S, Conceicao AS, and Raikhel NV (1995). An Arabidopsis
syntaxin homologue isolated by functional complementation of a yeast pep12
mutant. Pro. Nat. Acad. Sci. USA 92: 7262-7266.

Becherer KA, Rieder SE, Emr SD, and Jones EW (1996). Novel syntaxin
homologue, Pep12p, required for the sorting of lumenal hydrolases to the
Iysosome-like vacuole in yeast. Mol. Biol. Cell 7: 579-594.

Bednarek SY, Wiklins TA, Dombrowski JE, and Raikhel NV (1990). A
carboxy-terminal propeptide is necessary for proper sorting of barley lectin to
vacuoles of tobacco. Plant Cell 2: 1 145-1 155.

Bennett MK (1995). SNAREs and the specificity of transport vesicle targeting
Curr. Opin. Cell Biol. 7: 581-586.

Bennett MK, Calakos N, and Scheller RH (1992). Syntaxin: a synaptic protein
implicated in docking of synaptic vesicles at presynaptic active zones. Science
257: 255-259.

Bennett MK, Garcia-Arraras JE, Elferink LA, Peterson K, Fleming AM,

Hazuka CD, and Scheller RH (1993). The syntaxin family of vesiclar transport
receptors. Cell 74: 863-873.

62

Conceicao AS, Marty-Mazars D, Bassham DC, Sanderfoot AA, and Marty F,
Raikhel NV (1997). The syntaxin homologue AtPEP12p resides on a late post-
Golgi compartment in plants. Plant cell 9: 571 -582.

Darsow T, Rieder SE, and Emr SD (1997). A multispecificity syntaxin
homologue, Vam3p, essential for autophagic and biosynthetic protein transport to
the vacuole. J. Cell Biol. 138: 517-529.

Di Sansebastiano GP, Paris N, Marc-Martin S, and Neuhaus JM (1998).
Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-
terminal propeptide-mediated sorting pathway. Plant J. 15: 449-457.

Dombrowski JE, Schroeder MR, Bednarek SY, and Raikhel NV (1993).
Determination of the functional elements within the vacuolar targeting signal of
barley lectin. Plant Cell 5: 1113-1124.

Ferro-Novick S, and Jahn R (1994). Vesicle fusion from yeast to man Nature
370: 191-193.

Gomez L, and Chrispeel MJ (1993). Tonoplast and soluble vacuolar proteins
are targeted by different mechanisms Plant Cell 5: 1113-1124.

Hirai Y (1993). Molecular cloning of human epimorphin. Biochem. Biophys. Res.
Commun. 191: 1332-1337.

Hohl l, Robinson DG, Chrispeels MJ, and Him G (1996). Transport of storage
proteins to the vacuole is mediated by vesicles without a clathrin coat. J. Cell Sci.
109: 2539-2550.

Holwerda BC, Padgett HS, and Rogers JC (1992). Proaleurain vacuolar
targeting is mediated by short contiguous peptide interactions. Plant Cell 4: 307-
318.

lbaraki K, Horikawa HP, Morita T, Mori H, Sakimura K, Mishina M, Saisu J,
and Abe T (1995). Identification of four different forms of syntaxin 3. Biochemi.
Biophys. Res. Commun. 211: 997-1005.

Jagadish IIlIN, Tellam JT, Macaulay SL, Gough KH, James DE, and wsard
CW (1997) Novel isoform of syntaxin 1 is expressed in mammalian cells.
Biochem. J. 321: 151-156.

Jéintti J, Kerfinen S, Toikkanen J, Kuismanen E, Ehnholm C, deerlumd J,
and Olkkonen VM (1994) Membrane insertion and intracellular transport of
yeast syntaxin SsoZp in mammalian cells. J. Cell Sci. 107: 3623-3633.

63

Kirsch T, Paris N, Bulter JM, Beevers L, and Rogers JC (1994). Purification
and initial characterization of a potential plant vacuolar targeting receptor. Proc.
Nat]. Acad. Sci. USA 91: 3403-3407.

Krisch I, Sallbach G, raikhel NV, and Beevers L (1996). Interaction of a
potential vacuolar targeting receptor with amino- and carboxy-terminal targeting
determinants. Plant Physio]. 1 11 : 469-474.

Lauber MH, Waizenegger I,'Steinmann T, Schwarz H, Mayer U, Hwang I,
Lukowitz W, and Jiirgens G (1997). The Arabidopsis KNOLLE protein is a
cytokinesis-specific syntaxin. J. Cell Biol. 139: 1485-1493.

Lukowitz W, Mayer U, and Jiirgens G (1996) Cytokinesis in the Arabidopsis
embryo involves the syntaxin-related KNOLLE gene product. Cell 84: 61-71.

Matsuoka K, Bassham DC, Raikhel NV, and Nakamura K (1995). Different
sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of
distinct sorting machineries in tobacco cells. J. Cell Biol. 130: 1307-1318.

Matsuoka K, and Nakamura K (1991). Propeptides of a precursor to a plant
vacuolar protein required for vacuolar targeting. Proc. Nat]. Acad. Sci. USA 88:
834-838.

Murray MG, and Thompson WF (1980). Rapid isolation f high molecular weight
plant DNA. Nucleic Acids Res. 8: 4321-4325.

Makamura K, Matsuoka K, Mikumoto F, and Watanabe N (1993). Processing
and transport to the vacuole of a precursor to sweet potato sporamin in
transformed tobacco cell like BY-2. J. Expen'. Bot. 44 (Supplement): 331-338.

Neuhaus JM, Pietrzak M, and Boller T (1994). Mutation analysis of the C-
terminal vacuolar targeting peptide of tobacco transition between intracellular
retension and secretion into the extracellular space. Plant J. 5: 45-54.

Neuhus JM Sticher KL, Meins F, and Boller T (1991). A short C-terminal
sequence is necessary and sufficient for the targeting of chitinases to the plant
vacuole. Proc. Natl. Acad. Sci. USA 88: 10362-10366.

Newman T, de Bruiin FJ, Green P, Keegstra K, Kende H, McIntosh L,
Ohirogge K, Raikhel N, Sommerville S, Thomashow M, Retzel E, and
Somerville C (1994). Genes galore: a summary of methods for accessing results
from large -scale partial sequencing of anonymous Arabidopsis cDNA clones.
Plant Phys. 106: 1241-1255.

Paris N, Stanley CM, Jones RJ, and Rogers JC (1996). Plant cells contain two
functionally distinct vacuolar compartments. Cell 85: 563-572.

64

Rothman JE (1996). The protein machinery of vesicle budding and fusion
Protein Sci 5: 185-194.

Saalbach G, Jung R, Kunze G, Saalbach I, Adler K, and Miintz K (1991).
Different Iegumin protein domains act as vacuolar targeting signals. Plant Cell 3:
695-708. ’

Sambrook K, Frisch EF, and Maniatis T (1989). Molecular Cloning, a
laboratory manual 2"d ed. NY: Cold Spring Harbor Laboratory Press.

Sanderfoot AA, Ahmed SU, Marty-Mazars D, Rapoport I, Kirshhausen T,
Marty F, and Raikhel NV (1998). A putative vacuolar cargo receptor partially
colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots.
Proc. Nat]. Acad. Sci. USA 95: 9920-9925.

Sato MH, Nakamura N, Ohsumi Y, Kouchi H, Kondo M, Hara-Nishimura I,
Mishimura M, and Wada Y (1997). The AtVAM3 encodes a syntaxin-related
mulecule implicated in the vacuolar assembly in Arabidopsis thaliana. J. Bio.
Chem. 272: 24530-24535.

Schroeder MR. Borkhsenious ON, Matsuoka K, Nakamura K, and Raikhel
NV (1993). Colocalization of barley lectin and sporamin in vacuoles of trangenic
tobacco plants. Plant Physol 101: 451-458.

Simonsen A, Bremners B, Ronning E, Aasland R, and Stenmark H (1998).
Syntaxin -16, a putative Golgi t-SNARE. Eur. J. Cell Biol. 75: 223—231.

Sbllner I, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S,
Tempst P, and Rothman JE (1993). SNAP receptors implicated in vesicle
trageting and fusion. Nature 362: 318-324.

Tang BL, Low DYH, and Hong WJ (1998). Syntaxin 11: a member of the
syntaxin family without a carboxyl terminal transmembrane domain. Biochem.
Biophys. Res. Commun. 245: 627-632.

Von Schaewen A, and Chrispeels MJ (1993). Identification of vacuolar sorting
information in phytohemagglutinin, an unprocessed vacuolar protein. J. Expen'.
Bot. 44 (Supplement): 339-342.

Wada Y, Nakamura N, Obsumi Y, and Hirata A (1997). Vam3p, a new member
of syntaxin-related proteins, is required for vacuolar assembly in the yeast
Saccharomyces cerevisiae. J. Cell Sci. 110: 1299-1306.

Wang H, Frelin L, and Pevsner J (1997). Human syntaxin 7: a Pep12prs6p
homologue implicated in vesicle trafficking to lysosomes. Gene 199: 39-48.

65

Chapter III

The Plant V-SNARE AtVTI1a Likely Mediates Vesicle Transport from the TGN

to the PVC

This chapter was published in :
Haiyan Zheng, Gabriele Fischer von Mollard, Valentina Kovaleva, Tom H.
Stevens, and Natasha V. Raikhel (1999) The plant vesicle associated SNARE
AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the

prevacuolar compartment. Molecular Biology Of the Cell 10: 2251-2264.

Z.H. and G.F.M. contributed equally to this work. H.Z. has major contributions to
all the figures except yeast complementation experiments were done in
collaboration with G.F.M, and the immunocytochemical work was done in

collaboration with V.K.

66

Abstract

Membrane traffic in eukaryotic cells relies on recognition between v-
SNAREs on transport vesicles and t-SNAREs on target membranes. Here we
report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of
the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast.
AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32
and 30% identical to the yeast protein, respectively. By suppressing defects
found in specific strains of yeast vti1 temperature sensitive mutants, we show
that AtVTI1a can substitute for Vti1p in Golgi-to-PVC transport, whereas AtVTI1b
substitutes in two alternative pathways: the vacuolar import of alkaline
phosphatase and the so-called cytosol-to-vacuole pathway utilized by
aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs
of Arabidopsis. Using subcellular fractionation and immuno—electron microscopy,
we show that AtVTI1a co-localizes with the putative vacuolar cargo receptor
AtELP on the trans-Golgi network (TGN) and the prevacuolar compartment
(PVC). AtVTI1a also co-Iocalizes with the t-SNARE AtPEP12p to the PVC. In
addition, AtVTI1a and AtPEP12p can be co-immunoprecipitated from plant cell
extracts. We propose that AtVTI1a functions as a v-SNARE responsible for
targeting AtELP-containing vesicles from the TGN to the PVC, and that AtVTI1b

is involved in a different membrane transport process.

67

Introdcution

In the secretory and endocytic pathways, the movement of proteins and
membranes from one location to another relies mostly on vesicular transport.
One fundamental question is how the vesicles recognize the correct target
membrane. The SNARE hypothesis offers a widely accepted explanation of the
mechanism of specificity in vesicle targeting (Sbllner et al., 1993). SNAREs
(SNAP receptors) are membrane proteins found on both transport vesicles (v-
SNARE) and target organelles (t-SNARE). The specific interactions between t-
and v-SNAREs ensure that vesicles are targeted to the correct compartment and
lead to membrane fusion. The best-characterized SNARE complex consists of
syntaxin, SNAP25 (t-SNAREs on the presynaptic membrane), and VAMP-
1/synaptobrevin (v-SNARE on synaptic vesicles); it is involved in synaptic vesicle
exocytosis (Hanson et al., 1997; Sutton et al., 1998). Homologues of these
SNAREs are found to be involved in intracellular vesicle transport processes in
yeast and mammalian systems, further supporting this hypothesis (for review see
Hay and Scheller, 1997). Several t-SNAREs have been found in plant cells
recently (Bassham et al., 1995; Lukowitz et al., 1996; Sato et al., 1997; Zheng et
al., 1999), suggesting that the SNARE hypothesis also applies to plant cells.

Much of our knowledge about vesicular transport to the vacuole has been
gained from yeast studies. Several pathways to the yeast vacuole have been
described. The best characterized pathway for delivery of soluble proteins to the
vacuole is the carboxypeptidase Y (CPY) pathway. At the trans-Golgi or trans-

Golgi network (TGN), CPY is bound by its receptor (Pep1prs10p) and

68

packaged into transport vesicles. These vesicles then fuse with the prevacuolar
compartment (PVC)/Iate endosome. The PVC t-SNARE Pep12p is required for
correct sorting of CPY (Jones, 1977; Becherer et al., 1996). The v-SNARE Vti1p
interacts both genetically and biochemically with Pep12p (Fischer von Mollard et
al., 1997). It was thus proposed that Vti1p and Pep12p form a SNARE complex
that is involved in docking and fusion of TGN-derived transport vesicles with the
PVC. It has recently been reported that a subset of proteins, including alkaline
phosphatase (ALP), is transported to the vacuole by an alternative route,
independent of the CPY pathway, that bypasses the PVC (Cowles et al., 1997a;
Piper et al., 1997). This transport pathway requires the AP-3 adaptor complex
(Cowles et al., 1997b; Stepp et al., 1997) and Vam3p, the vacuolar t-SNARE
(Darsow et al., 1997; Piper et al., 1997; Wada et al., 1997; Srivastava and Jones,
1998). Vti1p has very recently been implicated as the v-SNARE that interacts
with Vam3p in the ALP pathway to the yeast vacuole (Fischer von Mollard and
Stevens, 1999). Another route to the vacuole, directly from the cytoplasm, has
recently been analyzed using the hydrolase aminopeptidase I (API) (Klionsky,
1998). This cytosol-to-vacuole transport (Cvt) pathway is blocked in vam3 mutant
cells (Darsow et al., 1997; Srivastava and Jones, 1998) as well as in vti1 mutant
cells (Fischer von Mollard and Stevens, 1999). Thus, in yeast, multiple pathways
are used for delivering vacuolar proteins, all of which require Vti1p. In addition to
a role in transport pathways to the vacuole, Vti1p also functions in retrograde
transport within the Golgi complex by interacting with the cis-Golgi t-SNARE

Sed5p (Lupashin et al., 1997; Fischer von Mollard et al., 1997). Furthermore,

69

 

 

prote

the r
terrr
Alth
plot
that
cor
Ar,-
Cor

log

Stri

195

101

pro

Holthuis et a]. (1998) reported the biochemical interaction of Vti1p with two
additional yeast Golgi/endosomal t-SNAREs, Tlg1p and TIgZp. Taken together,
these data suggest that Vti1p is a v-SNARE involved in multiple membrane
transport pathways in yeast.

In plants, three types of vacuolar sorting signals (VSSs) have been
identified (for review see Bassham and Raikhel, 1997). These VSSs can occur in
the form of a propeptide (either N-terminal or C-terminal) that is removed
proteolytically during or after transport to the vacuole, or they can form a part of
the mature protein. Interestingly, plant vacuolar proteins with N-terminal and C-
terminal VSSs appear to use independent pathways (Matsuoka et al., 1995).
Although very little information is available on the targeting signals of tonoplast
proteins in plants, it is known that they are transported by a different mechanism
than that of soluble vacuolar proteins (Gomez and Chrispeels, 1993). Several
components of the plant secretory machinery have been isolated as well. In
Arabidopsis, a Pep12p homologue, AtPEP12p, is found by its ability to
complement a yeast pep12 mutant (Bassham et al., 1995). AtPEP12p is
localized to a novel compartment by EM and biochemical analysis (Conceicao et
al., 1997; Sanderfoot et al., 1998). AtELP was identified in Arabidopsis by its
structural similarity to the EGF receptor and other cargo receptors (Ahmed et al.,
1997). AtELP is enriched in clathrin-coated vesicles (CCVs), it has been localized
to the TGN, and colocalized with AtPEP12p on the PVC by electron microscopy
(Ahmed et al., 1997; Sanderfoot et al., 1998). AtELP is homologous to BP-80, a

protein from pea CCVs that has been shown to bind a broad range of plant

70

 

VSSs
anofi:
sequr
daba

sonu
AfiDE
rnan-
TTSN

ther

encr
gen.
Year
cok:
Fmvc
AtPl
PTO;
Carg

Mat

Fuas

VSSs, but not to the C-terminal VSSs (Kirsch et al., 1994, 1996). Recently,
another AtELP-homologue from pumpkin has been found to recognize certain
sequence patches in some cargo proteins (Shimada et al., 1997). All of these
data support the notion that AtELP is a cargo receptor involved in transport of
some but not all vacuolar proteins. It is postulated that the compartment where
AtPEP12p resides is the equivalent of the PVC in yeast or the late endosome in
mammalian cells. This compartment accepts the transport vesicles formed at the
TGN as CCVs. Those vesicles contain at least a subset of vacuolar proteins and
the receptors (such as AtELP) involved in packaging them at the TGN.

We have identified two Arabidopsis genes (AtVTI1a and AtVTI1b)
encoding proteins homologous to yeast Vti1p. Although each Arabidopsis VTI1
gene can function in yeast, they function in different sorting pathways to the
yeast vacuole. By studying T7-epitope-tagged AtVTI1a, we found that AtVTI1a
colocalized with the putative vacuolar cargo receptor AtELP on the TGN and the
PVC, and with AtPEP12p on the PVC. Co-immunoprecipitation of AtVTI1a with
AtPEP12p suggested that these two proteins associate in the cell. Thus, we
propose that AtVTI1a is a plant v-SNARE involved in the transport of vacuolar

cargo from the Golgi to the PVC.
Materials and Methods
Plasmids, Yeast Strains and Growth Media
Mutant strains of vti1 were derived from the yeast strains SEY6210 (MA T_
]eu2-3,112 ura3—52 his3-A200 trp1-AQO1 ]y82-801 sch-AQ mel-) and SEY6211

(MATa ]eu2-3,112 ura3-52 his3-A200 trp1-A901 ade2-101 sch-AQ me]-

71

 

Iii...—

i'

(Robin
(FVMY;
been d
Steven
GALI-

mutatic

introdu
codon:
vector
AtVTI1
Open r
the sa
fusion
Subck
expres
flagmi
loo, F
the A
CYTOpl

Coll B

(Robinson et al., 1988). The strains vti1A(FvMY6), vti1-1 (FvMY7), vti1-2
(FvMY24), and vti1-11 (FvMY21) and the GAL1-VTI1 plasmid (vaM16) have
been described earlier (Fischer von Mollard et al., 1997; Fischer von Mollard and
Stevens, 1998). The vti1A yeast strain (FvMY6) was propagated carrying the
GAL1-VTI1 plasmid (vaM16) in the presence of galactose, because the WM
mutation is lethal to yeast cells.

To express AtVTI1a and AtVTI1b in yeast, BamHI and PstI sites were
introduced by PCR into AtVTI1a and AtVTI1b cDNAs flanking the start and stop
codons. The BamHl/Pst] fragments were inserted into the yeast expression
vector pVT102U (Vernet et al., 1987). To construct N-terminal T7-tagged
AtVTI1a, BamHI and Sal] sites were generated by PCR flanking the AtVTI1a
open reading frame. The BamHI/Sall fragment of AtVTI1a was then inserted into
the same sites of pET21a (Novagen, Madison, WI) to create a T7-N-terminal
fusion of AtVTI1a (pE'I'l'7-AtVTl1a). The T7-AtVT/1a fragment was then
subcloned into the Xbal and Xho] sites of the pVT102U vector for yeast
expression. To construct pBI-T7-AtVTI1a for plant transformation, the Xbal/Sac]
fragment of pETI'7-AtVTI1a was subcloned into pBl121 (Clontech Laboratories,
Inc., Palo Alto, CA). For E. coli overexpression of 6xHis-AtVTl1a, the Nde] site at
the ATG start codon and the BamHI site immediately downstream of the
cytoplasmic domain were introduced by PCR amplification. The NdeI/BamH]
fragment of AtVTI1a was then subcloned into pET14b and transformed into E.

coli BL21(DE3) cells for overexpression.

72

Yeast strains were grown in rich medium (1% yeast extract, 1% peptone,
2% dextrose, YEPD) or standard minimal medium (SD) with appropriate
supplements (Fischer von Mollard et al., 1997). To induce expression from the

GAL1 promoter, dextrose was replaced by 2% raffinose and 2% galactose.

Immunoprecipitation of 35S-Iabeled yeast proteins

CPY, ALP, and API were immunoprecipitated as described earlier
(Klionsky et al., 1992; Vater et al., 1992; Nothwehr et al., 1993). SEY6211 wild-
type cells and vti1 mutant cells were grown at 24°C and preincubated for 15 min
at 36°C before labeling at 36°C.

For CPY immunoprecipitations, log-phase growing yeast cells were

labeled for 10 min with 35S-Express label (10 III/0.5 on of cells) followed by a
30-min chase with cysteine and methionine. The medium was separated and the
cell pellet spheroplasted and Iysed. CPY was immunoprecipitated from the
medium and cellular extracts. For ALP immunoprecipitations, yeast cells were
labeled for 7 min and chased for 30 min. The cell pellet was spheroplasted. The
spheroplast pellet was extracted with 50 pl 1% SDS, 8M urea at 95°C, and
diluted with 950 pl 90 mM Tris-HCI pH 8.0, 0.1% Triton X-100, 2 mM EDTA; the

supernatant was used for immunoprecipitations. To investigate API traffic, 0.25

OD of yeast cells in 500 pl medium were labeled with 10 pl 35S-Express label for

each time point. After a 10 min pulse, cells were chased for 120 min. The cell
pellet was spheroplasted. Extracts for immunoprecipitations were prepared from

spheroplast pellets by boiling in 50 pl 50 mM sodium phospate, pH 7.0, 1% SDS,

3 M urea and diluted with 950 pl 50 mM Tris-HCI, pH 7.5, 0.5% Triton X-100, 150

73

 

mhll
Imnu
UgCh
radioi

RNAI

Baol
phen
moot
from
conc

S‘FL

Gan
use:
TAI
IAni
Pri
CC
thr

I“

(I

mM NaCl, 0.1 mM EDTA. The API antiserum was kindly provided by D. Klionsky.
lmmuno complexes were precipitated using fixed cells of Staphylococcus aureus
(IgGsorb). Immunoprecipitates were analyzed by SDS-PAGE and auto-
radiography.

RNA Preparation from Arabidopsis

Total RNA extraction from different plant organs was performed based on
Bar-Peled and Raikhel (1997), except that the RNA was further purified by
phenol:chloroformzisoamyl alcohol (25:24:1 by vol.) and chloroformzisoamyl
alcohol (24:1, v/v) extraction followed by ethanol precipitation. Purified total RNA
from one gram of tissue was resuspended in 200 pl DEPC-treated water. The
concentration of the RNA was determined by the ODzeo value.

5’ RACE (Rapid Amplification of cDNA 5’ Ends)

5’ RACE was performed according to the manufacturer’s (Gibco BRL,
Gaithersburg, MD) instruction. Total RNA (0.5 pg) from Arabidopsis roots was
used as a template and the required amount of Primer 1 (5’-GTG AGT TTG AAG
TAC AA-3’) was used for the first-strand cDNA synthesis. 5’ RACE Abridged
Anchor Primer (AAP) supplied by the manufacturer was used as a sense primer.
Primer 2 (5’- TGC GAT GAT GAT GGC TCC AA -3') and Primer 3 (5’- GTT CAT
CCT CCT CGT CAT -3’) were used as antisense primers for the first round and
the following nested PCR reactions, respectively. DNA fragments produced from
nested PCR were end-blunted, cloned into Bluescript SK(-) (Stratagene Cloning
Systems, La Jolla, CA) and manually sequenced using Sequenase Version 2.0

(United States Biochemical, Cleveland, OH).

74

 

RNA

each
by 82
(Arne
mote
Hybc

Anfit

taggi
Ifiadi
anfit
desc
seru
th

desr

55ub

Ce”

KOI

Phi

dlS(

RNA Blot Analysis

For northern analysis, 20 pg of Arabidopsis total RNA were applied to
each lane of a formaldehyde denaturing agarose gel and separated as described
by Sambrook et a]. (1989). Separated RNA was then transferred to a Hybond-N
(Amersham Life Science, Buckinghamshire, England) nylon membrane. For dot

blot analysis, various amounts of in vitro transcribed mRNA were applied to a

Hybond-N membrane. Blots were hybridized with 32P-UTP labeled RNA probes.
Antibody Production

6XHis-tagged AtVTI1a was overexpressed by IPTG induction. The His-
tagged protein was purified by passing through a Ni-NTA column (Novagen,
Madison, WI). The purified protein was then injected into a guinea pig for
antibody production. AtPEP12p rabbit antiserum and pre-immune serum were
described in Conceicao et a]. (1997). AtELP rabbit antiserum and pre-immune
serum were described in Ahmed eta]. (1997). HT-pyrophosphatase antibody is a
gift from Dr. S. Yoshida (Hokkaido University, Sapporo, Japan) and was
described in Maeshima and Yoshida (1989).
Subcellular Fractionation

To fractionate subcellular compartments based on their mass, differential
centrifugation was performed as follows: 0.5 grams of Arabidopsis root cultures
(21 days old) were homogenized in one milliliter extraction buffer (50 mM Hepes-
KOH, pH 7.5, 10 mM KOAc, 1 mM EDTA, 0.4 M sucrose, 1 mM D'I'I' and 0.1 mM
PMSF). The Iysate was centrifuged at 4°C, 1,000 g, for 10 min. The pellet was

discarded and the supernatant (S1) was then centrifuged at 4°C, 8,000 g, for 20

75

 

mil

Th

US

SU

A]

min. The pellet (P8) was resuspended in 200 pl of 2x Laemmli loading buffer.
This supernatant was ultracentrifuged at 4°C, 100,000 g, for 2 hrs. The pellet
(P100) was resuspended in 200 pl of 2x Laemmli loading buffer. The supernatant
(8100), P8, and P100 were analyzed by SDS-PAGE, followed by immunoblotting
using different antibodies.

Based on density differences, the microsomes were separated on a step
sucrose gradient as described in Sanderfoot et a]. (1998).
Arabidopsis Transformation

pBI-T7-AtVTI1a was introduced into Agrobacten'um tumefaciens LBA4404
by CaClz-based transformation. Arabidopsis Columbia plants were transformed
using vacuum infiltration as described in Bent et a]. (1994). Transformants were
selected by kanamycin, and the presence of T7-AtVTI1a was detected in several
independent lines by protein gel blot analysis using T7-monoclonal antibody
(Novagen, Madison, WI) and guinea pig polyclonal antiserum against AtVTI1a.
EM Procedure

The root tips of Arabidopsis plants transformed with T7-AtVTI1a were
fixed in a buffer containing 1.5% formaldehyde, 0.5% glutaraldehyde, and 0.05 M
sodium phosphate, pH 7.4, for 2.5 hrs at room temperature. The specimens were
rinsed in the same buffer and post-fixed in 0.5% 0304 for 1 hr at room
temperature. Dehydrated specimens were embedded in London Resin White
(Polysciences, Warrington, PA). Ultrathin sections were made with an Ultracut S
microtome (Reichert-Jung, Vienna, Austria) by a diamond knife and collected on

nickel grids precoated with 0.25% Fonnvar.

76

 

str
gri
us
PE
lat
ar

ar

(0

For immunolabeling, the protocol according to Sanderfoot et a]. (1998)
was used with modification. Primary mouse monoclonal antibody against T7
epitope tag (Novagen, Madison, WI) were detected by rabbit-anti-mouse IgG for
1 hr, followed by biotinylated goat anti-rabbit IgG for 1 hr, and then by
streptavidin conjugated to 10 nm colloidal gold particles. For double labeling, the
grids were first treated as above for T7 tag antibody, then a second fixation step
using 0.1% glutaraldehyde, followed by a second blocking step with 2% BSA in
PBST (PBS+0.1% Tween20) to prevent cross-reactivity of the T7 tag-antibody in
later steps (Slot et al., 1991). The grids were then incubated with specific rabbit
antiserum for AtELP for 4 hrs, followed by a 1-hr incubation with biotinylated goat
anti-rabbit IgG and then by streptavidin conjugated to 5 nm colloidal gold
particles. The control sections were treated with 2% BSA in PBST instead of
antibody against the T7-tag and with the AtELP preimmune serum. The grids
were washed in distilled water and stained with 2% uranyl acetate in H20 for 30
min and lead citrate for 10 min (Reynold’s Solution). The sections were examined
with a Philips CM 10 transmission electron microscope. All labeling experiments
were conducted several times each on independent sections. Fifty Golgi
complexes were analyzed for AtVTI1a distribution and forty complexes for
double-immunolabeling of AtVTI1a and AtELP.

Cryosections of Arabidopsis roots were utilized for investigation of
AtVTI1a and AtPEP12p co-localization. The sectioning procedure was described
in Sanderfoot et al. (1998). Immunolabeling was also performed as described in

Sanderfoot eta]. (1998) with some modifications. T7-AtVTI1a localization was

77

 

dete
with
and
was
acct

Imn

extr
mIt-l
(101
was
the

This

cen
butt
Glut
llov
imn
Re:

The

(All

detected as described above when LR White sections were used and visualized
with 10 nm colloidal gold. AtPEP12p was detected using AtPEP12p antiserum
and visualized with 5 nm colloidal gold. For final embedding, the grids were
washed and stained by a mixture of polyvinyl alcohol and uranyl acetate
according to Tokuyasu (1989).
Immunopurification of T7-AtVTI1a From Plant Extract

Three grams of 21-day-old plants were homogenized on ice in 6 ml of
extraction buffer (50 mM HEPES-KOH, pH 6.5, 10 mM potassium acetate, 100
mM sodium chloride, 5 mM EDTA, 0.4 M sucrose) with protease inhibitor cocktail
(100 pM PMSF, 1 pM pepstatin, 0.3 pM aprotinin, 20 pM Ieupeptin). The debris
was pelleted by centrifugation at 1,000 g for 10 min. Triton X-100 was added to
the supernatant to final concentration of 1% to solubilize membrane proteins.
This solubilized protein extract was incubated with 50 pl T7-tag antibody agarose
(Novagen, Madison, WI) at 4°C for 5 hrs. The agarose was then collected by
centrifugation at 4°C, 500 g, for one minute and washed 5 times in extraction
buffer + 1% Triton X-100. Protein purified by T7-tag antibody agarose was then
eluted in 50 pl 2x Laemmli buffer. Equal volume of total protein extract,
flowthrough or eluate were separated on a SDS-PAGE followed by
immunoblotting using different antibodies.

Results

There Are Two Highly Similar AVTI‘I Genes Found in Arabidopsis
A search of the Arabidopsis EST database using the Blast program

(Altschul et al., 1990) resulted in a partial sequence that showed similarity to

78

 

yeas

chl

sea!

(\0.

22-

SN

8?

yeast Vti1p. 5’ RACE was performed to obtain the upstream sequence of this
cDNA. With this 5’ RACE sequence, the Arabidopsis EST database was
searched again and two sets of EST clones were found. The clone (accession
no. T14238) containing an open reading frame of 221 amino acids was termed
AtVTI1a. The clone (accession no. T75644) containing an open reading frame of
224 amino acids was termed AtVTI1b (Figure 3-1). These two genes share
similarity at the nucleotide sequence level (58.4% identity) and the deduced
amino acid sequence level (59.5% identity; see Table 3-1). Hydropathy analysis
(Kyte and Doolittle, 1982) predicted similar structures for AtVTI1a and AtVTI1b
proteins (our umpublished results). The sequences predicted hydrophilic proteins
with a short hydrophobic region at their extreme C-termini (Figure 3-1,
underlined), possibly serving as a membrane anchor. The region immediately
preceding the probable membrane-spanning domain contains two heptad repeat
structures that would potentially form amphiphilic alpha helices. Predicted amino
acid sequences of these two AtVTI1 and Vti1 proteins found in other organisms
were compared using the J. Hein method in the MegAlign program (DNAStar
software package) (Figure 3-1 and Table 3-1). The alignment showed that Vti1
proteins exhibit significant similarities between yeast, mammals and plants
(thi1p and AtVTI1a: 32.4% identical, thi1p and AtVTI1b: 30.8% identical,
thi1p and thi1p: 23.9% identical, thi1p and thi1a: 33.8% identical, thi1p
and thi1b: 23.5% identical). All Vti1 proteins have a short hydrophobic region at
the C-terminus. The most conserved amino acid residues among Vti1 proteins

are concentrated in the heptad repeat region next to the transmembrane

79

 

Figure 3-1. Sequence comparison of AtVTI1a and AtVTI1b with other members
of the family including thi1p (Saccharomyces cerevisiae, accession no.
2497184), thi1p (Homo sapiens, accession no. 268740), thi1a (Mus
musculus, accession no. 3213227), thi1b (Mus musculus, accession no.
3213229). The sequence comparison was generated using the J. Hein method in
MegAlign (DNASTAR© program). Amino acids identical with thi1p are shaded
in black. The C-terminal hydrophobic domain is underlined.

8O

   
  
    
 
 
  
 
  
 
 
 
 
 
 
 
  
 
 
  
 
 

     
 

SKKCSSAIS -DG-- AtVI‘Ila
SRKCHSASV SNG—- AtVTI1b
---QAKAS EA-Pthil
NLQGVPERLLGthil
P
G

EITSKIIRV- thila
DLQGVPERLL thilb

Hi—H—IHHH

     
    
 
 

 

           

 

32 ----EQKKQKLSEIKSGLEN AtVTI1a
33 ———-EEKKGKIAQIKSGI AtVI‘Ilb
28 SQPLSQRNTTLKHIEQQ thil
35 TAGTEEKKKLIRDFDEKV‘TQ thil
28 PDEKKQMVANIEKQLE thila
35 ‘ thilb
66 KT ., AtVTI1a
67 KK vss AtVTI1b
68 Q5 PLQ thil
73 NYRKDLAKLHREVRSTPL thil
66 SYIQEMGKLETDF-SRI thila
73 NYRKDLAKLHREVRSTPL mm
105 ----- LEAGMADTKTAS-A AtVI‘Ila
106 ----- MESGMADLHAVS-A AtVTI1b
104 ----- FG--— - LNA-NID — D rm :;:, thil
]J2YGIYAVEN-—EHMN--RLQS . thi1
104 ----- AGN.--S—ENi- : ER-SR-LEthila
112YGTYT EHLN—-RLQS NRATQSIEthilb
139-s AtVI'Ila
140 ES AtVTI1b
136 thi1
148 thl
134 thila
148 thilb
179 T AtVI'Ila
180 K T AtVTI1b
176 K Wtil
188 9 R R - L L L s I thi1
174 .3. S T L L V I L G thila
188 R P R - L L L s thilb
216 YF- AtVI‘Ila
217 SY— AtVI‘Ilb
213 FS- thi1
226 YKFFRS-H thi1
213 FV---G—H thila
226 YKFFRH-H thilb

81

Table 3-1. Relative sequence identity between Vti1 protein homologues

 

 

AtVTI1a AtVTI1b thi1 thi1 thi1 thi1
AtVTI1a 100% 59.9 32.4 26.7 33.3 28.1
AtVTI1b 100% 30.8 27.5 31.8 28
thi1 100% 23.9 33.8 23.5
thi1 100% 30.1 91.9
thi1 100% 31 .5
thi1 100%

 

Amino acid sequence similarity shown as identical sequence percentage.
AtVTI1a, and thi1 p, Saccharomyces cerevisiae (accetion number 2497184);
HVti1 p, Homo sapiens (accession number 268740); thi1a, Mus musculus
(accession number 3213227); mVTl1b, Mus musculus (accession number

3213229). The optimal alignments were produced using the J. Hern method in
the DNAstar program.

82

domain, a region thought to be involved in interaction between t-and v-SNAREs
(Calakos et al., 1994; Hayashi et al., 1994; Fischer von Mollard and Stevens,
1998)
AtVTI1a and AtVTI1b Function in Different Trafficking Steps in Yeast

Next, we investigated whether either AtVTI1a or AtVTI1b could
functionally replace the yeast Vti1p in various membrane trafficking steps in
yeast. For this purpose the coding sequences of AtVTI1a or AtVTI1b were cloned
into a multicopy yeast expression vector behind the ADH1 promoter. In yeast the
VTI1 gene is essential for cell growth. Therefore, we determined whether
expression of the Arabidopsis Vti1 homologues would allow yeast cells to grow in
the absence of the yeast Vti1p. The expression of yeast VTI1 was placed under
the control of the GAL1 promoter. These cells (FvMY6/vaM16) were able to
divide on galactose plates (Figure 3-2A, Gal), but not on glucose plates (Glc).
Expression of either AtVTI1a or AtVTI1b allowed for growth on glucose medium.
Cells expressing AtVTI1b grew more slowly than cells expressing AtVTI1a. vti1A
cells (FvMY6) expressing AtVTI1a divided with a doubling time of 3.5 hrs and
vti1A cells expressing AtVTI1b had a doubling time of about 8 hrs, compared to
2.5 hrs for wild-type cells (our unpublished results). These data indicate that
either AtVTI1a or AtVTI1b could replace yeast Vti1p in its essential function,
although to different extents.

Various membrane trafficking steps in yeast can be analyzed by following

the fate of newly synthesized proteins in experiments involving pulse-chase

labeling with 358 followed by immunoprecipitation. The soluble vacuolar

83

 

the
the
the

VC-

8C
00

CF

fOL
se
ter
Ge
et

Se

30

lar

int

hydrolase carboxypeptidase Y (CPY) is glycosylated in the ER to produce the
p1CPY precursor (Stevens et al., 1982). Further modification in the Golgi
apparatus gives rise to p2CPY. CPY is sorted in the TGN and transported from
there through the prevacuolar/endosomal compartment (PVC) to the vacuole and
then cleaved to the mature mCPY (Bryant and Stevens, 1998). Transport from
the Golgi to the PVC is blocked in the temperature-sensitive vti1-1 cells (Fischer
von Mollard et al., 1997) . Compared to wild-type yeast where CPY was retained
in the vacuole as mature form (Figure 3-28, lane 1), the vti1-1 cells (FvMY7)
accumulated p2CPY within the cell (lane 3) and secreted p2CPY (lane 4) at the
non-permissive temperature. As indicated by the prevalence of mCPY (lane 5),
CPY was transported to the vacuole in vti1-1 cells expressing AtVTI1a as
effectively as in wild-type cells. By contrast, only low amounts of mCPY were
found in vti1-1 cells expressing AtVTI1b (lane 7), and most of the CPY was
secreted (lane 8). vti1-11 cells (FvMY21) accumulated p1CPY at the restrictive
temperature (Figure 3-2C, lane 1) due to a defect in retrograde traffic to the cis-
Golgi as well as a defect in traffic from the TGN to the PVC (Fischer von Mollard
et al., 1997). vti1-11 cells, but not vti1-1 cells, display a severe temperature-
sensitive growth defect, indicating that retrograde traffic to the Golgi is essential
(Fischer von Mollard et al., 1997). Expression of AtVTI1a suppressed the
accumulation of p1CPY and resulted in the appearance of mCPY (Figure 3-20,
lane 3). Expression of AtVTI1b also reduced the amount of p1CPY (lane 5); CPY
Was not directed to the vacuole, but was secreted instead (lane 6). These results

indicate that AtVTI1a can replace yeast Vti1p both in transport from the TGN to

84

 

Figure 3-2. Expression of either AtVTI1a or AtVTI1b allows yeast cells to grow in

the absence of Vti1p, but only AtVTI1a functions in TGN-to-PVC traffic.

A) Growth of the vti1A GAL1-VTI1 strain (FvMY6/vaM16) alone or expressing
AtVTI1a or AtVTI1b on plates containing galactose (Gal) or glucose (Glc).
The vti1A GAL 1-VT/1 strain could not grow on glucose after the expression of
VTI1 was shut off, but expression of either AtVTI1a or AtVTI1b supported
growth.

B) CPY traffic in wild-type, vti1-1 (FvMY7, and C) vti1-11 cells (FvMY21) alone (-
) or expressing AtVTI1a or AtVTI1b. Cells were grown at 24°C, preincubated
at 36°C for 15 min, pulse labeled for 10 min, and chased for 30 min at 36°C.
CPY was immunoprecipitated from cellular extracts (l) and extracellular
fractions (E) and analyzed by SDS-PAGE and autoradiography. The TGN to
PVC traffic block in vti1-1 cells (FvMY7) was suppressed by AtVTI1a
expression, as indicated by the presence of vacuolar mCPY, but not by
AtVTI1b expression.

C) vti1-11 cells (FvMY21) accumulated p1CPY due to a block in retrograde
traffic to the cis-Golgi. Expression of either AtVTI1a or AtVTI1b reduced the
amount of p1 CPY that accumulated.

85

A Gal Glc
GAL- VTI1

    

GAL-VTI1+AtVTI1a
GAL-VTI1+AtVT|1 b
E3 VVT vfl1-1

expression -— — AtVTI1 a AtVTI1b
l E

 

 

           

 

 

 

C vti1- 11
expression — AtVTI1 a AtVTI1 b
pchv. _
p1 CPY—

     

mCPY—

86

 

the
01’s

f€t

dlf

thr

pm

(:0
tr;
At

Al

07

th

the PVC (interaction with the t-SNARE Pep12p) and in retrograde traffic to the
cis-Golgi (interaction with the t-SNARE Sed5p). By contrast, AtVTI1 b functions in
retrograde traffic to the cis-Golgi but not in traffic from the TGN to the PVC.

The vacuolar membrane protein alkaline phosphatase (ALP) utilizes a
different transport pathway from the TGN to the vacuole and does not travel
through the PVC as does CPY (Bryant and Stevens, 1998; Odorizzi et al., 1998).
In pulse-chase labeling experiments, arrival at the vacuole is indicated by
processing of pALP to mALP (Figure 3-3A, Line 2) (Klionsky and Emr, 1989).
ALP traffic to the vacuole occurs with a half time of about five minutes in wild-
type cells. vti1-2 cells (FvMY24) accumulated pALP at the non-permissive
temperature (lane 4), demonstrating that Vti1p is also required for ALP transport
(Fischer von Mollard and Stevens, 1999). This trafficking defect was not
corrected by expression of AtVTI1a in vti1-2 cells (lane 6). By contrast, pALP was
transported to the vacuole and processed to mALP in vti1-2 cells expressing
AtVTI1b after a 30-min chase period (lane 8), indicating that AtVTI1b functions in
ALP traffic to the vacuole.

A third biosynthetic pathway to the vacuole is taken by aminopeptidase l
(API). API is synthesized as a cytoplasmic precursor, pAPI, and engulfed by a
double membrane that forms cytoplasm-to-vacuole transport (CVT) vesicles
(Klionsky, 1998). These CVT vesicles fuse with the vacuolar membrane and
pAPl is cleaved to vacuolar mAPI (Figure 3-3B, lane 2). Transport of API along
this pathway has a half time of about 45 min. Transport of API was blocked in

vti1-11 cells (FvMY21) at the restrictive temperature (Fischer von Mollard and

87

 

Figure 3-3. AtVTI1b but not AtVTI1a could replace yeast Vti1p in ALP and API
traffic to the vacuole, which are transported to the vacuole via two different
biosynthetic pathways.

A) VVIld-type and vti1-2 cells (FvMY24) alone (-) or expressing either AtVTI1a or
AtVTI1b were grown at 24°C, preincubated at 36°C for 15 min, pulse labeled
for 7 min, and chased for 0 min or 30 min at 36°C. ALP was
immunoprecipitated from cellular extracts and separated by SDS-PAGE. The
accumulation of pALP in vti1-2 cells was suppressed by expression of
AtVTI1b but not by AtVTI1a.

B) VVIld-type and vti1-11 cells (FvMY21) alone (-) or expressing AtVTI1a or
AtVTI1b were grown at 24°C, preincubated at 36°C for 15 min, labeled for 10
min, and chased for 0 min or 120 min at 36°C. API was immunoprecipitated
from cellular extracts and analyzed by SDS-PAGE. Vacuolar mAPI was found
only in vti1-11 cells expressing AtVTI1b, not in vti1-11 cells expressing
AtVTI1 a.

88

literal

’lltai
labelii
,P was
3E. ill

sion 1'

11111
mi
lpiafi
51011

A WT vti1-2
expression — — AtVTI1a AtVTI1b
chase [min] '0' J 30' 0' 30'. 0' '30" ‘0' 30'

 

 

  

pALP-

 
 

 

3 WT vti1-11
expression — — AtVTI1 a AtVTI1 b
chase [min] ‘ 0' 1' 0' 120' 0' 120' 0' 120'

 

 

    

89

Stevens, 1999), as indicated by the absence of mAPI after a 120-min chase
period (lane 4). Expression of AtVTI1a in vti1-11 cells did not suppress the API
traffic defect (lane 6). As indicated by the presence of mAPI in vti1-11 cells
expressing AtVTI1b (lane 8), AtVTI1b can partially fulfill the function of Vti1p in
API traffic along the Cvt pathway.

Taken together, these data indicate that whereas AtVTI1a can function in
traffic from the TGN to the PVC, AtVTI1a cannot replace Vti1p in traffic along
either the ALP or Cvt pathway to the vacuole. By contrast, AtVTI1b functions in
membrane traffic along the ALP and Cvt pathways to the vacuole but not in
transport from the TGN to the PVC.

Both AtVTI1a and AtVTI1b Transcripts Are Expressed in All Organs in
Arabidopsis

Finding that AtVTI1a and AtVTI1b function in different vacuolar transport
pathways in yeast prompted us to analyze their specific distribution in
Arabidopsis plants. To detect the expression pattern of these AtVTI1 genes, we
performed northern analysis of various Arabidopsis plant organs. Because of the
high similarity of AtVTI1a and AtVTI1b, an untranslated region of each clone was
used to prepare gene-specific RNA probes. The specificity of these two probes
was first checked by dot blot of in vitro translated AtVTI1a and AtVTI1b mRNA,
as revealed in Figure 3-4 A. The dot blot of in vitro transcribed AtVTI1a
hybridized with the AtVTI1a antisense RNA probe. Similarly, in vitro transcribed
AtVTI1b hybridized only with the AtVTI1b antisense RNA probe. These results

demonstrate that the probes are specific under stringent hybridization and

90

 

Figure 34. Northern blot analyses of AtVTI1a and AtVTI1b.

A) Dot blot for testing the specificity of the AtVTI1a and AtVTI1b probes. One
microliter of in vitro transcribed mRNAs of AtVTI1a and AtVTI1b in serial 10X
dilutions were applied to the Hybond N membrane and hybridized with in vitro
transcribed 01-32P UTP-labeled RNA probes at high stringency (65°C for 16 hrs).
The blot was washed under highly stringent consitions (2xSSC + 1% SDS for 30
min at 65°C, 0.2xSSC + 1% SDS 10 min for three times at room temperature).
The signal was then detected by autoradiography.

B) RNA gel blot analysis of AtVTI1 expression in several Arabidopsis organs.
Twenty micrograms of total RNA extracted from roots (R), stems (S), flowers (F),
and leaves (L) were separated on denaturing gel and transferred to Hybond N.
The membrane was then hybridized with probes described in A following the
same procedure.

91

RNA Dot

Dilution
factor

 

AtVTI1a ii. If IL]
AtVTI1b ,_
AtVTI1a
AtVTI1b
AtVTI1a ~
AtVTI1b

 

   

:1 ,0.

’l 105

 

 

 

 

1.5kb— ‘

Probes:

AtVTI1a AtVTI1b

RSFL RSFL

 

 

    

 

 

AtVTI1a AtVTI1b

92

 

washing conditions. These two gene-specific probes were used to hybridize RNA
blots of total RNAs from roots, stems, leaves, and flowers. As shown in Figure 3-
4B, AtVTI1a and AtVTI1b were expressed in all organs investigated. The
AtVTI1a probe also recognized a band that migrated at about 1.6 kb, however,
this band was found to be irrelevant to the AtVTI1a gene since another probe
toward AtVTI1a failed to recognize it (our unpublished results). The mRNA organ
distribution patterns of these two genes were similar to each other; however,
there was more mRNA in roots than in leaves, a pattern similar to the distribution
of AtPEP12 (Bassham et al., 1995) and AtELP (Ahmed et al., 1997). Thus, we
found no variation in distribution of AtVTI1a and AtVTI1b transcripts among plant
organs.
AtVTI1a Is an Integral Membrane Protein

To study the behavior of AtVTI1a, we raised antibodies towards the
cytosolic part of this protein in guinea pig. The antisera specifically recognized a
28 kDa band in leaves, roots, stems, and flowers of Arabidopsis (Figure 3-5A).
The molecular mass of this band agreed well with the deduced molecular mass
of AtVTI1a based on sequence information. The sequence analysis predicted
that AtVTI1a, like most other v-SNAREs, has a C-terminal hydrophobic domain
as a membrane anchor. Therefore, differential centrifugation experiments were
conducted to investigate whether AtVTI1a was associated with membranes. The
majority of the AtVTI1a protein was precipitated at 8,000 g and no AtVTI1a
remained in the supernatant after centrifugation at 100,000 9 (Figure 3-5B). To

confirm that AtVTI1a is an integral membrane protein, various treatments that

93

 

Figure3-5. AtVTI1a is an integral membrane protein.

A) Distribution of AtVTI1a in Arabidopsis organs. Equal amounts of total protein
from leaves (L), flowers (F), stems (S) or roots (R) were separated by SDS-
PAGE and immunoblotted with guinea pig antiserum against AtVTI1a. Molecular
weight is indicated on the left.

B) AtVTI1a fractionates with heavy membranes during differential centrifugation.
A post-nuclear supernatant from 0.5 g cultured roots was centrifuged at 8,000 g
for 20 min. The pellet (P8) was solubilized in 200 pl 2xLaemmli—loading buffer.
The supernatant (88) was further ultracentrifuged at 100,000 g for 2 hrs. The
pellet (P100) was solubilized in 200 pl of 2xLaemmli buffer. Equal amounts of the
supernatant (S100), P100, and P8 were separated by SDS-PAGE and
immunoblotted with anti-AtVTI1a antiserum. Molecular weight is indicated on the
left.

C) AtVTI1a is an integral membrane protein. Equal amounts of total membranes
from Arabidopsis cultured cells were treated with 2 M urea, 0.1% or 1% Triton X-
100, 0.1% or 1% SDS, 1 M NaCl, 0.1 M NaZC03 (pH 11), or extraction buffer
alone. All treatments were performed at room temperature for 30 min. An aliquot
of each treatment was saved as total. Membranes were pelleted by
centrifugation at 100,000 g for one hr after the treatments. Equal volumes of
supernatant or total were separated by SDS-PAGE and immunoblotted with anti-

AtVTl1a antiserum. S: supernatant, T: total.

94

P8 P1003
.L .F SR _. 97km "“"’ AtELP
31kD-;_,___ , 3....
1‘ e .----= AtPEP12p

31k
AtVTI1a

 

 

 

 

 

 

 

 

 

 

 

31kt)?

 

STSTSTST STSTSTST

 

 

.,,,._.,__..LM_.A_" -’ . .. a»
. 1 1

V

 

u -‘ I a . . ' ' ' v . w‘ h ' ~ ' :
31kD-{‘_ ~ .m '5 ”h“? ",' 1 -""l‘ m w m N. _.~ . .\ ”F W
l“ ‘ -.
n ‘, _»*;-‘;,;:.. A'Ek-I :32 ..;:.,‘

 

'“ ”~21: mm": “12:92:22; -.1'-'~ '

 

 

 

 

2M 0.1% 1% 0.1% 1% 1M 0.1M

Urea Triton mm 909 NaCl Na2C03 Bum”

95

 

affect the membrane association of peripheral proteins were applied to total
membranes from Arabidopsis suspension cells. The membranes were pelleted
aftenNards and the amounts of AtVTI1a in the supernatants were compared with
those in the starting material. AtVTI1a was not stripped from the membrane by 2
M urea, 1 M NaCl, or 0.1 M N82CO3, conditions that dissociate peripheral
proteins from membranes (Figure 3-5C). AtVTI1a was solubilized by detergents,
indicating that it is an integral membrane protein.
Cofractionation of AtVTI1a and Other Markers in Sucrose Density Gradients
To determine the subcellular localization of AtVTI1a, we performed a
sucrose density step gradient analysis. Post-nuclear supernatant from 3-week-
old Arabidopsis cultured roots was loaded on top of a step sucrose gradient
(15%, 24%, 33%, 40%, and 54% from top to bottom). The gradient was
equilibrated by ultracentrifugation at 100,000 g for 3 hrs at 4°C and fractions of
0.5 ml were collected from the top to the bottom. The sucrose density distribution
was close to linear after the centrifugation step (Figure 3-6B). Fractions were
then analyzed by immunoblotting. The fractionation of AtVTI1a was compared
with three other subcellular marker proteins, as shown in Figure 3-6A. AtVTI1a
co-fractionated with AtPEP12p, which peaked at 36.5% and 54.4%; AtELP
mostly co-fractionated with AtVTI1a, with peaks at densities of 36.5% and 54.4%.
A separate peak of AtELP was also observed at a sucrose concentration of
32.2%. The vacuolar tonoplast marker H*-pyrophosphatase (H+PPase;
Maeshima et al., 1994) fractionated at the top of the gradient, separated from

AtVTI1a and other marker proteins. These data suggest that AtVTI1a does not

96

 

Figure 3-6. Subcellular fractionation of AtVTI1a by step sucrose gradient. Post-
nuclear membranes of Arabidopsis roots were loaded on a step sucrose
gradient. After equilibrium by ultracentrifugation at 100,000 g for 3 hrs, 0.5 ml
fractions were collected from top (1) to bottom of the gradient (25). Equal
volumes of odd-numbered fractions were loaded on SDS-PAGE gel and
immunoblotted with anti-AtVTI1a, anti-AtELP, anti-AtPEP12p, and anti-
H+pyrophosphatase (H+PPase) antibodies. Blots were analyzed by densitometry
and the percentage of the total marker protein detected in each fraction for
AtVTI1a, AtPEP12p, AtELP, and H‘PPase was plotted in A. The sucrose

concentration of each fraction was determined by refractometry and plotted in B.

97

Sugar concentration (%)

0.35 -
0.3 -

 

 

20-

101

 

+ AtVTI1a
-—I- AtPEP" 2p
—0— AtELP

- --- - H+PPase

 

—.— % sucrose

 

98

reside on the tonoplast membrane, but rather co-fractionates with AtPEP12 on
the PVC or with AtELP on the TGN and the PVC.

T7-tagged AtVTI1a Behaves Similarly to Endogenous AtVTI1a in Yeast and
in Plants

To further differentiate the two AtVTI1 proteins and investigate AtVTI1a
specifically, an 11-amino-acid T7 tag (Novagen, Madison, WI) was fused at the
N-terminus of AtVTI1a. The behavior of this tagged version of AtVTI1a was first
compared with wild-type AtVTI1a in yeast and plants. T7-AtVTl1a was expressed
in yeast to determine whether the epitope-tagged protein retained function. The
growth behavior of vti1A cells (FvMY6) expressing either AtVTI1a or T7-AtVTl1a
was compared by measuring the optical density of cultures growing in logarithmic
phase (Figure 3-7A). These two strains grew at similar rates and had doubling
times of approximately 3.5 hrs. These data indicated that the T7-tagged AtVTI1a
was functional in yeast.

The T7-tagged AtVTI1a was transformed into Arabidopsis ecotype
Columbia. One of the transgenic lines expressing medium amounts of T7-
AtVTl1a was chosen for further study. On a western blot, in addition to
endogenous AtVTI1a migrating at 28 KDa, AtVTI1a antibodies also detected a
protein band migrating at about 29 KDa, which was also recognized by
monoclonal T7-antibody (Figure 3-7B). Thus this 29-kDa protein band was
determined to be T7-tagged AtVTI1a. T7-antibody did not recognize any other
protein bands in extracts from either the transgenic or wild-type line (Figure 3-

7B), suggesting that these antibodies were specific in Arabidopsis. Since we

99

 

Figure 3-7. T7-tag does not affect AtVTI1a function and is expressed in
transgenic plants.

A) Growth curves of vti1A cells expressing AtVTI1a or T7-AtVTl1a. vti1A cells
(FvMY6) expressing either AtVTI1 a or epitope-tagged T7-AtVTl1a grew at similar
rates, indicating that T7-AtVTl1a was functional. Cells were grown in a rich
medium at 30°C at logarithmic phase. The cell density was determined by
measuring the optical density at 600 nm.

B) T7-antibodies specifically recognized T7-AtVTl1a in transgenic plants. Equal
amounts of post-nuclear supernatant of Arabidopsis (T7-At\fl'l1a transgenic
plants or wild type) were separated on SDS-PAGE gel and immunoblotted with
monoclonal T7 antibody or polyclonal antiserum against AtVTI1a raised in guinea

pig-

IOO

5 expressed :1

113. villi ceis
, grew at sire-.312:
rown in a it

jetermined by

plants. E031
1a transgeii
toblotled it“
sed in 9in

In of cell density

 

 

 

 

1.5 “
,.<>
1 - .55"
.’§:’
0.5 "l ’0'3;.
”,0
_..g—_ vtifA+ AtVTI1a
....... 0...“... vti1A +17-AtVTl1a
0 L l I I
0 2 4 6 8
time [h]

th7th7

. anti-
a“"'T7 AtVTI1a

 

101

lacked any functional assay for AtVTI1a in plants, the fractionation patterns of
tagged and endogenous AtVTI1a on sucrose density gradients were compared.
No differences in fractionation patterns were observed between tagged and
endogenous AtVTI1a in transgenic plants, or between the fractionation pattern of
tagged AtVTI1a in transgenic plants and endogenous AtVTI1a in wild-type plants
(our unpublished results). There were also no observable phenotypic differences
between the transgenic plants and wild-type plants (our unpublished results).
These data indicate that T7-AtVTl1a expressed in plants behaves
indistinguishably from endogenous AtVTI1a and the expression of tagged protein
does not affect the physiology of the plant.
Cytochemical Analysis of T7-tagged AtVTI1a in Transgenic Plants

We have shown above that AtVTI1a co-fractionated with AtPEP12p and
AtELP on a sucrose step density gradient. Therefore, we attempted to further
investigate the subcellular localization of AtVTI1a and the relationship between
AtVTI1a and AtPEP12p or AtELP by immunocytochemistry. We found that
AtVTI1a antiserum was unsuitable for these studies, probably because of low
amounts of endogenous protein and loss of antigenicity during fixation. However,
the T7-tagged AtVTI1a transgenic plants allowed us to study the localization of
AtVTI1a in the cell, and to perform colocalization experiments with other
membrane markers. The majority of the T7-AtVTl1a-associated labeling was
found on the TGN (Figure 3-8A) and on electron-dense, uncoated vesicular
structures that were often found near the Golgi of the root cells (Figure 3-8B). We

performed statistical analysis of many independent micrographs showing T7-

102

AtVTI1a localization. This analysis indicated that the distribution of T7-VTl1a was
evenly split between TGN (51%) and dense vesicles (49%). The orientation of
the Golgi was determined based upon appearance and the more electron-dense
staining pattern of the trans-Golgi and the TGN. Almost no T7-AtVTl1a was
found on the cytoplasm, ER, nuclei or plasma membrane (our unpublished
results), and control sections showed almost no background (Figure 3-8C).

Our fractionation experiments indicated that AtVTI1a partially
cofractionated with AtELP, suggesting at least partial colocalization. To analyze
this possibility directly, we performed double-labeling experiments on T7-AtVTl1a
plants. AtVTI1a was first labeled with specific monoclonal antibody against T7,
and detected with 10 nm gold. A second fixation and blocking step was then
performed prior to incubating the sections with antiserum specific to AtELP,
followed by detection with 5 nm gold. It was observed that both T7 monoclonal
antibody and AtELP antiserum specifically labeled the TGN compartment (Figure
3-8D) and electron-dense structures (Figure 3-8E). In control experiments we
substituted pre-immune serum for one of the primary antibodies. An example of
one of these experiments is shown in Figure 3-8F. In this case, sections were
labeled with T7 antibody, followed by pre-immune serum instead of AtELP
antibody. No labeling of any structures with 5 nm gold was seen; however, T7-
AtVTl1a labeling was present on the TGN. The converse experiments were also
done omitting the T7 antibody. Again, no labeling with 10nm gold was seen (our

unpublished results). Also, no labeling of the TGN and dense structures was

103

 

Figure 3-8. In situ localization of T7-AtVTl1a and AtELP on ultrathin sections of
Arabidopsis roots from T7-AtVTl1a transgenic plants. T7-AtVTl1a and AtELP are
localized on the TGN and on dense vesicles.

A and B) Ultrathin sections were incubated with T7 monoclonal antibody
followed by rabbit anti-mouse lgG and biotinylated goat anti-rabbit secondary
antibody and were visualized with streptavidin conjugated to 10 nm colloidal gold.
C) Control. The ultrathin sections were treated with the same procedure as
described in A and B except T7 monoclonal antibody was substituted with 2%
BSA in PBS.

T7-AtVTl1a and AtELP are co-localized on the TGN (D) and on dense vesicles
(E).

D and E) Ultrathin sections were incubated with T7 monoclonal antibody followed
by rabbit anti-mouse lgG and biotinylated goat anti-rabbit secondary antibody
and were visualized with streptavidin conjugated to 10 nm colloidal gold. After the
second fixation step (see Materials and Methods), the same sections were
incubated with antiserum to AtELP, followed by biotinylated goat anti-rabbit
secondary antibody, then visualized with streptavidin conjugated to 5nm colloidal
gold.

F) Control section. The same procedures were used as in D and E except
preimmune serum was used in the place of AtELP antiserum.

G: Golgi; arrow: AtVTI1a; arrow head: AtELP; Bar=0.1 pm.

104

 

105

seen in the absence of both primary antisera, but with the secondary antibodies
decorated with 5 nm and 10 nm gold (our unpublished results).

We speculate that the electron-dense vesicles labeled with T7-AtVTl1a
are PVCs. AtPEP12p is the only known marker on the PVC. Therefore, similar
double EM immunocytochemistry was performed to colocalize T7-AtVTl1a and
AtPEP12p. For this localization, ultrathin cryosections were employed because
AtPEP12p could not be localized using embedment into conventional resin
(Conceicao et al., 1997). The incubation procedure was similar to that of the 17-
AtVTl1a and AtELP double labeling except that AtPEP12p antiserum were used
instead of AtELP antiserum. Analysis of sections revealed that T7-AtVTl1a and
AtPEP12p colocalized to the structures that are typical for the PVC (Figure 3-9A,
B) (Sanderfoot et al., 1998). No staining of the PVC was seen in control
experiments (Figure 3-9C). Together with the yeast complementation data, these
results strongly support our proposal that AtVTI1a is a v-SNARE involved in
traffic between the Golgi and the PVC.

AtVTI1 a Interacts with AtPEP12p

To further investigate whether AtVTI1a interacts with a t-SNARE in vivo,
we attempted to immunoprecipitate AtVTI1a from plant cell extracts and identify
the co-immunoprecipitated proteins. Cultured roots of T7-AtVTl1a plants or wild-
type plants were homogenized and the extract was clarified by centrifugation at
1000 g for 10 min at 4°C. Triton X-100 was added to the supernatant to a final
concentration of 1% to solubilize the membrane proteins. These lysates were

incubated with T7-antibody conjugated to agarose beads. The beads were

106

 

Figure3-9. T7-AtVTl1a and AtPEP12p colocalize on the PVC in cryosections of
Arabidopsis roots from T7-AtVTl1a transgenic plants.

A and B) Ultrathin sections were incubated with T7 monoclonal antibody
followed by rabbit anti-mouse lgG and biotinylated goat anti-rabbit secondary
antibody and were visualized with streptavidin conjugated to 10 nm colloidal gold.
After the second fixation step (see Materials and Methods), the same sections
were incubated with AtPEP12p antiserum, followed by biotinylated goat anti-
rabbit lgG, and then detected by streptavidin conjugated to 5 nm gold particles.
C) Control section. The same procedures were used as in A and B except first
antibodies were substituted with 2% BSA in PBS for T7 monoclonal antibody and
AtPEP12p preimmune serum for AtPEP12p antiserum.

G, Golgi; arrow: AtVTI1a; arrowhead: AtPEP12p; Bar=0.1 pm.

107

 

108

washed and the bound proteins were eluted. Samples of total extracts,
flowthrough, and eluate were separated on SDS-PAGE. The separated proteins
were then transferred to a nitrocellulose membrane and blotted by various
antibodies. T7-AtVTl1a bound to the T7 antibody agarose with high efficiency
(Figure 3-10). Significantly, a fraction of the total AtPEP12p was co-precipitated
with T7-AtVTl1a in the eluate. (Figure 3—10, right side) As we expected, in the
control experiment where wild-type plant extract was used (Figure 3-10, left
side), AtVTI1a did not bind to the T7-antibody. Accordingly, AtPEP12p was not
found in the eluate. Thus, our data indicate that AtPEP12p was associated
specifically with T7-AtVTl1a. In contrast, AtELP was not co-purified by T7-
antibody agarose. These co-immunoprecipitation experiments strongly suggest
that AtVTI1a forms a Triton X-100-resistant SNARE complex with AtPEP12p in

vivo.

Discussion

Several pathways to the vacuole have been identified in yeast. Vti1p, a
multifunctional v-SNARE, has been shown to be involved in numerous pathways
to the vacuole, including the CPY pathway via the PVC, the ALP alternative
pathway, and the CVT pathway for vacuole taking cytosolic proteins such as API
(Fischer von Mollard et al., 1997; Holthuis et al, 1998; Fischer von Mollard and
Stevens, 1999). We have identified two Arabidopsis VTI1 homologues. The
deduced amino acid sequences of these two genes share significant similarity to
Vti1p found in yeast and mammals (Fischer von Mollard et al., 1997; Lupashin ef

al., 1997; Advani et al., 1998; Fischer von Mollard and Stevens 1998; Li, et al.,

109

T7-AtVTl1a Wild-type
T FT E f T FT E
AtVTI1a im‘ ”up... ‘ .

 

 

 

 

 

AtPEP12p W. W a.a.-PW... W m ”I ,

 

 

 

 

 

AtELP WW... .......... WWW..-

 

 

Figure 3-10. AtVTI1a associates with AtPEP12p.

Post-nuclear supernatant from three grams of T7-AtVTl1a transgenic or wild-type
Arabidopsis plants (21 days old) were treated with 1% Triton X-100 to solubilize
membrane proteins. An aliquot was saved as total protein. The Triton X-100
solubilized protein extract was then incubated with 100 pl T7-antibody agarose
(Novagen, Madison, WI) for 5 hrs. Beads were pelleted and,the flowthrough
collected. After being washed, proteins associated with the T7-antibody agarose
were eluted. Equal volumes of total (T), flowthrough (FT) and elute (E) were
separated by SDS-PAGE, followed by immunoblotting with antibodies against
AtVTI1a, AtPEP12p or AtELP.

110

1998). We have found that AtVTI1a and AtVTI1 b were able to substitute for yeast
Vti1p in different membrane transport pathways. AtVTI1a efficiently suppressed
the CPY mistargeting and the growth defect in one set of vti1 temperature-
sensitive mutants and in vti1 null mutants, suggesting that AtVTI1a could
substitute functionally for yeast Vti1p in these pathways. On the other hand,
rather than rescuing the CPY missorting phenotype, AtVTI1b was found to
restore transport of (1) the vacuolar protein ALP that is transported through the
Golgi but bypasses the PVC; and (2) the hydrolase API, which utilizes the CVT
pathway from the cytoplasm to the vacuole. By contrast, AtVTI1a does not
function in the ALP or API transport pathway in yeast.

Whereas there is only one VTI1 gene in yeast, two VTI1-related genes
have been identified in Arabidopsis, mouse and human. It is speculated that the
existence of two paralogues reflected greater complexity of the endomembrane
system in higher organisms compared to yeast. In other words, various members
of the Vti1 gene family probably have different functions. This notion is
supported by the recent report that the two mouse VTI1 genes are expressed
ubiquitously and the mouse Vti1 proteins may be localized on different
compartments (Xu et al., 1998). Whereas the mouse paralogues share only 30%
amino acid identity (Lupashin et al., 1997), the plant paralogues are more closely
related and share 60% amino acid identity. RNA analysis in plants using gene-
specific probes did not detect any expression pattern difference between these
two genes, indicating that both genes are expressed in the same cells and do not

represent organ-specific isoforms. However, the intracellular location of the

111

AtVTI1b protein is not yet known. In yeast, the two Arabidopsis Vti1 homologues
have functionally substituted for thi1p in different vesicle transport steps. In
plants, AtVTI1a most likely functions in a transport pathway analogous to the
CPY pathway (see below). Based on yeast complementation data, we propose
that AtVTI1b is involved in different vacuolar transport pathways in plants.
However, the specific function of the two VTI1 genes in plants will be revealed
only when we are able to investigate their products at the protein level.

In plants, several components of the vacuolar targeting pathway
machinery have been identified. AtPEP12p is a t-SNARE that resides on the
PVC (Conceicao et al., 1997). AtELP is proposed to be a vacuolar protein-
sorting receptor. In previous studies it has been demonstrated that AtPEP12p
and AtELP colocalize on the PVC; AtELP has also been found in the Golgi and
TGN (Sanderfoot et al., 1998). Since it is highly probable that both of these
proteins are involved in mediating transport of soluble vacuolar proteins, their
intracellular distribution in relation to AtVTI1a was very revealing. Under EM, T7-
AtVT|1a was localized on the TGN and on vesicular structures that most likely
compose the PVC. By double Iabeling, AtVTI1a was found to co-localize with
AtELP at the TGN and with AtPEP12p at the PVC. The colocalization of these
three proteins suggests that AtVTI1a, AtELP, and AtPEP12p are most likely
involved in the same pathway, the pathway responsible for the transport of a
subset of vacuolar proteins at the step between the TGN and the PVC. Co-
immunoprecipitation of AtVTI1a and AtPEP12p strongly support the hypothesis

that AtVTI1a, as a v-SNARE, is responsible for the docking of vesicles from the

112

TGN to the PVC by interacting with AtPEP12p, the PVC t-SNARE. It will be
interesting to characterize the vesicles whose fusion is controlled by AtVTI1a and
to define the branches of the plant membrane traffic pathways in which AtVTI1a
is involved. Further investigations should also reveal the membrane traffic

pathways regulated by AtVTI1 b.

113

References

Advani RJ, Bae HR, Bock JB, Chao DS, Doung YC, Prekeris R, Yoo JS, and
Scheller RH (1998). Seven novel mammalian SNARE proteins localize to distinct
membrane compartments. J. Biol. Chem. 273: 10317-10324.

Ahmed SU, Bar-Peled M, and Raikhel NV (1997). Cloning and subcellular
location of an Arabidopsis receptor-like protein that shares common features with
protein-sorting receptors of eukaryotic cells. Plant Physiol. 114: 325-336.

Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ (1990). Basic local
alignment search tool. J. Mol. Biol. 215: 403-410.

Bar-Peled M, and Raikhel NV (1997). Characterization of AtSEC12 and
AtSAR1. Proteins likely involved in endoplasmic reticulum and Golgi transport.
Plant Physiol. 1 14: 315-324.

Bassham DC, and Raikhel NV (1997). Molecular aspects of vacuole biogenesis.
Adv. Bot. Res. 25: 43-58.

Bassham DC, Gal S, Conceicéo AS, and Raikhel NV (1995). An Arabidopsis
syntaxin homologue isolated by functional complementation of a yeast pep12
mutant. Proc. Natl. Acad. Sci. USA 92: 7262-7266.

Becherer KA, Rieder SE, Emr SD, and Jones EW (1996). Novel syntaxin
homologue, Pep12p, required for the sorting of lumenal hydrolases to the
Iysosome-like vacuole in yeast. Mol. Biol. Cell 7: 579-594.

lent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, Giraudat J, Leung
J, and Staskawicz BJ (1994). RPS2 of Arabidopsis thaliana: a leucine-rich
repeat class of plant disease resistance genes. Science 265: 1856-1860.

Bryant NJ, and Stevens TH (1998). Vacuole biogenesis in Saccharomyces
cerevisiae: protein transport pathways to the yeast vacuole. Microbiol. Mol. Biol.
Rev. 62: 230-247.

Calakos N, Bennett MK, Peterson KE, and Scheller RH (1994). Protein-protein
interactions contributing to the specificity of intracellular vesicular trafficking.
Science 263: 1146-1149.

Conceicao SA, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, and
Raikhel NV (1997). The syntaxin homolog AtPEP12p resides on a late post-
Golgi compartment in plants. Plant Cell 9: 571-582.

Cowles CR, Snyder WB, Burd CG, and Emr SD (1997a). Novel Golgi to

vacuole delivery pathway in yeast: identification of a sorting determinant and
required transport component. EMBO J. 16: 2769-2782.

114

 

Cowles CR, Odorizzi G, Payne GS, and Emr SD (1997b). The AP-3 adaptor
complex is essential for cargo-selective transport to the yeast vacuole. Cell 91:
109-1 18.

Darsow T, Rieder SE, and Emr SD (1997). A multispecificity syntaxin
homologue, Vam3p, essential for autophagic and biosynthetic protein transport to
the vacuole. J. Cell Biol. 138: 517-529.

Fischer von Mollard G, and Stevens TH (1998). A human homolog can
functionally replace the yeast vesicle-associated SNARE Vti1p in two vesicle
transport pathways. J Biol. Chem. 273, 2624-2630.

Fischer von Mollard G, and Stevens TH (1999). The Saccharomyces
cerevisiae v-SNARE Vti1p is required for multiple membrane transport pathways
to the vacuole. Mol. Bio. Cell 10: 1719-1732.

Fischer von Mollard G, Nothwehr SF, and Stevens TH (1997). The yeast v-
SNARE Vti1p mediates two vesicle transport pathways through interactions with
the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137: 1511-1524.

Gomez L, and Chrispeels MJ (1993). Tonoplast and soluble vacuolar proteins
are targeted by different mechanisms. Plant Cell 5: 1113-1124.

Hanson Pl, Heuser JE, and Jahn R (1997). Neurotransmitter release—four
years of SNARE complexes. Curr. Opin. Neurobiol. 7: 310-315.

Hay JC, and Scheller RH (1997). SNAREs and NSF in targeted membrane
fusion. Curr. Opin. Cell Biol. 9: 505-512.

Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Sudhof TC, and
Niemann H (1994). Synaptic vesicle membrane fusion complex: Action of
clostridial neurotoxins on assembly. EMBO J. 13: 5051-5061.

Holthuis JC, Nichols BJ, Dhruvakumar S, and Pelham HR (1998). Two

syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 17: 113-
126.

Jones EW (1977). Proteinase mutants of Saccharomyces cerevisiae. Genetics
85: 23-33.

Kirsch T, Paris N, Butler JM, Beevers L, and Rogers JC (1994). Purification
and initial characterization of a potential plant vacuolar targeting receptor. Proc.
Natl. Acad. Sci. USA 91: 3403-3407.

115

 

Kirsch T, Saalbach G, Raikhel NV, and Beevers L (1996). Interaction of a
potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting
determinants. Plant Physiol. 111 : 469-474.

Klionsky DJ (1998). Nonclassical protein sorting to the yeast vacuole. J. Biol.
Chem. 273: 10807-10810.

Klionsky DJ, and Emr SD (1989). Membrane protein sorting: biosynthesis,
transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 8:
2241-2250.

Klionsky DJ, Cueva R, and Yaver DS (1992). Aminopeptidase l of
Saccharomyces cerevisiae is localized to the vacuole independent of the
secretory pathway. J. Cell Biol. 119: 287-299.

Kyte J, and Doolittle RF (1982). A simple method for displaying the hydropathic
character of a protein. J. Mol. Biol. 157: 105-132.

Li HC, Tahara H, Tsuyama N, and lde T (1998). A thi1 homologue: its
expression depends on population doubling levels in both normal and SV40-
transformed human fibroblasts. Biochem. Biophys. Res. Commun. 247: 70-74.

Lukowitz W, Mayer U, and Jijrgens G (1996). Cytokinesis in the Arabidopsis
embryo involves the syntaxin-related KNOLLE gene product. Cell 84: 61-71.

Lupashin VV, Pokrovskaya ID, McNew JA, and Waters MG (1997).
Characterization of a novel yeast SNARE protein implicated in Golgi retrograde
traffic. Mol. Biol. Cell 8: 2659-2676.

Maeshima llll, and Yoshida S (1989). Purification and properties of vacuolar
membrane proton-translocating inorganic pyrophosphatase from mung bean. J.
Biol. Chem. 264: 20068-20073.

Maeshima M, Hara-Nishimura l, Takeuchi Y, and Nishimura, M (1994).
Accumulation of vacuolar H"-pyrophosphatase and H"-ATPase during

reformation of the central vacuole in germinating pumpkin seeds. Plant Physiol.
106: 61-69.

Matsuoka K, Bassham DC, Raikhel NV, and Nakamura K (1995). Different
sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of
distinct sorting machineries in tobacco cells. J. Cell Biol. 130: 1307-1318.

Nothwehr SF, Roberts CJ, and Stevens TH (1993). Membrane protein

retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by
a cytoplasmic signal containing aromatic residues. J. Cell Biol. 121: 1197-1209.

116

 

Odorizzi G, Cowles CR, and Emr SD (1998). The AP-3 complex, a coat of
many colors. Trends Cell Biol. 8: 282-288.

Piper RC, Bryant NJ, and Stevens TH (1997). The membrane protein alkaline
phosphatase is delivered to the vacuole by a route that is distinct from the VPS-
dependent pathway. J. Cell Biol. 138: 531-545.

Robinson JS, Klionsky DJ, Banta LM, and Emr SD (1988),. Protein sorting in
Saccharomyces cerevisiae: isolation of mutants defective in the delivery and
processing of multiple vacuolar hydrolases. Mol. Cell Biol. 8: 4936-4948.

Sambrook J, Fritsch EF, and Maniatis T(1989). Molecular Cloning, a
Laboratory Manual (2nd Edition). Cold Spring Harbor Laboratory Press,
Plainview, NY. pp7.37-7.57 .

Sanderfoot AA, Ahmed SU, Marty-Mazars D, Rapoport l, Kirchhausen T,
Marty F, and Raikhel NV (1998). A putative vacuolar cargo receptor partially
colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots.
Proc. Natl. Acad. Sci. USA 95: 9920- 9925.

Sato MH, Nakamura N, Ohsumi Y, Kouchi H, Kondo M, Hare-Nishimura I,
Nishimura M, and Wada Y (1997). The AtVAM3 encodes a syntaxin-related
molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J. Biol.
Chem. 272: 24530-24535.

Shimada T, Kuroyanagi M, Nishimura M, and Hara-Nishimura l (1997). A
pumpkin 72-kDa membrane protein of precursor-accumulating vesicles has
characteristics of a vacuolar sorting receptor. Plant Cell Physiol. 38: 1414-1420.

Slot JW, Geuze HJ, Gigengack S, Lienhard GE, and James DE (1991).
Immuno—Iocalization of the insulin regulatable glucose transporter in brown
adipose tissue of the rat. J. Cell Biol 113: 123-135.

Séllner T, Bennett MK, Whiteheart SW, Scheller RH, and Rothman JE (1993)
A protein assembly-disassembly pathway in vitro that may correspond to

sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75: 409-
418.

Srivastava A, and Jopnes EW (1998) Pth1Nam3p is the syntaxin homolog at
the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of
vacuolar hydrolases. Genetics 148: 85—98.

Stepp J, Huang K, and Lemmon SK (1997). The yeast adaptor protein

complex, AP-3, is essential for the efficient delivery of alkaline phosphatase by
the alternate pathway to the vacuole. J. Cell Biol. 139: 1761-1764.

117

Stevens T, Esmon B, and Schekman R (1982). Early stages in the yeast
secretory pathway are required for transport of carboxypeptidase Y to the
vacuole. Cell 30: 439-448.

Sutton RB, Fasshauer D, Jahn R, and Brunger AT (1998). Crystal structure of
a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature
395: 347-53.

Tokuyasu KT (1989). Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for
cryoultramicrotomy. Histochem. J. 21; 163-171.

Wada Y, Nakamura N, Ohsumi Y, and Hirata A (1997). Vam3p, a new member
of syntaxin related protein, is required for vacuolar assembly in the yeast
Saccharomyces cerevisiae. J. Cell Sci. 110: 1299—1306.

Vater C.A., Raymond C.K., Ekena K, Howald-Stevenson I, and Stevens TH
(1992). The VPS1 protein, a homolog of dynamin required for vacuolar protein
sorting in Saccharomyces cerevisiae, is a GTPase with two functionally
separable domains. J. Cell Biol. 119: 773-786.

Vernet T, Dignard D, and Thomas DY (1987). A family of yeast expression
vectors containing the phage f1 intergenic region. Gene 52: 225-233.

Xu Y, Wong SH, Tang BL, Subramaniam VN, Zhang T, and Hong W (1998). A
29-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein
receptor (Vti1-rp2) implicated in protein trafficking in the secretory pathway. J.
Biol. Chem. 273: 21783-21789.

Zheng H, Bassham DC, Conceicao AS, and Raikhel NV (1999). The syntaxin

family of proteins in Arabidopsis: a new syntaxin homologue shows
polymorphism between two ecotypes. J. Exp. Bot. 50: 915-924.

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Chapter IV

The Comparison of Two AtVTI1 Proteins and Characterization of

Atvti12, a T-DNA Insertion Mutant of AtVTI12

119

Abstract

In Arabidopsis, SNAREs are frequently encoded by gene families
composed of two or more highly homologous genes. This phenomenon may be a
reflection of the complexity of the endomembrane system. AtVTI11 and AtVTI12
are about 65% identical at the amino acid sequence level. AtVTI11 has been
cha racterized in detail previously. It is localized on the TGN and the PVC. On the

T G N, AtVTI11 co-localizes with AtELP, the NTPP cargo receptor, and sporamin,
a vacuolar soluble protein with an NTPP sorting signal. AtVTI11 also forms a
SNARE complex with SYP2 and SYP5 at the PVC. AtVTI12, however, is found
mostly in the SNARE complex with SYP4 and SYP6. In this chapter, I further
compared the difference between these two proteins. Based on density
gradients, AtVTI11 and AtVTI12 have different subcellular locations. Atvti12, a
null mutant of AtVTI12 has been characterized in detail. This mutant has no
obvious phenotype. However, the apparent normal phenotype may be explained

by the fact that AtVTI11 was found to form a SNARE complex with SYP4 and
SYP6 in the mutant.

120

Introduction

The plant secretory pathway plays a variety of roles in every step of plant
development. This pathway is composed of a series of membrane-bound
organelles. Each organelle has its own unique biochemical and physiological
features and performs different functions. Thus, it is important to maintain
homeostasis of each organelle. Vesicle transport is the major means by which
proteins are delivered to and from these organelles. Although it is believed that
the basic machinery for operating vesicular transport is well conserved between
all eukaryotic cells, the uniqueness of the plant endomembrane system can not
be ignored. For instance, in mammals and yeast, each cell has only one type of
vacuole whereas in plants, different types of vacuoles frequently co-exist in one
cell (Paris et al., 1996; Jauh et al., 1999). For soluble vacuolar proteins, there are
at least three separate delivery routes based on the location and peptidal
sequence of the sorting signal. Some proteins, represented by sweet potato
sporamin and barley aleurain, carry a sorting signal with a typical NPIR motif,
e.g. an N-terminal propeptide (NTPP) signal, at the N-terminus of the protein
(Matsuoka and Nakamura, 1991). These proteins first enter the endomembrane
system at the endoplasmic reticulum and are then transported through the Golgi
apparatus to the trans-Golgi network (TGN). At the TGN, a membrane bound
cargo receptor (AtELP is one of the examples, see Ahmed et al., 2000)
presumably recognizes the NTPP and directs those proteins into clathrin-coated

vesicles (CCV). These vesicles deliver their cargo to the prevacuolar

121

compartment (PVC), which further fuses with the vacuole. AtVTI11 and SYP21
most likely are involved in this process (Zheng et al., 1999; Bassham et al., 1995;
Conceicao et al., 1997). On the other hand, some proteins carry their vacuolar
sorting signal at the C-terminus of the protein. These signals are called C-
terminal propeptides (CTPP). CTPP cargos are delivered through a route
completely different from the NTPP pathway (Matsuoka et al., 1995). None of the
molecular components involved in this pathway have been identified yet. Very
often, different types of sorting signals also determine the destination to a
specific vacuole. Most NTPP cargos are delivered into the lytic vacuole, while
CTPP cargo is sent to the pH neutral storage vacuoles. Sometimes, the lytic and
the protein storage vacuoles fuse to form one central vacuole (Di Sansebastiano
et al., 2001). It is possible that this complex vacuolar system requires more
sophisticated transport machinery than that used in yeast. Based on genome
analysis, there are 21 SNAREs in Saccharomyces cerevisiae (Bock et al., 2001).
In Arabidopsis, there are 55 SNAREs that can be subgrouped into several
families (Sanderfoot et al., 2000). These multi-gene families could be the
reflection of a more complex endomembrane system.

The VTI1 type SNARE has multiple functions. In yeast, although encoded
by a single gene, it can form SNARE complexes with 5 of 8 syntaxins (Lupashin
et al., 1997; Fischer von Mollard et al., 1997; Abeliovich et al., 1998; Fischer von
Mollard and Stevens, 1999). In mouse and human, there are 2 VTI1 genes that
encode proteins about 30% identical to each other and 25% to 33% to yeast

Vti1p (Lupashin et al., 1997; Fischer von Mollard and Stevens 1998). In mouse,

122

 

VTl1a is localized to the Golgi and involved in intra-Golgi trafficking. Vti1b is
found on Golgi, TGN, and posSibly also on the endosome (Xu et al., 1998).
These two proteins do not completely co-localize when expressed in mammalian
cells (Xu et al., 1998).

In Arabidopsis, the VTI1 family is composed of three closely related
members (65% to 75% identical at the amino acid sequence level). They are all
about 25% identical to yeast Vti1p and are closer to mammalian VTl1a than to
mammalian VTl1b (Sanderfoot et al., 2000). AtVTI11 and 1 2 are highly
expressed, while AtVTl13 produces only very low level of mRNA that can not be
detected on northern blot (data not shown). Previously, we have described the
characterization of AtVTI11 (AtVTI1a, Zheng et al., 1999). AtVTI11 is localized on
the TGN, together with the NTPP cargo receptor AtELP, and on the PVC,
together with SYP2 and SYP5 groups of syntaxins (Zheng et al., 1999;
Sanderfoot et al., 2001). It also physically interacts with SYP2 and SYP5, as has
been shown by co-immunoprecipitation experiments (Zheng et al., 1999;
Bassham et al., 2000; Sanderfoot et al., 2001). We suggest that AtVTI11 is
involved in vesicle trafficking between the TGN and PVC by forming a SNARE
complex with SYP2 and SYP5 groups of syntaxins (Zheng et al., 1999;
Sanderfoot et al., 2001). It is possible that AtVTI11 is involved in NTPP cargo
transport based on its co-localization with AtELP and sporamin (Zheng et al.,
1999; chapter V). AtVTI12, a close relative of AtVTI11 (65% identical at the

peptide sequence level), is also expressed in Arabidopsis (Zheng et al., 1999).

123

AtVTI11 and AtVTI12 are expressed in all the tissues that have been
investigated. The expression patterns of these two genes are also similar.
However, there is some evidence supporting the notion that AtVTI11 and
AtVTI12 function in different pathways. First, AtVTI11 and AtVTI12 complement
different yeast vti1 mutant alleles that block different vacuolar protein transport
pathways (Zheng et al., 1999). In plant cells, in contrast to the SNARE complex
composed of AtVTI11, SYP2 and SYP5, AtVTI12 forms a SNARE complex with
SYP4 and SYP6 groups of syntaxins (Bassham et al., 2000; Sanderfoot et al.,
2001). These results indicate that AtVTI12 may play a role in vesicle trafficking
different from AtVTI11. However, although there is some indication that AtVTI11
is involved in NTPP cargo transport, no clue has been found about the function of
AtVTI12. Thus, we decided to search for individual knockout mutants of AtVTI11
and AtVTI12. Here, I describe a T-DNA insertion mutant in the promoter region of
AtVTI12. Although AtVTI12 protein is abolished from this mutant, Atvti12 has a
normal phenotype. Co-immunoprecipitation results suggest that the apparent
normal phenotype may be due to the presence of AtVTI11. I propose that
although AtVTI11 has different functions in the wild type, it could substitute for

AtVTI12 in the Atvti12 mutant.

Materials and Methods

Plasmids, Transgenic Plants and Arabidopsis Mutants
For generating glutathione-S-transferase (GST) fusion constructs of

AtVTI11 or AtVTI12, the same PCR fragments used to construct His-AtVTI11

124

(Zheng et al., 1999) or His-AtVTI12 (Bassham et al., 2000) were instead fused
with pGEX5x-3.

Arabidopsis plants expressing T7-AtVTl11 were described by Zheng et al.,
1999. Arabidopsis plants expressing HA-AtVTl12 were described by Bassham et
al. (2000).

Arabidopsis T-DNA insertion mutant Atvti12 was identified with the help of
Mendel Biotechnology. The details of identification of mutant plants in T-DNA
insertion mutant pools were described by Sanderfoot et al. (2001).

Antibodies and Columns

The same antigen used to generate anti-AtVTI11 in guinea pig (Zheng et
al., 1999) was used to raise rabbit antiserum against AtVTI11. Rabbit antiserum
raised against AtVTI12 was described by Bassham et al. (2000). AtVTI12
antiserum was further affinity purified against E. coli expressed GST—AtVTl12.
Recombinant GST-AtVTl12 was over-expressed and purified with the use of
glutathione-Sepharose beads (Amersham-Pharmacia Biotech AB). Five mg of
overexpressed GST—ATVTI12 was then bound to 1 ml of glutathione—Sepharose
4B and equilibrated with 200 mM boric acid, pH 9.0. Dimethyl pimelimidate was
added to reach the final concentration of 4 mglml. After incubation at room
temperature for 30 min, the cross-linker was quenched with 0.2 M ethanolamine
(pH 8.0) and washed with phosphate-buffered saline (PBS). Anti-AtVTI12 crude
serum was applied to this column. After washes with 5 ml 0.1 M Tris, pH 8.0,
followed by another wash with 5 ml of 0.01 M Tris, pH 8.0, the antibodies bound

to the column were eluted with 10 ml 100 mM glycine, pH 2.5. The eluate was

125

collected in 1-ml fractions. The fractions containing antibodies were pooled and
protein concentration determined by the value of O.D.280. One percent BSA and
0.02% azide was added before storage at 4°C. Antibodies from pre-immune
serum or AtVTI11 serum were purified by incubating 1 ml serum with 1.5 ml
Protein A Sepharose 6MB (Amersham Pharmacia Biotech AB) for 30 min at
room temperature. The beads were washed with 5 ml 0.1 M Tris, pH 8.0,
followed by another wash with 5 ml of 0.01 M Tris, pH 8.0. The bound antibody
was eluted by 2x25 ml 0.1M glycine, pH 2.5, for 10 min each. The two fractions
were pooled. The protein concentration was determined by the value of O.D.230,
For the anti-AtVTI12 column, 2 mg of the GST-AtVTl12 affinity purified antibody,
were incubated with 1.5 ml protein A 6MB beads for 1 hour at room temperature.
For anti-AtVTI11, pre-immune of AtVTI11 or pre-immune of AtVTI12 columns,
two mg of the protein A affinity-purified antibodies were incubated with 1.5 ml
protein A 6MB beads for one hour at room temperature. After wash with 2x5 ml
200 mM borate acid, pH 9.0, the crosslinker dimethyl pimelimidate was added to
reach the final concentration of 5 mglml. The reactions were kept at room
temperature for 30 min before quenching by 0.2 M ethanolamine (pH 8.0). The
beads were stored in PBS with 0.02% azide at 4°C.
Immunoprecipitation

Twenty gm of 21-day-old liquid cultured Arabidopsis roots were
homogenized on ice with 5 ml Extraction Buffer (50 mM HEPES-KOH, pH 6.5, 10
mM potassium acetate, 100 mM sodium chloride, 5 mM EDTA, 0.4 M sucrose)

with CompleteTM protease inhibitor tablets (Roche). To prepare the total

126

membrane, the homogenate was centrifuged at 1000 g for 15 min and the
supernatant was subjected to 100,000 g ultracentrifugation for 3 hours. The pellet
was homogenized in 3 ml TBS (Tris balanced buffer; 0.14 M NaCl, 2.7 mM KCI,
25 mM Tris, pH 8.0) with miniCompleteTM tablets (Roche). The total membrane
was solubilized by adding Triton X-100 to 1%. The solubilized membrane was
further cleared by ultracentrifugation at 100,000 g for 30 min. The supernatant
was incubated with antibody or pre-immune columns for 2 hours. After 5 x .
washes with 5 ml TBST (TBS + 1% Triton X-100) each, the bound protein was
eluted with 4 ml 0.1 M glycine (pH 2.5). The antibody columns were neutralized
by washing with 10 ml PBS. The eluted protein was precipitated by 10%
trichloroacetic acid. After two acetone washes, the protein pellet was solubilized
in 50 pl 2X Laemmli buffer. The protein was separated by SDS-PAGE and
different proteins were detected by western blot.
Pulse-Chase Labeling

Fifty mature Arabidopsis rosette leaves were cut to thin strips and
immersed in 10% cellulase (Onozuka R10), 0.5% macerozyme R10 (Yakult
Honsha Co., Ltd Japan) and 0.08% BSA in enzyme solution (0.5 M betaine, 10
mM CaClz and 1.5 mM MES, pH 5.7). Vacuum was applied for 30 min to facilitate
penetration of the solution to the leaf tissues. After incubation for 3 hours in the
dark, the protoplasts were separated from undigested tissues as described in
Bednarek and Raikhel (1991). The protoplasts were diluted to a final
concentration of 100,000 cells/ml in incubation medium (Gamborg's B5 basal

medium with mini organics (Sigma) supplemented with 0.3 M betaine, 0.1 M

127

glucose, 1 mg/L 2,4-D). The protoplasts were labeled with 100 pCi 35SExpress
(Dupont) for four hours. The labeling was stopped by adding 100 pl of chase mix
(165 mM methionine and 110 mM cysteine in incubation buffer) to the cells. After
30 min chase, the protoplasts were then separated from the incubation buffer
and lysed in TBSE (0.14 M NaCl, 2.7 mM KCI, 25 mM Tris, pH 8.0, 0.5mM
EDTA) + 1% Triton-X 100. The lysates were cleared of insoluble debris by brief
centrifugation at 10,000 g for 30 sec. Protein A beads bound with equal amount
of affinity-purified aleurain antibody or pre-immune antibody were added to the
lysates and incubated for 2 hours at 4°C. After the beads had been washed 3
times with TBSE + 1% Triton-X 100, 50 pl 2X laemmli loading buffer were added
to the beads. The eluted proteins were then separated by 12% SDSA-PAGE and
visualized by autofluorography.
Subcellular Fractionation

Twenty-one-day old Arabidopsis cultured roots were used to prepare total
membrane as described in "Immunoprecipitation". The total membrane was then
layered on top of a discontinuous Accudenz® (Accurate chemicals and Scientific
Corp., NY) gradient (1.5 ml of each: 2%, 5%, 9%, 12%, 15%, 20% and 30% [wlw]
from the top to the bottom). The gradient was then equilibrated by
ultracentrifugation at 100,000 g for 16 hours. Fractions of 0.5 ml each were taken
from the top to the bottom and proteins were separated by SDS-PAGE. Western
blots were used to visualize different subcellular markers.

PCR and RT-PCR

128

 

Genomic DNA was isolated using the CTAB extraction procedure
described in Sanderfoot et al. (2001). The following primers were used for
screening T-DNA inserted in the gene of AtVTI12: 5' end gene-specific primer: 5'
TAT TTC CTG GAC GAG TAA TCT TGG TTC TGC 3', 3' end gene-specific
primer: 5' TCT GAC GTG ACA GTG GGT CTC CTG CCT GCG 3'. T-DNA left
border: 5' CTC ATC TAA GCC CCC ATT TGG ACG TGA ATG 3'. T-DNA left
border nested: 5' TTG CTT TCG CCT ATA AAT TAC GAC GGA TCG 3'. T-DNA
right border: 5' TGG GAA AAC CTG GCG TTA CCC AAC TTA AT 3'. The PCR
reaction condition for amplifications of genomic DNA was the standard condition
described in manufacturer's recommendations (Gibco-BRL, Rockville, MD).
Reverse transcription reactions for RT-PCR were done using the Superscript II
reverse transcriptase system following recommendations from the manufacturer
(Gibco-BRL, Rockville, MD). The following gene-specific primers were used to
generate first-strand cDNAs: for AtVTI12: 5' GAG CCA CGA TTA CCG ATGT 3';
for NPSN-12: 5' AGT GTA ATA TGC ACC AAA CC 3'. These reverse primers
and forward primers (for AtVTI12: 5' GAA AAT GTC ACT CTG CAT CG 3'; for
NPSP12: 5' GAG CCT GAA ATA ATC CGG CAG AT 3') were used for PCR
amplification of the reverse transcript products with the standard conditions

described in the manufacturer's recommendations (Gibco-BRL, Rockville, MD).
Results

The Specificity of Antibodies Raised Against AtVTI11 and AtVTI12.
Instead of being concentrated in certain domains, the similarities between

AtVTI11 and AtVTI12 are found throughout the sequence (Zheng et al., 1999).

129

 

Thus, it is difficult to generate specific antibodies using a particular peptide
sequence. Instead, anti-VTI11 and anti-12 rabbit anti-sera were raised against
His-tagged N-terminal part of the proteins just before the transmembrane domain
(Zheng et al., 1999; Bassham et al., 2000). Previously, we have shown that when
these two antibodies are used on western blots to visualize T7-AtVTl11 or HA-
Ath12 in total protein extracts from transgenic plants, anti-AtVTI11 specifically
recognizes T7-AtVTI11 and anti-AtVTI12 specifically recognizes HA-AtVTI12
(Bassham et al., 2000). However, because the expression levels of T7-AtVTl11
and HA-12 are different in transgenic plants, the quantities of AtVTI11 and
AtVTI12 are difficult to control and thus the specificity of the antibodies is still not
clearly evaluated. Here, two different approaches were used to address the
specificity of these two antibodies. First, cytosolic domains of AtVTI11 and 12
(identical to the part that fused with 6XHis to make antigens) were fused with
GST and over-expressed in E. coli. The recombinant protein was affinity purified
through a glutathione column. The amount of purified proteins was visually
determined by Coomassie Brilliant Blue stain after SDS-PAGE. As shown in
Figure 4-1A, when equal amounts of GST-AtVTI11 and GST-AtVTI12 were
loaded on the gel, western blot Indicates that the affinity of anti-AtVTI11 to the
GST-AtVTI11 is about 10 times higher than to the GST-AtVTI12. AtVTI12
antibody, however, shows almost equal sensitivity towards GST-AtVTI11 or GST-

AtVTl12 proteins.

130

 

 

Figure 4-1. Characterization of the specificity of Anti-AtVTI11 and Anti-AtVTI12.
A) The GST-fusion of N-terminal AtVTI11 or 12 were purified by glutathoine
columns. Equal amount of purified GST-fusion proteins of Ath11 and 12
(loading quantities were indicated on the top) were separated by SDS-PAGE.
AtVTI11 and 12 antisera were used for western blots. Molecular weight marker is
shown at the left. “>”: the full length GST-fusion protein. B) Total membrane
from 10 gm of root culture (21-day old) were solubilized by 10% Triton X-100.
Equal volumes of these solubilized proteins were passed through anti-AtVTI12,
11 or pre-immune columns. After extensive wash, protein bound to the columns
was eluted by 0.1 M glycine (pH 2.5) and precipitated by 10% TCA. 1/10 eluate
from anti-AtVTI11 (V11), anti-AtVTI12 (V12), their pre-immune columns (pre) or
1/300 of total membrane protein (T) were separated by SDS-PAGE. Anti-AtVTI11
or 12 were used for western blot. “>”: AtVTI11, “*”: AtVTI12

131

 

A GST-12
ng of protein ’ 1 . 10 100

 

fli‘ ,w

   

45kD- *: ----~- w {3 Anti-AtVTI11

 

 

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Tlll ail: 45kDa
SDSPIGE -
ghlmaiie’s

al merit:

Triton 1.1: B
art-All: V12 Pre V11 Pre T
teCOézfiu . , ,
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imns iii”
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132

Because immunoprecipitation is the major means by which we can
address the question of SNARE complex composition, the specificities of these
two antibodies when used for immunoprecipitation were evaluated. The
antibodies from anti-AtVTI11 or pre-immune sera were affinity purified through
protein A beads and eluted using 0.1 M glycine (pH 2.5). The amount of eluted
antibodies was visually determined by SDS-PAGE followed by Coomassie
Brilliant Blue staining. The antibodies were then cross-linked to a Protein A 6MB
column by dimethyl pimelimidate with the ratio of 1 mg antibody/1 ml beads. The
same procedure was used to generate anti-AtVTI12 column and its pre-immune
control column except the anti-AtVTll12 was affinity purified through a GST-
AtVTl12 column. Total membrane prepared from 10 gm of 21-day-old
Arabidopsis cultured roots was used as starting material. The post-nuclear total
membranes were solubilized with 1% Triton X-100 and cleared by
ultracentrifugation at 100,000 g for 30 min before being divided into equal
volumes and incubated with pre-immune columns or anti-AtVTI11 or AtVTI12
columns for 2 hours. After washing, the proteins bound to the columns were
eluted with 0.1 M glycine, pH 2.5. The total membrane or the eluates from
immune or pre-immune columns were separated by SDS-PAGE and western
blots were used to detect AtVTI1 proteins. In the lanes where total membrane
protein was loaded, AtVTI12 antibody detected two bands, whereas only the
lower band was detected by AtVTI11 antisera (Figure 4-1 B). Since AtVTI11 anti-
sera were more specific than AtVTI12, it was concluded that the lower band

detected by both antisera was AtVTI11. The band recognized only by AtVTI12

133

anti-serum was AtVTI12. Although not easy to distinguish, these two proteins
migrated with about 1 to 3 kDa difference on SDS-PAGE. Based on the
assumption that AtVTI12 antiserum has equal sensitivity towards both AtVTI11
and 12, there may be more AtVTI12 than AtVTI11 in the total membrane proteins
from cultured roots (Figure 4-1 B). Although the ratios of these two proteins vary
slightly, there were always more AtVT12 proteins than AtVTI11 in all tissues
examined (data not shown). When used for immunoprecipitation, AtVTI11
antibody had higher affinity toward AtVTI11 protein than AtVTI12. However,
AtVTI12 antibody, although it would not distinguish AtVTI11 and 12, also
precipitated more AtVTI12 owing to a larger amount of AtVTI12 in the sample
(Figure 4-1 B).

In conclusion, when used for western and immunoprecipitation, AtVTI11
and AtVTI12 antibodies are not absolutely specific to one or the other, although
AtVTI11 are relatively more specific towards AtVTI11. However, there is a
molecular weight difference between the two proteins. When carefully examined,

these two proteins can be distinguished on western blots.

AtVTI11 and AtVTI12 Have Different Fractionation Patterns

To further analyze the functional difference between AtVTI11 and
AtVTI12, the subcellular localizations of these two proteins were studied. Neither
AtVTI11 nor AtVTI12 antibodies work well in immunocytochemistry. Therefore,
T7-AtVTl11 and HA-AtVTl12 double transgenic plants were used for EM study.

The root tip cells were double labeled with T7- and HA- monoclonal antibodies to

134

visualize the locations of AtVTI11 and 12. Both AtVTI11 and AtVTI12 were found
on the TGN or the PVC membrane. Sometimes these two proteins were co-
localized on the same membrane, but at other times, they were localized on
separate membranes (Zheng H, Kovaleva V and Raikhel NV, unpublished). To
address the question of the relative location of AtVTI11 and AtVTI12 accurately,
a discontinuous Accudenz gradient was used to fractionate total microsomes
from Arabidopsis roots. Post-nuclear supernatant from 21-day-old Arabidopsis
cultured roots was loaded on top of a step Accudenz gradient (2, 5, 9, 12, 15, 20,
and 30% from top to bottom). The gradient was equilibrated by ultracentrifugation
at 100,000 g for 16 hours at 4°C, and fractions of 0.5 ml were collected from the
top to the bottom. These fractions were then analyzed by western blot. As shown
in Figure 4-2A, after ultracentrifugation, the gradient became almost linear with a
slower slope between 1.075 mglml to 1.175 mglml. We chose to look at the
fractionation patterns of several well-studied markers. Using the experimental
conditions described above, microsomes harboring SYP2 (the PVC membrane)
and SYP6 (the TGN membrane) can be clearly distinguished. SYP2 fractionated
at lower density (around 1.125 mglml), whereas SYP6 peaked at the high-density
(around 1.175 mglml) fractions. AtELP, which labels the TGN and the PVC, and
SYP4, which labels TGN by immunocytochemistry, mostly fractionated with
SYP6 (AtELP see Figure 4-2, data for SYP4 is not shown). SYP5 co-fractionated
with SYP2 (data not shown). AtVTI11 completely co-fractionated with SYP2.

However, using AtVTI12 anti-sera, we saw two bands peaked at different

135

 

Figure 4-2. Subcellular fractionation of AtVT11 and AtVTI12 by discontinuous
Accudenz gradient. Post-nuclear membranes of Arabidopsis cultured roots (21-
day-old) were loaded on a step Accudenz gradient. After equilibrium by
ultracentrifugation at 100,000 g for 16 hours, 0.5-ml fractions were collected from
the top (1) to the bottom of the gradient. The densities of the fractions were
determined by refractometry and plotted in A. Equal volumes of even-numbered
fractions were loaded on an SDS-PAGE and immunoblotted with anti-sera of
interested proteins as shown in B.

136

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137

fractions. The band with higher molecular weight co-fractionated with SYP6 and
peaked at high-density fractions. This band most likely represented AtVTI12. The
lower molecular band has the same molecular weight as the band recognized by
anti-AtVTI11 and was found to co-fractionate with SYP2 at relatively lower
density. This band is most likely AtVTI11. The Accudenz gradient thus offered
the first clear evidence that AtVTI11 and AtVTI12, two very closely related
SNAREs, might have different subcellular locations. The different location in the
cell may reflect the fact that these two proteins are involved in the formation of
different complexes.
Characterization of the Mutant Atvti12

A reverse genetics approach was taken to further address the function of
AtVTI12. To identify a T-DNA insertion mutant of AtVTI12, pools of Arabidopsis
Col-gl seeds mutagenized with T-DNA were screened using gene-specific
primers and T-DNA border primers (see Materials and Methods). One mutant
plant with a T-DNA inserted at —76nt upstream of transcription start (Figure 4—3A)
was identified at the heterozygous stage. This allele was named atvti12 for its
mutation in AtVTI12. The seeds of this plant were collected and the individual
plants from the next generation (F1) were examined in detail. Genomic PCR was
performed using a 5' gene-specific primer and T-DNA left border primer to detect
the insertion; while 5' and 3' gene specific primer pairs were used to detect the
intact AtVTI12 gene without the insertion. Among 42 F1 plants, 12 were
homozygous for the T-DNA insertion and 10 were homozygous wild-type plants.

The total RNA prepared from a mixture of leaves, stems, and flowers of

138

W ....

 

Figure 4-3. Atvti12 is a null mutant for AtVTI12. A) The diagram of the T-DNA
insertion in Atvti12 gene. T-DNA is inserted at -76 nt upstream from transcription
start of AtVTI12 gene on chromosome l. B) RT-PCR of AtVTI12. Total RNA from
seedlings of wild-type (Col-O), heterozygous plants (20, 39) or homozygous
insertion mutant (21, 29) were used as templates. AtVTI12 cDNA was generated
by reverse transcription with gene specific primers at the 3’ end of the AtVTI12
mRNA. 5’ and 3’ primer pairs were used to amplify the cDNA. The amplified DNA
was separated by 1% agarose gel and visualized by 1% ethdium bromide. The
molecular weight is indicated at the left. C) RT-PCR of NPSN-12 using the same
template described in B with gene specific primers for NPSN-12 mRNA. D)
Western blots of total protein extracted from wild-type (7, 27) or Atvti12 (21, 29)
seedlings. The proteins were separated by SDS-PAGE and immunoblots were
used to visualize AtVTI11 or 12. Arrows indicate the AtVTI12 protein band that
was missing in Atvti12 plants.

139

tram of the lit
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'2. Total RN12
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SN- 1 2 leh'. l
or Atvti12 (2‘. i.
:mmunohlohxre
protein hatch

 

 

 

 

 

 

 

 

 

140

o AtVTI12
0-5Kb- AtVTI12
0.7Kb- AtNPSN12
20 21 29 39 Col-O
7 21 27 29 7 21 27 29
Anti-ATVTI12 Anti-AtVTI11

wild-type plants, heterozygous plants (20, 39) and homozygous Atvti12 plants
(21, 29) were used as templates for RT-PCR. The condition of the RT-PCR was
designed to detect even very low levels of mRNA (see Materials and Methods).
As shown in Figure 4-3B, RT-PCR from RNA of homozygous Atvti12 plants did
not produce a band that represents AtVTI12 mRNA. In comparison, using RNA
from wild type or heterozygous plants, RT-PCR produced a band at around
500bp. This band indicates that AtVTI12 mRNA was present in the total RNA
sample and thus the gene was expressed in those plants. RT-PCR of an
unrelated gene (NPSN12) using the same RNA samples was used as a control
to indicate the quality of the total RNA sample (Figure 4-3C). Two plants with
homozygous insertion (21 and 29) and two plants with homozygous wild-type
AtVTI12 genes (7 and 27) were chosen for further characterization. Their seeds
were germinated on plates containing solid medium. The protein extracts from
these seedlings were further analyzed. As shown in Figure 4-3D, on western blot,
AtVTI12 antibody recognized a band at around 28 to 30 kDa that was missing
from the homozygous ATVti12 plants but present in wild-type plants. This band is
putatively the AtVTI12 protein. In contrast, when AtVTI11 antibody was used for
western blot analysis, there was no significant difference between proteins of the
wild type and the Atvti12 homozygous plants. Proteins extracted from other
tissues (flowers, mature leaves, and roots) have been analyzed with the same
results (data not shown). This experiment indicated that there is no significant
amount of AtVT12 protein in Atvti12 mutant plants. At the same time, the AtVTI11

protein level is not changed between Atvti12 mutant and wild type plants.

141

The Atvti12 plants were then checked for any defect in NTPP vacuolar
protein transport. It has been shown that Arabidopsis aleurain has a typical
NTPP signal and is most likely transported through the route common for NTPP
proteins (Ahmed et al., 2000). Thus, the processing of aleurain protein was
examined in Atvti12 plants. When the vacuolar cargo transport routes are
blocked, we expect the marker protein is either targeted to the wrong place and
degraded (reduced amount) or accumulated in an intermediate compartment
(indicated by appearance of higher molecular variants due to lack of processing).
In total protein extract, no difference in total amount of mature aleurain was found
between wild type or Atvti12 plants (Figure 4-4A). There is no obvious abnormal
accumulation of higher molecular variants of aleurain in the Atvti12 plants either.
To examine the dynamics of aleurain transport, pulse-chase labeling of aleurain
was performed. Protoplasts generated from mature leaves of the wild type and
the Atvti12 plants (Number 30 line) were labeled with 35SExpress for 4 hours.
The cells were lysed and total proteins were incubated with the pre-immune or
anti-aleurain columns (1 mg pre-immune or affinity purified aleurain antibody/ 1
ml protein A 4MB). After washing, the retained proteins were eluted and
separated by SDS-PAGE. Since the pre-immune antibody precipitated strong
background bands, we have to use the affinity-purified aleurain antibody reveal
the purified aleurain. ln autofluorographs that were exposed for 20 days, the
weak aleurain bands were revealed. However, there was no difference in the
amount of precursor or mature protein between the wild type or Atvti12 plants

(Figure 4-48). The homozygous Atvti12 plant also had no visible phenotype as

142

A

 

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a eura‘n- M"""'“"‘~ ~‘N-u . xiv W‘w mm

 

     

e > ' - -
'.' ' ‘ .r 13?} él‘t‘ti

wt 21

y . n... -, ~_ , M I -. .'.r- '-
‘e'a..--~. v

 

Figure 4-4. Aleurain processing in Atvti12 cells. A) Western blot of aleurain.
Total protein extracts from wild type (7, 27) or atvti12 homozygous mutant (21,
29) seedlings were separated by SDS-PAGE. Aleurain was detected by immuno-
blot. Molecular weight marker was marked at the right. B) Pulse-chase labeling of
aleurain. Protoplasts from wild type (wt) or homozygous Atvti12 (21) mature leafs
were labeled with 358 Express for 4 hours and chased for 30 min with excess
cold cysteine and methionine. Pre-immune (Pre) or affinity-purified aleurain
(aleu) antibodies were used to immuno-precipitate the radio-labeled proteins.
One forth of the precipitated proteins were analyzed by SDS-PAGE followed by
autofiuorogarphy. The aleurain bands were marked at the left. Molecular weight
marker was labeled at the right.

143

 

Figure 4-5. Phenotype of Atvti12. The plants were grown in standard conditions
with 16-hour light/8—hour dark. The picture was taken at 21 days after
germination. Number 14 at left is a homozygous Atvti12 mutant. Number 28 at
the right is a plant segregated from the same parental line with homozygous
intact AtVTI12 genes. The Atvti12 plant has no apparent phenotype.

shown in Figure 4-5. Under various physiological conditions, no difference
between the wild type and the Atvti12 plants was observed (data not shown). In
conclusion, Atvti12 mutant has no obvious phenotype and the absence of
AtVTI12 protein caused no obvious defect to the plants.

We have shown that the AtVTI1 family of proteins, although closely
related, may form SNARE complexes with different sets of syntaxins (Bassham
et al., 2000; Sanderfoot et al., 2001). Thus, they may have functional differences
as well. The genes encoding other members of those SNARE complexes, e.g.
SYP2, SYP5, SYP4, and SYP6 are all absolutely required for the plant; since no
homozygous knock-out mutants has been recovered. Thus, it is a surprise to us
that the plants lacking AtVTI12 protein do not have any physiological or
developmental defect. One hypothesis is that AtVTI11 and AtVTI12 can
functionally substitute for each other 'when one is missing. To evaluate this
possibility, I analyzed the SNARE complexes of AtVTI11 in the wild type and
Atvti12 mutant. As shown in Figure 4-6, when AtVTI11 antibody was used for
immunoprecipitation, SYP2 and SYP5 families of syntaxins were co-precipitated
in both the wild type and Atvti12 mutant plants. There was no difference in the
amount of SYP2 and SYP5 brought down from the wild type or Atvti12 mutant
plants. This observation suggests that AtVTI11 forms a SNARE complex with
SYP2 and SYP5 and its ability to perform its functions are not changed when
AtVTI12 is missing. However, there was much more SYP4 and SYP6 co-

precipitated in Atvti12 than in the wild-type plants (Figure 4-6). Most likely,

145

IP: AtVTI11 IP: AtVTI11

 

 

 

 

mutant wt mutant
.7 F'- e T 5'- - E'- ..

 

 

 

 

 

 

 

 

 

. (AtVTI11)

 

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g 5
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!

 

 

 

 

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Figure 4w6. Immunoprecipitation of AtVTI11 from wild-type and Atvti12 mutant
plants. Total membarne from 10 gm of 21-day old wild type or Atvti12 root culture
were treated with 1% Triton X-100 to solublize the membrane proteins. An aliquot '
of 1/300 was saved as total membrane protein samples (T). The Triton X-100
solubilized membarne proteins were then passed through anti-AtVTI12 or pre-
immune columns. After extensive wash, the proteins associated with the columns
were eluted by 0.1 M Glycine (pH 2.5) and precipitated by 10% TCA. 1/5 of the
eluates (EL) or 1/300 of total membarne proteins (T) were separateed by SDS-
PAGE. Proteins of interest were visualized by immuno—blotting Arrow head

indicates AtVTI12 band in wild-type plants.

146

AtVTI11 formed a SNARE complex with SYP4 and SYP6 and performed the
function of AtVTI12.

Based on the data described above, I propose that in wild type plants,
AtVTI11 interacts with SYP2 and SYP5. One function of this SNARE complex
might be to facilitate the transport of NTPP vacuolar proteins because AtELP (a
vacuolar cargo receptor) also co-localizes with AtVTI11 and SYP2. AtVTI12,
however, interacts with SYP4 and SYP6 to form a completely different SNARE
complex. The function of this SNARE complex requires further investigation. In
the mutant where AtVTI12 is absent, AtVTI11 performs dual functions and forms
a noncognate SNARE complex with SYP4 and SYP6 (Figure 4-7) and leads to

normal plants.
Discussion

Although the basic machinery for vesicle transport is believed to be well
conserved among all eukaryotic organisms, plant secretory pathways are much
more complicated than single-celled yeast. Plant cells often have multiple
vacuoles that require multiple pathways for vacuolar protein transport. At the
whole plant level, different cell types might require cell-specific functions
performed by these pathways that may not be necessary for yeast or mammals.
For the components of the secretory machinery, it is possible that gene families
are developed from one single gene to accommodate the complex duties they
face. The AtVTI1 family proteins are proven to be a good example. In yeast, the
VTI1 protein is encoded by a single gene but The protein is involved in several

SNARE complexes and thus has diverse functions (review see Gotte and Fischer

147

 

Figure 4-7. Summary of results. AtVTI11 and AtVTI12 are highly homologous
SNAREs. Although they are both localized on the TGN and the PVC membranes,
Their ratios between two membranes are different and can be separated by
biochemical means. Moreover, AtVTI11 forms SNARE complexes with SYP2 and
SYP5, whereas AtVTI12 forms complexes with SYP6 and SYP4. In the AtVTI12
mutant Atvti12, AtVTI11 forms an additional SNARE complex with SYP4 and
SYP6.

148

 

Wild-type

  
   

 

 

 

 

 

 

 

 

 

 

 

AtELP
AtELP
AtVTI11
AtSYP6 AtSYPz ._
AtVTI11
AtSYP4 2 . - “SYP5
AtELP
—->
atvti12 mutant
T
Gl
fl AtELP
“ AtELP
E AtVTI11
AtSYP6 AtSYPz
AtVTI11 AtVTI11 .
AtSYP4 ' . “SYP5
AtELP
‘—*
SQ t-SNARE _.I'- v-SNARE
>- NTPP cargo receptor

0 NTPP cargo

 

 

 

149

 

von Mollard, 1998). When expressed in the yeast vti1 mutant, AtVTI11 facilitates
the transport of soluble carboxy-peptidase Y to the vacuole. AtVTI12, however, is
more effective in the transport of vacuolar membrane proteins and in the
autophagy pathway (Zheng et al., 1999). In plants, AtVTI11 is in a SNARE
complex with SYP2 and SYP5, whereas AtVTI12 is in a SNARE complex
composed of SYP4 and SYP6. (Bassham et al., 2000; Sanderfoot et al., 2000).
SYP2 is localized on the PVC, and SYP4 on the TGN. Using transgenic plants
expressing T7- or HA- tagged AtVTI11 or AtVTI12, we have shown by
immunocytochemistry that these two proteins are both localized on the TGN and
the PVC and sometimes on the same membrane. However, when separated by
an Accudenz gradient, AtVTI11 and AtVTI12 membranes are fractionated
differently. EM, as a snapshot, reflected that AtVTI11 and AtVTI12 are localized
on both the TGN and the PVC. However, density gradient fractionation reflected
the ratios of marker proteins on different membranes. AtVTI11 co-fractionated
with SYP2 and AtVTI12 with SYP4 and SYP6. It is likely that in vivo, after the
CCV fuses with the TGN, AtVTI11 spend majority of its time on the PVC before
being recycled back. Most AtVTI12 proteins remain on the TGN after mediating
the fusion between a yet unknown vesicle with the TGN. SNAREs are localized
on the membrane where they function. It thus safe to speculate that AtVTI12 may
also play a role in vesicle transport between the TGN and the PVC. However, in
Atvti12 mutant, although AtVTI11 forms a SNARE complex with SYP4 and SYP6,
the fractionation pattern of AtVTI11 was still the same as in wild type plants (data

not shown). This can be explained by the observation that the subcellular

150

 

 

localization of a SNARE is determined by its transmembrane domain sequence
(Rayner and Pelham, 1997; Watson and Pessin, 2001) instead of by SNARE
partners it interacts with.

There is no indication about the function of AtVTI12 in plants yet except
that when expressed in yeast, it functioned in membrane vacuolar membrane
protein transport and the cytoplasm-to-vacuole (Cvt) pathway (Zheng et al.,
1999). Although it has been shown that membrane protein vacuolar transport is
separated from soluble protein transport, it is a poorly characterized pathway in
plants. Except for electron microscopy description of plant autophagy, little is
known about the molecular basis of autophagy and the related Cvt pathway. In
yeast, besides Vti1p, Tngp and Tlg1p are also required for the Cvt pathway and
for vacuolar membrane protein transport. SYP4 family of proteins are the
homologues of Tngp in Arabidopsis. SYP6 is a Tlg1 homologue. Thus, it is likely
that the transport steps in which SYP4, SYP6 and AtVTI12 play roles are evolved
from the membrane vacuolar protein transport or the Cvt pathways in yeast. We
hope the study about these Arabidopsis SNAREs will shade light on the exact
nature and the functions of these pathways in plants.

Reverse genetics is a powerful way to address the question regarding the
functions of AtVTI12 and its SNARE complex. However, SYP41, SYP42 and
SYP6 mutations have been shown to be lethal to the plants (Sanderfoot et al.,
2000). These observations indicate that the AtVTI12-SYP4-SYP6 complex is
involved in a function that is critical for the cell. Unfortunately, those SYP mutants

are not very helpful for our understanding of the exact nature of this complex.

151

 

 

 

 

 

Surprisingly, the situation of mutation at the AtVTI12 gene is quite
different. In Atvti12, where the AtVTI12 is not expressed, the plant appeared
normal. When AtVTI11 SNARE complex was precipitated, we found besides the
complex formed with SYP2 and SYP5, AtVTI11 also formed a complex with
SYP4 and SYP6. It is likely that AtVTI11 can fully substitute for AtVTI12 in this
mutant.

A similar situation is observed in the AtVTI11-SYP2-SYP5 pathway. As
previously shown, SYP21 and 22, although closely related, can not functionally
substitute for each other. SYP21 and SYP22 null mutants are both lethal possibly
at as early as the pollen stage (Sanderfoot et al., 2001). Recently, an AtVTI11
null mutant zig-1 has been identified. The complete absence of AtVTI11 protein
causes a shoot gravitropic defect, wrinkled leaves, fragmented vacuoles, and
some other light phenotypes (Morita et al., 2001; Kato et al., 2001). It is
speculated that the gravitropic defect is a secondary defect caused by certain
changes in the physical or chemical environment of the vacuole (Morita et al.,
2001). At least in roots, AtVTI12 was found in the complex with SYP21 and
SYP22 (Zheng and Raikhel, unpublished data). AtVTI12 seems to take the place
of AtVTI11 and generates relatively normal plants.

It seems that SYP2 and SYP4 group of syntaxins, although are composed
of closely related family members, in the cell, each member has unique functions
and cannot take each other's place. The reason might rely on the dynamic
difference between syntaxins and VTI1 type SNAREs. In a cell, the localization of

a SNARE reflects its functional site. Syntaxins are SNAREs with less flexible

152

 

locations. The subcellular localization of a syntaxin is determined by its
transmembrane domain sequence (Rayner and Pelham, 1997; Watson and
Pessin, 2001). SYP41 and SYP42 have been localized on different domains of
the TGN. Although SYP21 and SYP22 are both localized on the PVC in root tips,
SYP22, but not SYP21, has been found on tonoplast of small vacuoles in shoot
meristems (Sato et al., 1997) and in vacuole-enriched membranes prepared from
suspension cells (Rojo E and Raikhel NV, unpublished data). When one SYP is
absent, it is impossible for its homologue to take its place unless the two are
localized at the same place. However, the VTI1-type SNAREs are more mobile.
lmmunocytochemical study localized both AtVTI11 or AtVTI12, on the TGN and
the PVC, although their distribution ratios between the TGN and the PVC might
be different as reflected by their density gradient fractionation pattern difference.
AtVTI11 and AtVTI12 may both shuttle between the TGN and the PVC. When
one is missing, the other one has the chance to meet its SNARE partner and to
fulfill its functions. Thus, although VTI1-type proteins might have functions critical
for the plant, the loss of one is well tolerated. Only in the double mutant, we
might have the opportunity to understand the real function of AtVTI1 family of
proteins.

The phenomenon that two functionally different SNARE proteins substitute
for each other in vivo has not been reported before. However, noncognate
SNAREs have been shown to form complexes in arbitrary combinations in vitro
(Fasshauer et al., 1999). It is not difficult to imagine that more closely related

AtVTI11 and AtVTI12 could be exchanged in their SNARE complexes in vitro. In

153

vivo, this possiblity of substitution also exists because both AtVTI11 and AtVTI12
are localized on the TGN and the PVC membrane (Zheng H, Kovaleva V and
Raikhel NV unpublished data). In Arabidopsis, there are quite a number of
reports of mutated genes causing no phenotype. Normally, this is attributed to
gene redundancy. However, when we start to understand the machinery of the
plant cell function in more detail, sometimes, the "redundant" genes might be
found to have functional differences. This mechanism for related but not
redundant proteins to stand in for each other reflects one facet of flexibility of

plant cells.

154

References

Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE,
Matsuoka K, and Raikhel NV (2000). The plant vacuolar sorting receptor AtELP
is involved in the transport of NH2-terminal propeptide-containing vacuolar
proteins in Arabidopsis thaliana. J. Cell Biol. 149: 1335-1344.

Abeliovich H, Darsow T, and Emr SD (1999). Cytoplasm to vacuole trafficking
of aminopeptidase l requires a t-SNARE-Sec1p complex composed of Tlg2p and
Vps45p. EMBO18: 6005-6016.

Bassham DC, Gal S, Conceicéo AS, and Raikhel NV (1995). An Arabidopsis
syntaxin homologue isolated by functional complementation of a yeast pep12
mutant. Proc. Natl. Acad. Sci. USA 92: 7262-7266.

Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, and Raikhel NV (2000).
AtVPS45 complex formation at the trans-Golgi network. Mol. Biol. Cell 11: 2251-
2265.

Bednarek SY, and Raikhel NV (1991). The barley lectin carboxyl-terminal
propeptide is a vacuolar protein sorting determinant in plants. Plant Cell 3: 1195-
1206.

Bock JB, Matern HT, Peden AA, and Scheller RH (2001). A genomic
presepective on membrane compartment organization. Nature 409: 839-841.

Conceicao AS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, and
Raikhel NV (1997). The syntaxin homologue AtPEP12p resides on a late post-
Golgi apparatus in plants. Plant Cell 9: 571-582.

Di Sansebastiano GP, Paris N, Marc-Martin S, and Neuhaus JM (2001).
Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be

visualized by green fluorescent proteins targeted to either type of vacuoles. Plant
Physiol. 126: 78-86.

Fasshauer D, Antonion W, Margittai M, Pabst S, and Jahn R (1999). Mixed
and non-cognate SNARE complexes. J Biol. Chem. 274: 15440-15446.

Fischer von Mollard G, and Stevens TH (1998). A human homolog can
functionally replace the yeast v-SNARE Vti1p in two vesicle transport pathways
J. Biol Chem. 273: 2624-2630.

Fischer von Mollard G, and Stevens, TH (1999). The Sacchromyces cerevisiae
v-SNARE Vti1p is required for multiple membrane transport pathways to the
vacuole. Mol. Biol. Cell 10: 1719-1732.

155

 

Fischer von Mollard G, Northwehr SF, and Stevens TH (1997). The yeast v-
SNARE Vti1p mediates two vesicle transport pathways through interactions with
the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137: 1511-1524.

Gotte M and Fischer von Mollard G (1998). A new beat for the SNARE drum.
Trends Cell Biol. 8: 251-218.

Jauh GY, Philips TE, and Rogers JC (1999). Tonoplast intrinsic protein
isoforms as markers of vacuolar functions. Plant Cell 11: 1867-1882.

Kato T, Morita Ml, Fukaki H, Yoshiro Y, Uehara M, Nihama M, and Tasaka M
(2001). SGR2, a phospholipase-like protein, and ZlG/SGR4, a SNARE, are
involved in the shoot gravitropism of Arabidopsis. Submitted.

Lupashin VV, Pokrovskaya ID, McNew JA, Waters MG (1997).
Characterization of a novel yeast SNARE protein implicated in Golgi retrograde
traffic. Mol. Biol. Cell 8: 1379-1388.

Matsuoka K, and Nakamura K (1991). Propeptide of a precursor to a plant
vacuolar protein required for vacuolar targeting. Proc. Natl. Acad. Sci USA 88:
834-838.

Matsuoka K, Bassham DC, Raikhel NV, and Nakamura K (1995). Different
sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of
distinct sorting machineries in tobacco cells. J. Cell Biol. 130: 1307-1318.

Morita MT, Kato T, Nagafusa K, Saito C, Ueda T, Nakano A, and Tasaka M
(2001). Involvement of the vacuoles of the endodermis in early process of shoot
gravitropism in Arabidopsis. Submitted

Paris N, Stanley CM, Jones, RL, and Rogers JC (1996). Plant cells contain two
functionally distinct vacuolar compartments. Cell 85: 563-572.

Rayner JC, and Pelham HR (1997). Transmembrane domain-dependent sorting
of proteins to the ER and plasma membarne in yeast. EMBO J. 16: 1832-1841.

Sanderfoot AA, Assad, FF, and Raikhel NV (2000). The Arabidopsis genome.
An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein
receptors. Plant Physiol. 1 24: 1 558-1569.

Sanderfoot AA, Pilgrim M, Adam L, and Raikhel NV (2001). Disruption of
individual members of Arabidopsis syntaxin gene families indicates each has
essential functions. Plant Cell 12: 659-666.

Sato MH, Nakamura N, Ohsumi Y, Kouchi H, Kondo M, Hara-Nishimura I,
Nishimura K, and Wada Y (1997). The atVAM3 encodes a syntaxin-related

156

molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J Biol.
Chem. 272:24530-24535.

Watson RT, and Pessin JE (2001). Transmembrane domain length determines
intracellular membarne compartment localization of syntaxins 3, 4, and 5. Am. J.
Physiol. Cell Physiol. 28: C215-0223.

Xu Y, Wong SH, Tang BL, Subramaniam VN, Xhang T, and Hong W (1998). A
29-kilodalton Golgi soluble N-ethylmaeimide—sensitive factor attachment protein

receptor (Vti1-rp2) implicated in protein trafficking in the secretory pathway. J.
Biol. Chem. 273: 21783-21789.

Zheng H, Fischer von Mollard G, Kovaleva V, Stevens TH, and Raikhel NV
(1999). The plant vesicle associated SNARE AtVTI1a likely mediates vesicle
transport from the trans-Golgi network to the prevacuolar compartment. Mol. Biol.
Cell 10: 2251-2264.

157

 

 

Chapter V

Characterization of AtVTI1-Containing Vesicles Purified by

Immuno-affinity Columns

158

 

 

Abstract

The Pre-vacuolar compartment (PVC) is a recently identified compartment
of the endomembrane system in plants which function is still not clear. The
syntaxin SYP21 is the only known marker that localizes exclusively in the PVC.
Several other membrane proteins, including AtELP, AtVTI11 and 12, have been
found on this compartment as well as on the TGN. However, none of the soluble
proteins present in the lumen of the PVC have been identified. l have taken a
biochemical approach to characterize this organelle and investigate its function. I
have purified the PVC based on the presence of AtVTI11 and 12 are localized on
it. An affinity column with conjugated anti-AtVTI12 antibodies was used to purify
AtVTI1-containing vesicles. Sporamin, an NTPP cargo marker was found to co-
purify in the vesicle fraction. This co-localization was confirmed by immuno-
electron microscopy. To identify novel cargo, the proteins in the immunopurified
fraction were separated by 2-D gel electrophoresis. These proteins will be

identified by mass spectrometry techniques.

159

Introduction

The prevacuolar compartment (PVC) is defined as an intermediate
organelle in the vesicular trafficking pathway from the TGN to the vacuole. It is
most prominent in cells where new vacuoles are actively formed. It has been
extensively characterized morphologically by electron microscopy (Marty, 1978).
It was speculated that provacuoles were involved in protein transport to the
vacuole both through the biosynthetic pathway and through autophagy (Marty,
1978; Marty, 1980). However, no resident protein of the PVC was known and
thus it was hard to study this organelle. Recently it was shown that SYP2 is
localized in the PVC. SYP2 is an Arabidopsis syntaxin homologue of yeast
Pep12p. In yeast Pep12p resides on the prevacuolar compartment (PVC) and is
critical for vacuolar transport of carboxy-peptidase Y from the TGN to the vacuole
(Becherer et al., 1996). In Arabidopsis root tips, SYP2 group proteins decorate a
tubular-vesicular electron dense structure between the TGN and the vacuole.
AtELP, a vacuolar cargo receptor, can be observed on the TGN and on this
compartment but not in the vacuole (Conceicao et al., 1997, Sanderfoot et al.,
1998). Based on these results, we speculate that this organelle is a PVC that
functions as a midstation for protein transport between the TGN and the vacuole
and allows recycling of the trafficking machinery before arrival at the lytic
vacuole. To characterize the PVC in more detail, we have purified it by
biochemical means and analyzed its protein content. The detailed catalog of

protein content of this organelle will offer us insight into its traffic dynamics, and

160

 

 

will supply us with more tools to study it by genetic and biochemical means. The
identification of the PVC content also will yield clues about the function of SYP2.

The purification methods based on the unique physical features of the
organelle have not proved to be very efficient for PVC purification, in contrast to
those for vacuole, nuclei or chloroplast isolation. A major constraint is that the
PVC is most abundant in vacuolating cells of the meristem which are difficult to
harvest and thus tissues with less accumulation of PVCs must be used.
Moreover, the PVC has similar physical characteristics as a lot of other
organelles and thus it is difficult to separate to high purity (Zheng and Raikhel,
unpublished). Therefore, the only available method to obtain highly enriched PVC
fractions relies on affinity purification.

AtVTI11and 12 and SYP2 have been found on the PVC by immuno-
electron microscopy (Zheng et al., 1999; Zheng, Kovaleva and Raikhel,
unpublished data). Moreover, AtVTI11 and 12 are found to co-fractionate with
SYP2 on various types of density gradients (Zheng et al., 1999; Sanderfoot and
Raikhel, unpublished data). Our results indicate that most of AtVTI11 and 12 is
present on the PVC membrane. Thus, it is possible to use AtVTI11 or 12
antibodies to purify the PVC. The AtVTI12 antibody can be purified from other
lgGs in the serum with relative ease and it recognizes AtVTI11 ands 12 with
similar sensitivity (See chapter IV). I have used this antibody to affinity purify the
membranes containing AtVTI1 (AtVTI11 and 12) in large scale. The purified
microsomes were eluted with detergent and separated by 2-D gel

electrophoresis. The specific spots will be identified using matrix-assisted laser

161

 

desorption ionization- time of flight (MALDI-TOF)- or electrospray ionization
(ESl)- mass spectrometry (MS). After the proteins have been identified, the
interesting ones will be studied further by immunocytochemistry to confirm their
localization.

Few vacuolar soluble markers endogenous to Arabidopsis have been
characterized to date. Exogenous markers like sweet potato sporamin are
properly targeted to the vacuole when expressed in Arabidopsis (Matsuoka et al.,
1991). Recently, it has been shown AtELP specifically interacts with the
sporamin NTPP peptide and that AtELP and sporamin co-localize on the same
domain of the TGN (Ahmed et al., 2000) indicating that may travel through the
PVC, as AtELP, in route to the vacuole. Thus, sporamin can be used as a marker
to evaluate the quality of the isolated PVC vesicles. Here, I describe preliminary

results in the characterization of the protein content of the PVC.

Materials and Methods:

Arabidopsis Transgenic Lines

The generation of Arabidopsis transgenic plants expressing sporamin was
described in Matsuoka et al., 1995. The generation of Arabidopsis expressing
T7-AtVTI11 is described in Zheng et al., 1999.

An Arabidopsis plant expressing T7-AtVTl11 was crossed with pollen from
a plant expressing sporamin. The F2 generation from the cross was screen for
plants stably expressing T7-AtVTI11 and sporamin.

Electron Microscopy

162

 

The procedure used to prepare grids for thin plastic sections followed by
immunolabeling with the appropriate antibodies was described in Zheng et. al
(1999) and Ahmed et al. (2001).

Antibody Purification and Preparation of Affinity Columns

The AtVTI12 antibody was subject to affinity purification against E. call
over expressed GST-fusion of AtVTI12. Antibody and pre-immune column were
made as described in Chapter IV with equal amounts of purified lgG.

AtVTI11 Vesicle Enrichment:

Two hundred grams of Arabidopsis cultured roots were homogenized in
tissue extraction buffer (50 mM HEPES.KOH pH6.5, 10mM KOAc, 100mM NaCl,
5mM EDTA and 13.7% Sucrose) with complete protease inhibitor cocktail
(Roche) using a food processor at high speed 10 sec for three times. The
homogenized material was filtered through Miracloth (Calbiochem) and spun at
10009 for 15 min to get rid of the debris. The supernatant was filtered through
Miracloth again and total membrane fraction was pelleted by ultracentrifugation
at 100,000 g for 30 min. The pellet containing total membrane was homogenized
by douncing with 16ml tissue extraction buffer. The homogenate was divided into
halves and loaded on top of two sucrose gradients (12 ml 24%, 12ml 36%, 3 ml
56% sucrose in tissue extraction buffer). The gradient was spun at 100,000 g for
two hours in a swing rotor and the interface between 24% and 36% sucrose was
collected. The sugar concentration of this AtVTI11 enriched fraction was adjusted

to 13-14% by adding 5 volumes of tissue extraction buffer without sucrose.

163

 

Affinity Purification

A volume of tissue extraction buffer + 10% BSA was added to the AtVTI11
enriched fraction described above to achieve a BSA concentration of 5%. This
membrane fraction was then incubated overnight at 4°C with 1ml protein-A beads
conjugated with antibodies that had been pre-incubated two hours with Tissue
Extraction Buffer +10% BSA. The beads were then washed with 6X5ml tissue
extraction buffer for 10 min each. The protein in the vesicles was eluted with 5ml
Tissue Extraction Buffer + 1% Triton X-100. The protein in the eluate was
precipitated with trichloroacetic acid (final concentration 10%) and washed with
acetone twice. The beads were regenerated by treating with 5m| 0.1M glycine
(pH2.5), washed with PBS twice and stored in PBS with 1% azide.
2-D Electrophoresis

The 2-D gel separation procedure was performed as in Peck et al. (2001)
with minor changes. Detergent eluted proteins from antibody columns were
mixed with an equal volume of Tris-buffered phenol, pH 8 (Gibco-BRL, Rockville,
MD) and back extracted twice with Back Extraction Buffer (100mn Tris, pH8.4,
20nM KCI, 10mM EDTA and 0.4% B-mercaptoethanol). Two to five volumes of
100mM ammonium acetate in methanol was added to the phenol phase and
proteins were precipitated by centrifugation at 10,000 g for 30 min at 4°C. After
washing the pellet with 80% acetone twice, the protein pellet was resuspened in
370 pl of 2-D loading buffer (7 M urea, 2 M thiourea, 4% CHAPs, 0.5% IPG-
Buffer (Amersham-Pharmacia), 2% DTT, and 0.1% bromphenol blue). This

loading buffer with solubilized proteins was used to rehydrate the 18cm 3-10NL

164

IPG strips (Amershan Pharmacia) on IPGphor (Amersham Pharmacia) at 80 V
for 12 hours. Subsequently, the isoelectric focusing was performed under the
following condition: 500 V for 2 hours, 1000 V for 2 hours, gradient to 5000 V in
12 hours, 5000 V for 12 hours. After isoelectric focusing, IPG strips were
equilibrated in the equilibration buffer (50 mM Tris, pH 6.8, 6M urea, 30%
glycerol, 1% SDS, and 0.01% bromophenol blue) plus 1% DTT and 15 min in
equilibration buffer plus 4.5% iodoacetamide. The strips were then loaded on
12% SDS-PAGE gel (Protean ll, Bio-Rad) for separation in the second

dimension. Proteins were visualized by silver nitrate staining.
Results

Enrichment of AtVTI1 Vesicles by Sucrose Gradient

The antibody raised against AtVTI12 can be purified from the serum by
affinity purification through GST-AtVTI12 columns. The resulting AtVTI12
antibody recognizes both AtVTI11 and 12 as shown in Chapter IV. However, this
purified antibody also recognizes other background proteins (Figure 5-1 B, line2).
The lack of detergents in the buffer required for purification of vesicles increased
the background level. Thus, additional purification steps for reduction of the
background were implemented. We separated total microsomes on a
discontinuous sucrose gradient. After a short spin (100,0009 for 3 hours), most
microsomes were concentrated at the interphases between the different gradient
steps. These microsomes were collected and examined by western. As shown in
Figure 5-1, the interphase between 24 and 36% sucrose had the highest amount

of AtVTI1. Moreover, the unspecific background was also dramatically reduced.

165

sample I -1 32%?)
'2 -45KD
24%
-31KD
-3
36% 7
‘4 ‘ -21kD
55%

 

Figure 5-1. AtVTI1 vesicles were enriched though a step sucrose gradient. A)
Scheme of the sucrose gradient used for enrichment of AtVTI1 vesicles. Total
microsome (sample) was loaded on the step gradient with sucrose concentration
of 24%, 36% and 55% from top to bottom. The microsomes were separated on
the gradient by ultracentrifugation 100,000 g for three hours. The top (where the
sample was loaded), and interphases between different steps were collected. B)
Western blot of the fractions taken indicated in A. Equal amounts of proteins of
different fractions were loaded on the SDS-PAGE. The AtVTI1 was visualized by
western blot. Although AtVT12 antibody recognized many background bands as
shown in fraction 2, which was from interphase between sample and the 24%
gradient. Fraction 3, which was from the interphase of gradient 24% and 36%,
enriched majority of the AtVTI1 vesicles with less background bands. Molecular
weights are indicated at the right.

166

This enriched AtVTI1-containing membrane fraction was used as the starting
material for affinity purification of AtVTI1 vesicles.
The Characterization of Affinity Purified AtVTI1 Vesicles

The AtVTI1 enriched fraction from the discontinuous sucrose gradient was
passed through affinity columns containing beads cross-linked to affinity purified
AtVTI12 antibody or pre-immnune antibody. After extensive washing, the vesicles
retained on the beads were eluted with 1% Triton X-100. The eluates from the
immune or preimmune columns were examined by western blot analysis with
antibodies for markers that were expected to localize in those vesicles. Previous
evidence suggests that sporamin is recruited by AtELP at the TGN and is
transported into the vacuole via the PVC (Ahmed et al., 2000). AtELP has been
shown to colocalize with AtVTI11 (Zheng et al., 1999) on the TGN and can also
be found on the PVC membrane (Sanderfoot et al., 1998). As shown in Figure 5-
2, we detected AtVTI1, AtELP and sporamin in the detergent eluate from immune
column but not from pre-immune column, indicating that we had successfully
affinity purified AtVTI1 vesicles that contained all three markers. In contrast, Bip,
an ER residential chaperone, was not present in the purified vesicle. Although
1% Triton X-100 should not dissociate the bond between the antibody and the
antigen, some free AtVTI1 molecules may reside on the same membrane where
other AtVTI11 molecules were bound to the antibody and account for the eluted
AtVTI1. In the case of sporamin, multiple bands were observed, probably caused
by changes in the glycosylation states of the protein. The 28 kDa band in the

eluate from immune column is also slightly bigger than reported for mature

167

 

 

 

AtVT11 *“"""

 

 

 

fol-“:1 ." s
.
*1
‘I
'I
‘ , ‘1‘. _ 9 .u .

sporamin I

 

 

 

 

BIP

 

 

 

Figure 5-2. Characterization of affinity purified AtVTI1 vesicles. 1/1000 of total
AtVTI1 enriched membrane fraction (T), 1/5 of eluate from pre-immune column
(Pre) or 1/5 of Triton X-100 eluate from immune column (Im) were separated by
SDS-PAGE. Different protein markers were visualized by western blots.
Molecular weights were indicated at the right.

168

sporamin (24 kDa) or its precursor (26 kDa) (Matsuoka et al., 1995b).
Conceivably, the TGN or the PVC harbor a particular form of glycosolated
sporamin. However, the exact nature of the larger molecular weight of the
sporamin band needs to be examined more carefully before any conclusion can
reached. The evaluation of the eluates from the immune and pre-immune
columns indicated that intact AtVTI1 vesicles were purified using this procedure.
AtVTI11 and Sporamin Colocalize on an Area Close to the Golgi.

The N-terminal propeptide (NTPP) from sporamin has been shown to
interact with AtELP in vitro. In vivo, AtELP and sporamin are found on the same
domain of the TGN (Ahmed et al., 2000). AtVTI11 also co-localizes with AtELP
on the TGN (Zheng et al., 1999). However, no direct evidence on the
colocalization of sporamin and AtVTI11 was available. Here, I show that
sporamin is present in purified AtVTI1 vesicles. To confirm the co-localization of
sporamin and AtVTI1, an immunocytochemical experiment was performed using
an Arabidopsis line that expresses both T7-AtVTl11 and sporamin (Figure 5-3).
Thin plastic sections of root tips were used to carry out double-labeling
experiment. Sections were first labeled with monoclonal anti-T7 antibody
(Novagen) and detected with 10 nm gold, Subsequent to a second fixation and
blocking step, the sections were incubated with anti-sporamin followed by
detection with 5 nm gold. Representative photomicrographs are shown in Figure

5-4A-D. T7-AtVTI11 co- localizes with sporamin at the trans-Golgi and in other

169

 

Spo Spo+T7 T7

 

31kD-
Anti—Sporamin

 

 

 

 

31'0“ ,_____,_.. J; Anti—T7

 

 

 

Figure 5-3. The plants crossed between transgeneic plant expressing T7-
Ath11 and sporamin. Protein extracts from the parental plants expressing T7-
AtVTl11 (T7) or sporamin (Spo) or the crossed progeny (Spo + T7) were
separated by SDS-PAGE. Antibodies were used to detect T7-AtVTl11 or
sporamin by western blots. Molecular weight was indicated at the left.

170

 

 

 

Figure 5-4. Immuno-localization of T7-AtVTI11 and sporamin on thin sections
from Arabidopsis roots. Thin sections of root tips from transgenic plant
expressing both T7-AtVTI11 and sporamin were first incubated with T7
monoclonal antibody followed by rabbit anti-mouse lgG and biotinylated goat
anti-rabbit secondary antibody and were visualized with streptavidin conjugated
to 10nm colloidal gold (arrow). After a second fixation step, the same sections
were incubated with anti-sproamin antiserum and the bound antibody was
visualized with protein-A conjugated to 5 nm colloidal gold (arrowheads). A-D)
The colocalization of T7-AtVTl11 and sporamin at the structure near the Golgi
(G). E) The predominant staining of sporamin in the vacuole (V). F) Control
experiment where first antibodies against T7 or sporamin were omitted in the
procedure. Ch: chloroplast. Bars represent 0.1 pm.

171

 

Table 5-1. Relative distribution of T7-AtVTI11 and sporamin in intracellular
compartments of roots from transgenic Arabidopsis.

 

 

 

 

Outside of vacuole Vacuole
Together Separately T7-AtVTI1 1 Sporamin
T7-AtVTI11 sporamin
41 7 3 O 56

 

 

 

 

 

 

 

173

 

structures near the vacuole. Those structures resemble the ones where
sporamin was found to co-Iocalize with AtELP (Ahmed et al., 2000). Sporamin
also accumulates in the vacuole, but AtVTI11 does not reach the vacuolar
membrane (Figure 5-4E). The statistics of the double labeling experiment are
shown in Table 5-1. These results are consistent with the analysis of the affinity
purified AtVTI1 vesicles. Taken together, these data indicates that the AtVTI1
family of proteins is involved in NTPP cargo transport.

Specific Proteins Can be Visualized by 2-D Gel and Silver Staining.

After purification of intact AtVTI1 vesicles from Arabidopsis, we undertook
the identification of the protein content of those vesicles. The resolving power of
SDS-PAGE was proven inadequate (Zheng and Raikhel, unpublished data).
Thus, I separated the eluted proteins by 2-Dimensional gel electrophoresis and
visualized the proteins by silver nitrate staining. Due to the low yield of the
purification, the eluates from 5 isolations were pooled before loading. Comparing
eluate from pre-immune and immune column, several spots were reliably
determined to be specific to the eluate from the immune column in three different
experiments (Figure 5-5). Due to the inherent limitations of the technique, large

proteins or highly hydrophobic proteins may have been missed.

Discussion

We have determined the subcellular localization of AtVTI11 and 12, and
the complexes they form with other SNAREs (Zheng et al., 1999; Bassham et al.,

2000). However, functional data on the AtVTI1 family is still scarce. Here, I found

174

 

Figure 5-5. Representative 2-D gels showing protein profiles of affinity-purified
AtVTI1 vesicles. From five affinity purification experiments, Triton-X 100 eluate
from pre-immune (A) or immune (B) columns were pooled (about 100 ug
protein). Samples were then separated by 2-D gel. Three pairs of gels were
compared. The spots reproducibly present in the eluate of immune column but
not in pre-immune column are indicated with circles.

175

rofiles plalflnilym 30 [(0.
nts. mm 100 a:
pooled (aboul ills;
tree pairs of gels: 25 kD-
l of immune calm 20 kD"

 

120 kD-
90 kD-

60 kD-
50 kD-

40 kD-

30 kD-

25 kD-
20 kD-

 

pH3>>>>>>>>>>pH1o

176

that sporamin, an NTPP-containing protein, is present in the affinity purified
AtVTI1 vesicles. Furthermore, sporamin and T7-AtVTl11 were found on the same
subcellular location by EM studies. Previous experiments have shown that the
TGN in plants has different domains. AtELP co-localizes with sporamin but not
with barley lectin at the TGN (Ahmed et al., 2000), suggesting that NTPP-
sporamin and CTPP-barley lectin are recruited to different domains of the
compartment. SPY41 and SYP42, two closely related syntaxin homologues, are
also localized on different domains of the TGN (Bassham et al., 2000). Although
on separate experiments, sporamin and AtVTI11 have each been co-Iocalized
with AtELP by EM, biochemical and immunocytochemical methods taken
together provided strong evidence that AtVTI11 also colocalizes with sporamin.
These data also support the notion that at least one member of the AtVTI1
family, AtVTI11, is involved in NTPP cargo transport. However, it is not clear
whether AtVTI11 and sporamin also co-localize on the PVC. The purified AtVTI1
vesicles are a mixture of vesicles from the TGN, the PVC, and maybe other
membranes that contain AtVTI11 or 12. On EM, the PVC can be identified by
immunolabeling with SYP2 antibodies. We have found that AtVTI11 and SYP2
co-localize on the PVC. Double labeling experiments with SYP2 and sporamin
have to be done to test whether sporamin is transported to the PVC. The
presence in the PVC of another NTPP-containing protein, Arabidopsis aleurain,
will also tested. If that is the case, it will offer direct evidence that the PVC is the
midstation for the biosynthetic pathway of NTPP-containing vacuolar cargo

transport.

177

As mentioned above, the PVC contains the marker proteins SYP2, AtELP,
AtVTI11, 12 as well as the recently identified syntaxins SYP5 and 6 (Sanderfoot
et al., 1998; Zheng et al., 1999; Sanderfoot et al., 2001). However, only SYP2 is
exclusively localized in the PVC, whereas the other proteins can also be found
on the TGN. Therefore the PVC appears to be closely related to the TGN. To
further characterize the PVC and address its function we have undertaken the
identification of its protein content. In recent years, great advances have been
made in the techniques required for separation and identification of individual
proteins in complex protein mixtures. The improved reproducibility of 2-D gel
electrophoresis, the development of new mass spectrometry technologies and
the complete genome sequence from several model organisms such as
Arabidopsis allows the identification of proteins in high throughput fashion
(review see Mann et al., 2001). It is now possible to characterize all the protein
species and their relative quantities within a given organelle at this so-called
"proteomic" scale. This proteomic approach will be especially useful when
characterizing novel organelles such as the PVC where molecular markers are
scarce.

SYP21 is the only marker present exclusively on the PVC. However, the
available SYP21 antibodies are not suitable for vesicle purification as the signal
to noise ration is low (Sanderfoot and Raikhel, unpublished). Instead, we used
AtVTI12 antibodies, which potentially would purify both AtVTI11 and 12
containing vesicles, due to the cross-reactivity of the antibody. The purified

vesicles were genuine and intact based on the presence or absence of several

178

markers from which the intracellular localization is known. However, the identity
of these vesicles needs further investigation. Firstly, we must establish whether
they are a mixture of AtVTI11 and AtVTI12 vesicles. Secondly, as AtVTI11 and
AtVTI12 are localized both on the TGN and the PVC, we need to show the origin
of the purified vesicles. Once the proteins in the purified vesicles have been
identified, their subcellular location needs to be studied before assigning them to
either the TGN or the PVC.

In many cases, the amount of individual protein from purified AtVTI1
vesicles was lower than the limit of detection. When the sensitivity of the
identification techniques increases, It may become possible to identify them.
Alternatively, a novel approach to purify the PVC could be used. As shown in
Figure 5-3, the amount of vesicles purified by the AtVTI1 antibody is low. The low
yield may be due to the inherent nature of the antibody or to the conformation of
AtVTI1 protein on the membrane that may prevent a higher affinity interaction
with the antibody. Raising the scale of the purification would also increase the
background level. As an alternative, antibodies against other markers on the
PVC, such as SYP5 and SYP6, may be used (Sanderfoot et al., 2001).
Ultimately, the purification of the PVC and the characterization of its "proteome"

will shed light into the function of this organelle.

179

 

References

Ahmed SU, Rojo E, Kovaleva V, Venkatarmam S, Dombrowski JE, Matsuoka
K, and Raikhel NV (2000). The plant vacuolar sorting receptor AtELP is involved
in the transport of NH2-terminal propeptides-containing vacuolar proteins in
Arabidopsis thaliana. J. Cell Biol. 149: 1335-1344.

Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, and Raikhel NV (2000).
AtVPS45 complex formation at the trans-Golgi network. Mol. Biol. Cell 11: 2251-
2265.

Becherer KA, Rieder SE, Emr SD, and Jones EW (1996) Novel syntaxin
homologue, Pep12p, required for the sorting of lumenal hydrolasese to the
Iysosome-like vacuole in yeast. Mol. Biol. Cell 7: 579-594.

Conceicio AS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, and
Raikhel NV (1997). The syntaxin homologue AtPEP12p resides on a late post-
Golgi apparatus of Saccharomyces cerevisiae Mol. Biol. Cell 10: 2407-2423.

Mann M, Hendrickson RC, and Pandey A (2001). Analysis of proteins and
proteomes by mass spectrometry. Annu. Rev. Biochem. 70: 437-473.

Marty F (1978). Cytochemical studies on GERL, provacuoles and vacuoles
inroot meristematic cells of Euphorbia. Proc. Nat. Acad. Sci. USA 75: 852-856.

Marty F (1980). High voltage electron microscopy of membrane interactions in
wheat. J. Histochemistry and cytochemistry 28: 1 129-1 132.

Matsuoka K, and Nakamura K (1991). Propeptide of a precursor to a plant
vacuolar protein required for vacuolar targeting. Proc. Natl. Acad. Sci USA 88:
834-838.

Matsuoka K, Bassham DC, Raikhel NV and Nakamura K (1995). Different
sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of
distinct sorting machinery in tobacco cells. J. Cell Biol. 130: 1307-1318.

Matsuoka K, Watanabe N, and Nakamura K (1995). O-glycosylation of a

precursor to a sweet potato vacuolar protein sporamin, expressed in tobacco
cells. Plant J. 8: 877-889.

Peck SC, Niihause TS, Hess D, Iglesias A, Meins F, and Boller T (2001).
Directed proteomics identifies a plant-specific protein rapidly phosphorylated in
response to bacterial and fungal elicitors. Plant Cell 13: 1467-1475.

Sanderfoot AA, Ahmed SU, Marty Mazars D, Rapopot l, Kirchhausen I,
Marty F, and Raikhel NV (1998). A putative vacuolar cargo receptor partially

180

colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis Roots.
Proc. Natl. Acad. Sci. USA 95: 9920-9925.

Sanderfoot AA, Kovaleva V, Bassham DC, and Raikhel NV (2001).
Interactions between syntaxins identify at least five SNARE complexes within the
Golgi/prevacuolar system of the Arabidopsis. Mol. Biol. Cell 12:

Zheng H, Fischer von Mollard G, Kovaleva V, Stevens TH, and Raikhel NV
(1999). The plant vesicle associated SNARE AtVTI1a likely mediates vesicle
transport from the trans-Golgi network to the prevacuolar compartment. Mol. Biol.
Cell 10: 2251-2264.

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Chapter VI

Conclusions and Future Directions

182

Endomembrane system is critical for an eukaryotic cell. Vesicle transport
is the major means for traffic flows between different membrane bound
compartments where SNARE proteins are important players. However, until
recently, most of the knowledge regarding the function of this system was
acquired by studying single celled yeast or mammalian cell cultures. In vascular
plants, the endomembrane system is evolved to suit plant specific challenges.
For example, plant cells have developed complex vacuolar system that are
composed of different types of vacuoles and multiple pathways to deliver proteins
to these vacuoles. Different cells also need specialized membrane system for
their particular functions. For example, it is very likely that the vacuolar protein
transport system in developing cotyledon will be quite different from the one used
by the guard cells. For SNARE genes, one single gene in yeast always found to
have evolved into a family of one to five members in Arabidopsis. Plants also
have novel SNAREs with no homologues in other kingdoms (Sanderfoot et al.,
2000). This phenomenon of gene duplications and novel genes reflects the
complexity and uniqueness of the plant secretory machinery. The understanding
of their specific features will facilitate our understanding of all aspects of plant
life.

AtVTI1 is a family of SNARE proteins in Arabidopsis that have
homologues in yeast and mammals. In yeast, Vti1p is a multi-functional SNARE
encoded by a single gene. It has been found to interact with 5 out of 8 of yeast
syntaxins (Gotte and Fischer van Mollard, 1998). In Arabidopsis, VTI1 family is

composed of three members. AtVTl13 is expressed in very low level and can not

183

be detected by northern analysis (data not shown). AtVTI11 and AtVTI12 are
highly expressed and present in every tissue examined. Their amino acid
sequences share about 65% identity. Cytosolic part of the protein was tagged
with 6XHis tag and over-expressed in E.coli. These recombinant proteins were
used to raise rabbit antisera. When these two anti-sera are carefully evaluated, I
found that anti-AtVTI11 has higher affinity towards AtVTI11 protein, while anti-
AtVTI12 recognizes both AtVTI11 and AtVTI12 to the same extent (Chapter IV).
In addition, these two proteins can be distinguished by a slight molecular weight
difference on western blot. Using anti-AtVTI11 in combination with transgenic
plants expressing T7-tagged AtVTI11, l characterized AtVTI11 substantially
(Chapter III). AtVTI11 is an integral membrane protein associated with several
membrane fractions. It is localized on the Golgi, the TGN (trans-Golgi network)
and the prevacuolar compartment (PVC). It co-Iocalizes with AtELP, the NTPP
cargo receptor, at the same domain of the TGN. It also physically interacts with
SYP2 and 5 (Chapter II, IV). In a transgenic plant simultaneously expressing T7-
AtVTI11 and sporamin, an NTPP vacuolar protein, these two proteins have been
co-localized to the same organelles (Chapter V) by EM double immunolabeling.
All these data suggest that AtVTI11 is involved in NTPP cargo transport from the
TGN to the PVC.

Although AtVTI11 and 12 are both expressed in most tissues of the plant
with similar mRNA tissue distribution of pattern (Chapter III), they differ in several
other aspects. When expressed in yeast, AtVTI11 can functionally replace yeast

Vti1 p in the transport step of carboxy-peptidase Y from the TGN to the PVC. On

184

the other hand, AtVTI12 is more efficient in facilitating the vacuolar transport of a
membrane protein alkaline phosphatase (ALP) and in the cytosol-to-vacuole
transport pathway (Chapter III). In plants, AtVTI11 forms complex with SYP2 and
SYP5, while AtVTI12 with SYP4 and 6 (Bassham et al., 2000; Sanderfoot et al.,
2000; Chapter IV). These two proteins also have distinct subcellular locations
based on Accudenz density gradient (Chapter IV). AtVTI11 has been found to co-
fractionate with SYP2 and SYP5, while AtVTI12 with SYP4 and SYP6. All these
results suggest that these two VTI1 proteins have different functions in plant
(summarized in Figure 6-1). A null mutant of AtVTI12 (Atvti12) has been
identified and characterized in detail in Chapter IV. Although this mutation
caused the absence of AtVTI12, there is no observable phenotype different from
wild type plants. This can be explained by the fact that SYP4 and 6 can be co-
precipitated with AtVTI11 by anti-AtVTI11 antibody in Atvti12 but not in the wild-
type plant extracts. This substitution of AtVTI12 with 11 allowed the plant to
perform its normal function without disturbance (Chapter IV). However, the
function of AtVTI12 and the SNARE complex it being a part of remains unclear.
zig-1 (sgr-4) is an Arabidopsis shoot gravitropic defect mutant was
identified from a pool of fast-neutron treated seeds (Fukaki et al., 1996). Just
recently, the gravitropic defect in sgr-4 has been found to be due to a disruption
in AtVTI11 gene. zig-1 has a DNA rearrangement that leads to a deletion from
the 5th intron to downstream of AtVTI11 gene. Inflorescence shoots and etiolated
hypocotyl of zig-1 failed to response to gravity and grow in random directions.

(Yamauchi et al., 1997; Kato et al., 2001). Except occasional irregular sizes and

185

TGN
GOLGI

* AtELP ,.
W

 

 

 

    

 

 

 

 

 

 

AtSYP6 _ , AtSYP2 . _
" AtVTI11
AtSYP4 _ AtSYP5
AtELP
——->

 

 

 

 

 

:35.» t-SNARE _,r- v-SNARE
>I NTPP cargo receptor

0 NTPP cargo

 

 

 

 

Figure 6-1. Schematic model for the AtVTI1 protein functions in plant cells. A
diagram of the late secretory pathway between the Golgi and the PVC is shown.
On the TGN, AtVTI11 is packaged into the vesicles together with NTPP cargo
and AtELP. Once reach the PVC, AtVTI11 interacts with SYP2 and SYP5 to form
a SNARE complex. This complex facilitates the fusion of vesicles and the PVC
membrane and the NTPP cargo is delivered to the PVC. AtVTI12 is also found
on the TGN. It interacts with SYP6 and SYP4. The significance of this SNARE
complex is not clear. Presumably, it is involved in vesicles targeting to the TGN.

186

shapes of endodermal cells, this mutant has normal tissue organization in
the stem with one epidermal cell layer, three cortex layers and one endodermal
cell layer arranged in a concentric manner from the outside to the core. The
amyloplasts, a special type of plastids containing starch granules, act as statolith
for gravity sensing and sediment at the bottom in the direction of gravity (Sack
1997). They are located in Columbia cells of root caps and the endodermis or
starch sheets of the shoots. ln wild type plants, the amyloplasts are sedimented
at the bottom and are able to move downward in response to the change of the
gravity stimulation (Konings, 1995; Blancaflor, et al., 1998). However, in zig-1
mutant, they are localized on the top, the bottom and the side of the endodermal
cells in the inflorescent stem and do not respond towards the directional change
in gravity. The amyloplasts in Columbia cells of root cap are sedimented
correctly, which correlates with the fact that the roots of zig-1 respond to gravity
normally (Kato et al., 2001; Morita et al., 2001). Beside the gravitropic defect, the
shoots also elongate in zigzag fashion and the leaves are wrinkled and smaller
than wild type plants (Yamauchi et al., 1997). At the cellular level, besides the
abnormal amyloplasts position in endodermal cell layers, aberrant vacuolar or
vesicular structures are occasionally observed in the cytoplasm (Morita et al.,
2001). The fragmented vacuoles are observed in the epidermis of the hypocotyl
using y—TIP-GFP as a tonoplast visual marker (Dr. Morita, personal
communication). This array of phenotypic abnormalities are complemented by
reintroduction of the genomic fragment containing AtVTI11 (MUL8.15; Kato et al.,

2001). The shoot gravitropic defect, but not the zigzag shape of the stems or

187

wrinkled and shrunken leaves, are also complemented by AtVTI11 expressed
under control of pSCR, an endodermis specific promoter (Kato et al., 2001).
Besides zig-1, two other alleles of zig are also found to have gravitropic defect.
zig-2 is a complete deletion mutant and zig-3 is a point mutant with a 61510
point mutation. More recently, another shoot gravitropic mutant, sgr-3, is found to
be attributed by a point mutation in SYP22 gene (Dr. Tasaka, personal
communication). It is likely the vesicle trafficking step of which AtVTI11 and
SYP22 are involved in gravity sensing in stems.

The shoot gravitropism defect in zig-1 may also be a second effect caused
by abnormal vacuolar protein trafficking that leads to physical or chemical
changes of the vacuole. Thus, the transport of vacuolar marker proteins should
also be investigated in zig-1 mutant. For example, the endogenous NTPP marker
aleurain, carboxy-peptidase Y homologue, VPE (vacuolar processing enzymes)
and other markers can be pulse-chase labeled in zig-1 protoplasts to investigate
their transport process. GFP-fusion markers for the study of vacuolar protein
targeting have been developed rapidly. GFP-AtELP may be used to monitor the
dynamics of vesicle transport between the TGN and the PVC. S-TlP-GFP, a
tonoplast marker (Cutler et al., 2000) can be used for observe vacuolar
morphology changes and vacuolar membrane protein transport. GFP fused to a
C-terminal vacuolar sorting determinant (VSD) from tobacco chitinase A has
been shown to be correctly targeted to neutral vacuolar compartment but
excluded from lytic vacuoles in tobacco cells (Di Sansebastiano et al., 1998).

GFP fused with VSD from barley aleurain (an NTPP containing sequence) is

188

 

 

 

correctly delivered to the lytic vacuole (Di Sansebastiano, 2001). In tobacco and
other species, vacuoles have been shown to regenerate from devacuolated
protoplasts (Wu and Tsai 1992). In this system, the GFP fused with C-terminal or
N-terminal VSD are targeted correctly to the neutral pH or lytic vacuoles
respectively (Di Sansebastiano, 2001). Using this vacuole regeneration system in
combination with GFP markers, we can start to study the vacuolar protein
targeting in double mutants. From a different approach, vacuoles from zig-1
should be compared with the ones from wild type plants. Vacuoles have been
easily purified by flotation on a Percoll gradient (Gomez L, and Chrispeels MJ,
1993). The difference in metabolite profile can be compared by nuclear-magnet
resonance (NMR) or GC (gas chromatography)-mass spectrometry. 2-D gel
profiles or isotope-coded affinity tags (ICAT; Gygi et al., 1999) treated samples
followed by mass spectrometry can be used to access the vacuolar protein
content changes. The phenotype of zig-1 is not as severe as we assumed based
on its role in the vacuolar protein transport. It is unlikely vacuolar proteins are
completely blocked from entering vacuole as AtVTI12 is capable to substitute
AtVTI11 (Zheng H, and Raikhel NV, unpublished). However, it is possible when
both VTI1 proteins are absent, the transport will be dramatically changed. In the
double mutant, there might be other phenotypes that are unrelated to
gravitropism. In that case, the experiments mentioned above should be also used
to evaluate the double mutant. The study in double mutant will yield rich
information regarding the role of AtVTl family in the vacuolar protein transport

and in the whole plant wellbeing. However, if the functions of AtVTI11 and 12 are

189

critical for the plant, it is possible that the double mutant will be lethal. In that
case, zig-3, the point mutant of AtVTI1 should used to cross with Atvti12. It is
more likely to yield viable plants that allow us to conduct meaningful analysis.
The correlation between vacuolar protein transport and the gravitropic
signal perception is significant for us to understand endomembrane system at the
whole plant level. The root and shoot gravitropism mechanism is related in the
sense that both of them use amyloplasts indicator for gravity. The sedimentation
of amyloplasts to the direction of the gravity induced certain unknown chemical
signals that lead to redistribution of auxin and asymmetry growth (Sack 1997).
Roots grow towards the direction of the gravity and the shoots grow in the
opposite direction. If the vacuolar transport or homeostasis defect is related to
amyloplasts sedimentation abnormalty and thus gravitropic defects in shoots, it is
very likely similar mechanism also exists in roots. However, amyloplasts of zig-1
roots are still able to sediment according to the direction of gravity and there is no
gravitropic defect in roots (Morita et al., 2001). Furthermore, in zig-1 roots, we
failed to find any abnormality in the Golgi and vacuolar morphology or the
aleurain transport (Zheng H, Kovaleva V and Raikhel NV, unpublished results).
Based on preliminary results, when AtVTI12 was immunoprecipitated from zig-1
roots but not wild type roots, SYP21 and SYP22 were both co-precipitated. It is
likely that AtVTI12 can substitute AtVTI11 in zig-1 roots and interacts with both
SYP21 and SYP22. The apparent normal roots may be the result of this
substitution. It is imaginable that when the vacuolar transport in roots is also

disturbed, the root gravity response might be reduced or diminished. It is thus

190

 

interesting to see the root gravitropism response in double mutant of zig-1 and
Atvti12. Of course, it is also possible that the mechanism of gravity perception in
roots is different from shoots and does not require AtVTI11 or AtVTI12 functions.
The fact that Atvti12 resembles the wild type while zig-1 has abnormal
phenotypes worth our investigation. In single mutants, both proteins seem to be
able to form SNARE complexes with syntaxins that normally only interacts with
the other one. It appears that the AtVTI11 is efficient at forming the complex with
SYP4 and SYP6 and resulted in a normal plant in Atvti12. On the other hand, at
least in stems, the complex AtVTI12 formed with SYP2 and 5 seems not enough
for the proper function. One possible explanation is that there are three VTI1
genes in Arabidopsis. AtVTI13 is more closely related to AtVTI12 (75% identical)
than to AtVTI11 (65% identical). AtVTI13 expression level is significantly lower
than the other two genes, and it can not be detected by anti-AtVTI12 antibody on
western (the more general antibody). However, we can not rule out that AtVTI13
is expressed highly in certain cells or the very low level of protein is enough to
functionally complement AtVTI12 in Atvti12. The gene expression pattern of
these three VTI1 genes should be investigated in more detail. Another
explanation about the different phenotypes between zig-1 and Atvti12 lies in the
difference between SYP2 and 4. SYP41 and 42 both are localized on the TGN
but at different domains. It is possible AtVTI12 form complex with SYP41 and 42
to lead different vesicles recycling back to the TGN. As we know, AtVTI11 is
localized on both the PVC and the TGN membrane. In Atvti12, AtVTI11 still has

the opportunity to interacts with both SYP41 and SYP42 and fulfill the functions

191

of AtVTI12. The situation for SYP2 group syntaxins is different. In root tips, both
SYP21 and SYP22 are found on the PVC (Sanderfoot et al., 1999). The
interaction between AtVTI11 and SYP2 may help vesicle trafficking from the TGN
to the PVC. AtVTI12 is also observed on both the PVC and the TGN membrane
in root tips. Thus, in the roots of zig-1, AtVTI12 may be able to transport vesicles
from the TGN fonrvard to the PVC by interacting with SYP21 and SYP22 and
allows the roots grow without defect. However, in shoot meristem, SYP22 has
been observed on small vacuolar membranes (Sato et al., 1997). SYP22, but not
SYP21, is also found to be enriched in vacuoles prepared from protoplasts of
suspension cultured Arabidposis cells (Rojo E, and Raikhel NV, unpublished
data). Because AtVTI12 does not travel beyond the PVC, it is not able to interact
with SYP22 on the vacuole and stand in for AtVTI11 in that step of membrane
fusion in shoot tissues of zig-1. To see if this model is true, we should consider
the plant as truly a multicellular organism with different tissues and organs
requiring different specialized cellular functions. Most information regarding the
subcellular locations of the SNAREs is acquired by studying root tips. However,
the locations of these SNAREs may be different in soil grown plants. Using
gradient fractionation or immunocytochemistry, the subcellular localization of
AtVTI11, AtVTI12, SYP21, SYP22, SYP41, and SYP42 should be revisited with
tissue specificity in mind. Preliminary results indicate that on Accudenz gradient,
vacuoles float on top of the gradient. AtVTI11 peak is shifted to lighter fractions in
stems compares to the same experiment using root materials (data not shown).

SYP21 and SYP22 have identical fractionation pattern in roots, but it will be

192

interesting to see whether SYP21 and SYP22 are localized differently in stems.
To study the tissue specific expression pattern of these SNARE genes, promoter
fused with GFP or GUS should also used to tranfonn Arabidopsis.

In our lab, there are several lines of T-DNA mutants of AtELP and other
VSR (Vacuolar sorting receptor) genes. None of those mutants has no obvious
phenotype (Avila-Teeguarden E, and Raikhel NV, unpublished results). Since
AtELP is in the same pathway with the AtVTI11 in transport of NTPP proteins, it
is possible other VSRs also have related functions. Thus, it will be interesting to
examine the phenotype of those mutants with zig-1 or Atvti12.

In general, the mutants of AtVTI11 and 12 offer good tools to understand
the function of this family of SNAREs. They are also useful in our attempt to
understand the roles they play in the endomembrane system and in the

development and physiology of plants.

193

 

References

Blancaflor EB, fasano JM, and Gilroy S (1998) Mapping the functional roles of
cap cells in the response of Arabidopsis primary roots to gravity. Plant Physiol.
116: 213-222.

Cutler SR, Ehrhardt DW, Griffitts, JS, and Somerville CR (2001) Random
GFP :: cDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at high frequency. Proc. Natl. Acad. Sci. USA 97: 3718-3723.

Di Sansebastiano GP, Paris N, Marc-Martin S, and Neuhaus JM (1998)
Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-
terminal propeptide-mediated sorting pathway. Plant J. 15: 449-457.

Di Sansebastiano GP, Paris N, Marc-Martin S, and Neuhaus JM (2001).
Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be

visualized by green fluorescent proteins targeted to either type of vacuoles. Plant
Physiol. 126: 78—86.

Fukaki H, Fujisawa H, and Tasaka M (1996) SGR1, SGR2, SGR3: novel
genetic loci involved in shoot gravitropism in Arabidopsis thaliana. Plant Physiol.
110: 945-955.

Gomez L, and Chrispeels MJ (1993). Tonoplast and soluble vacuolar proteins
are targeted by different mechanism. Plant Cell. 5: 1113-1124.

Gétte M and Fischer von Mollard G (1998) A new beat for the SNARE drum.
Trends Cell Biol. 8: 251 -21 8.

Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, and Aebersold R (1999)
Quantitative analysis of complex protein mixtures using isotope-coded affinity
tags. Nat Biotechnol 17: 994-999.

Kato T, Morita Ml, Fukaki H, Yoshiro Y, Uehara M, Nihama M, and Tasaka M
(2001) SGR2, a phospholipase-Iike protein, and ZIG/SGR4, a SNARE, are
involved in the shoot gravitropism of Arabidopsis (Submitted).

Konings H (1995). Gravitropism of roots, an evaluation of progress during the
last three decades. Acta Bot. Need. 44: 195-223.

Morita MT, Kato T, Nagafusa K, Saito C, Ueda T, Nakano A and Tasaka M
(2001). Involvement of the vacuoles of the endodermis in early process of shoot
gravitropism in Arabidopsis (Submitted).

Sack FD (1997) Plastides and gravitropic sensing. Planta 203: 863-868.

194

 

Sanderfoot AA, Kovaleva V, Zheng H, and Raikhel NV (1999) The t-SNARE
AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells.
Plant Physiol. 121: 929-938.

Sanderfoot AA, Assaad, FF, and Raikhel NV (2000), The Arabidopsis genome.
An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein
receptors. Plant Physiol. 124: 1558-1569.

Sato MH, Nakamura N, Ohsumi Y, Kouchi H, Kondo M, Hare-Nishimura l,
Nishimura K, Wada Y (1997). The atVAM3 encodes a syntaxin-related molecule
implicated in the vacuolar assembly in Arabidopsis thaliana. J Biol. Chem. 272:
24530-24535.

Wu FS, and Tsai YZ (1992) Evacuolation and enucleation of mesophyll
protoplasts in self-generating Percoll gradients. Plant Cell Environ. 15: 685-692.

Yamauchi Y, Fukaki H, Fujisawa H and tasaka M (1997). Mutations in the

SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism.
Plant Cell Physiol. 38: 530-535.

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