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FUNCTIONAL ANALYSIS OF A PHOSPHATIDIC ACID
TRANSPORT SYSTEM IN ARABIDOPSIS THALIANA

presented by

Binbin Lu

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5/08 Kthroj/Accanpres/CIRC/Dateoue.indd

 

FUNCTIONAL ANALYSIS OF A PHOSPHATIDIC ACID TRANSPORT
SYSTEM IN ARABIDOPSIS T HALIANA

By

Binbin Lu

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Biochemistry and Molecular Biology

2009

ABSTRACT

FUNCTIONAL ANALYSIS OF A PHOSPHATIDIC ACID TRANSPORT
SYSTEM IN ARABIDOPSIS T HALIANA

By
Binbin Lu

Membranes consisting of lipid bilayers are indispensable structures of all
organisms. Membranes of lipid assembly are often distinct from final destination
membranes, which require substantial polar lipid trafficking and lipid transport. A
striking example is the biogenesis of the photosynthetic membranes (thylakoids) in
plastids of plants. Many lipid biosynthetic enzymes at the endoplasmic reticulum (ER)
and the inner and outer plastid envelope membranes are involved in this process.
Arabidopsis thaliana mutants that are disrupted in the incorporation of ER—derived lipid
precursors into thylakoid lipids are available due to systematic genetic screening and
bioinformatics studies. Three proteins identified in three of these mutants,
TRIGALACTOSYLDIACYLGLYCEROL l (TGDl), TGDZ, and TGD3, are postulated
to be the permease, substrate binding component, and ATPase, respectively, of a
proposed lipid translocator at the inner chloroplast envelope membrane. In chapter 2, I
describe the identification and characterization of TGD3, a small ATPase proposed to be
part of this translocator. The TGD3 protein shows basal ATPase activity when expressed
as a maltose-binding protein fusion and is localized inside the chloroplast beyond the
inner envelope membrane. Similar to the tgdl and tgd2 mutants, a tgd3 mutant carrying a
T-DNA insertion just 5’ of the TGD3 coding region (tgd3-1) also accumulates
triacylglycerols and trigalactolipids. However, the TGD3 gene expression level is only

slightly reduced in this tgd3-1 mutant. Mutants fully disrupted in TGD3 were unavailable

to analysis at the time of the study. By utilizing RNA silencing, Arabidopsis mutant
plants with more severely disrupted TGD3 function were generated and analyzed, as
described in chapter 3. The phenotype of these RNAi mutant plants are nearly identical to
tgd3-1, and do not show the expected severe growth defect and embryo lethality seen in
other tgd mutants, raising the possibility of an additional ATPase associated with the
proposed TGD complex. Chapter 4 focuses on the in-depth biochemical studies of the
TGD2 protein, whose C-terminal domain (TGD2C) was previously shown to specifically
bind phosphatidic acid (PtdOH). The PtdOH binding site was further narrowed down to a
25 amino acid segment, which by itself is sufficient to mediate specific binding of TGD2
to PtdOH. However, binding characteristics of this 25-mer were different from that of
wild-type TGD2C suggesting that additional sequences of TGD2 are needed for this 25-

mer to provide the proper context for wild-type like PtdOH binding.

ACKNOWLEDGMENTS

My time at Michigan State will be the most fruitful and unforgettable five years of
my life and that fact is partially due to many wonderful people I have known and
interacted with. First, I would like to give my thanks to my thesis advisor, Dr.Christoph
Benning. During my rotation in his lab, he inspired me to develop my scientific skills,
fostered my interest to become an independent scientist as well as improved my English
(since he always spoke so fast that I had to learn really hard to catch up with him and
understand what he was saying). He also worked hard to get the National Science
Foundation grant that initiated the membrane lipid trafficking project involving TGD
proteins to insure my place in his lab. After I started my thesis work, he always guided
me in the right directions and pushed me to work hard and intelligently to get my two
publications. I also learned a lot fiom him about how to write a scientific paper in correct
English.

I would also like to thank my other two mentors, Dr. Koichiro Awai and Dr.
Changcheng Xu, who directly guided me through my rotation and the first couple of
months in the Benning lab to help me get used to the new environment and become an
independent scientist as well as an active thinker. They both are great scientists and
wonderful people and I hope they continue to succeed in their own careers.

My thesis advisory committee, Dr. Michael Garavito, Dr. Jianping Hu, Dr. Ken
Keegstra, and Dr. Beronda Montgomery also deserve my thanks. They provided me a lot

of feedbacks and contributed thoughtful insights about my research during every

iv

discussion meeting. Special thanks will be given to Dr. Beronda Montgomery, who
allowed me to use her spectrophotometer/plate reader that generated data for my first
publication in JBC (Figure 2.7) and Dr. Michael Garavito who shared with me his
DsRed-plel-His vector, which was used to clone our TGD2 gene and greatly improved
solubility and expression of resulting proteins. These work laid the basis of results
described in Chapter 4 and directly led to my second I BC paper.

I am also thankful to the Department of Biochemistry and Molecular Biology and
MSU US. Department of Energy Plant Research Laboratory for accepting me as a
graduate student and provide support for my study, travel to scientific meetings as well as
easy access to numerous facilities. I also benefited from the community here at MSU
where I had countless discussions with graduate students and postdocs, especially the
Garavito lab members, who offered me great technical support and friendship and the
Benning lab members, who always made the lab a pleasant work place and a warm
family during my stay in the United States.

All of my co-workers and friends have been wonderful, and the Benning lab and
MSU has been my second home, so I am happy that my lab mates and friends could
witness my recent marriage, since you are like my family members away from China.
Leaving is bittersweet, but knowing that I will stay in University of Michigan after I
leave MSU makes me feel a little better. Thanks for all the people and hope to see you in

the future.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii
LIST OF FIGURES ................................................................................. ix
CHAPTER 1 -Galactoglycerolipid biosynthesis and lipid trafficking between the
endoplasmic reticulum and the plastid in plants ................................ 1
1.1. Introduction ................................................................................... 2
1.2. Galactoglycerolipids are the most abundant lipids in plants ........................... 3
1.3. Three enzyme systems are involved in galactoglycerolipid biosynthesis in plants.7
1.4. The two-pathway hypothesis of galactoglycerolipids biosynthesis .................. 11
1.5. Basic polar lipid transfer mechanisms involving plastids ............................. 15
1.5.1. Flip-flop movement ................................................................ 15
1.5.2. Interrnembrane transport and transfer by membrane contact sites ......... 21
1.5.3. Vesicular lipid transfer ............................................................ 24
1.6. Current progress in the study of lipid trafficking in Arabidopsis .................... 26
Reference list ........................................................................................ 30
CHAPTER 2 - Identification and characterization of TGD3 .................................. 39
2.1. Abstract ...................................................................................... 40
2.2. Introduction ................................................................................. 41
2.3. Experimental procedures .................................................................. 43
2.3.1 Plant materials and growth conditions ........................................... 43
2.3.2 Genotyping and complementation .............................................. 44
2.3.3 Lipid assays ........................................................................ 45
2.3.4 Mass spectrometry ................................................................ 45
2.3.5 Subcellular localization assays .................................................. 46
2.3.6 Recombinant MBP-ATGD3 protein production and purification ........... 46
2.3.7 Assay for ATPase activity ....................................................... 47
2.4. Results ....................................................................................... 49
2.4.1 Identification of TGD3 by genomics and reverse genetics .................. 49
2.4.2 The tgd3 mutant shares a complex lipid phenotype with other tgd
Mutants ............................................................................. 57
2.4.3 The TGD3 protein is Imported into the chloroplast ........................... 67
2.4.4 Recombinant TGD3 protein has ATPase activity ............................. 70
2.5. Discussion ................................................................................... 74
Reference List ....................................................................................... 78

CHAPTER 3 -Germination, growth and lipid analysis of Arabidopsis plants disrupted in
TGD3 ............................................................................... 81
3.1. Abstract ...................................................................................... 82

vi

3 .2. Introduction ................................................................................. 83

3.3. Experimental Procedures .................................................................. 85
3.3.1. Plant materials and growth conditions .......................................... 85
3.3.2. Lipid and fatty acid analysis ..................................................... 85
3.3.3. Root elongation assays ............................................................ 86
3.3.4. Generation and analysis of TGD3 RNAi plants ............................... 86
3.3.5. Dominant-negative mutation analysis .......................................... 91
3.3.6. Chlorophyll quantification ....................................................... 92

3.4. Results ........................................................................................ 92
3.4.1. Oligogalactolipids accumulate in TGD3i leaves .............................. 92
3.4.2. The phenotype of TGD3i and tgd3-1 are very similar ........................ 94
3.4.3. Impaired growth and development of TGD3i ................................. 98
3.4.4. Overexpression of TGD3F94A cDNA in wild type causes dominant

negative effect .................................................................... 103

3.4.5. Inducible silencing of TGD3 in Arabidopsis ................................. 106
3.5. Discussion ................................................................................. 109
Reference List ...................................................................................... 112

CHAPTER 4 Biochemical analysis of the phosphatidic acid binding site in Arabidopsis

TGD2 protein ..................................................................... 114
4.1. Abstract ..................................................................................... 115
4.2. Introduction ................................................................................ 116
4.3. Experimental procedures ................................................................ 118
4.3.1. Expression and purification of DR-TGD2 fusion proteins ................. 118
4.3.2. Phylogenetic analysis of TGD2 ................................................ 122
4.3.3. Protein-lipid overlay assay ...................................................... 122
4.3.4. Liposome-association assay .................................................... 123
4.3.5. Quantitative analysis of immunoblots ........................................ 123
4.4. Results ...................................................................................... 124
4.4.1. TGD2 orthologs in plants and Cyanobacteria ................................ 124
4.4.2. Specific binding of the DR-TGD2C WT fusion protein to PtdOH ....... 130
4.4.3. Identification of TGD2C amino acid sequences necessary for PtdOH
binding ............................................................................ 135
4.4.4. A TGD2 minimal domain sufficient for PtdOH binding. 141
4.4.5. PtdOH binding properties of the minimal PtdOH binding domain ....... 144
4.5. Discussion .................................................................................. 150
Reference List ...................................................................................... 154
CHAPTER 5 - Conclusions and perspectives ................................................... 157
5.1. Major Conclusions ........................................................................ 158
5.2. Remaining questions and future directions ............................................ 159
5.2.1. Demonstration of direct interaction of the three TGD proteins. . . . . . . . ....160
5.2.2. Investigation of the function of MCE domain in TGD2 .................... 161
5.2.3. Reconstitution of TGD transporter and in vitro uptake assay .............. 163
5.3. Summary. ............................................................................... 165
Reference List ...................................................................................... 169

vii

LIST OF TABLES

Supplemental table 4.1 PCR primers used for generation of DR-TGDZ fusion

proteins .............................................................................................. 1 20

viii

LIST OF FIGURES

Figure1.1. Chemical structures of plant galactoglycerolipids ................................... 5

Figure1.2. Three enzyme systems involved in galactoglycerolipid biosynthesis in
plants .................................................................................................... 9

Figurel .3. Two-pathway hypothesis of galactoglycerolipid biosynthesis in plants ........ 14

Figure1.4. Postulated lipid transfer processes involved in ER-to-plastid lipid trafficking in

plants .................................................................................................. 17
Figure 2.1. Isolation of the TGD3 gene by reverse genetics ................................... 50
Figure 2.2. Identification of the T GD3 locus (At1g65410) .................................... 55
Figure 2.3. Lipid phenotype of the tgd3 mutant compared with the tng, tgdZ mutant, and
Col-2 wild type ...................................................................................... 58
Figure 2.4. Liquid chromatography/MS analyses of lipid extracts of tgd3 mutant and Col-
2 wild type Arabidopsis leaves .................................................................... 60
Figure 2.5 . Polar lipid composition and fatty acid content in the tgd3 mutant .............. 64
Supplemental Figure 2.1. Fatty acid composition of lyso-MGDG (A) and lyso-DGDG (B)
in the tgd3 mutant and Col 2 wild type lines .................................................... 66
Figure 2.6. Subcellular localization of TGD3 ................................................... 68
Figure 2.7. Purification and ATPase activity of recombinant TGD3 protein ............... 72
Figure 2.8. Model of the proposed TGD lipid transporter complex .......................... 77
Figure 3.1. Generation of T 6031' constructs ..................................................... 89
Figure 3.2. Oligogalactolipids accumulate in T GD3-RNAi plants ............................ 93
Figure 3.3. Polar lipid composition and fatty acid content in the tgd3 mutant .............. 96
Figure 3.4. Delayed root growth in tgd3 mutants ............................................... 99
Figure 3.5. Delayed germination in tgd3 mutants .............................................. 101

ix

Figure 3.6. Impaired growth and slight chlorosis phenotype of tgd3 mutants. . . . . . . . l 02

Figure 3.7. Dominant negative effect caused by expression of T GD3F 94A mutant cDNA

in Col-2 wild type ................................................................................. 105
Figure 3.8. Inducible silencing of T GD3 in Arabidopsis ..................................... 108
Figure 4.1. Sequence analysis of TGD2 ........................................................ 126

Supplemental figure 4.1. Binding of DsRed-TGDZC WT fusion protein to PtdOH. . 1 31

Figure 4.2. Binding of the DR-TGDZC WT fusion protein to PtdOH as a function of
weight percent of PtdOH in a PtdOH/PtdCho mixture ....................................... 132

Supplemental figure 4.2. SDS—PAGE of the proteins used for liposome binding assay. 1 37

Figure 4.3. Identification of a PtdOH binding domain in TGD2C .......................... 139
Figure 4.4. Fine mapping of a TGD2 minimal PtdOH binding sequence .................. 142
Figure 4.5. Reduced affinity of the minimal PtdOH binding domain for PtdOH ......... 146
Figure 4.6. Additional TGD2 sequences contributing to PtdOH binding .................. 149

Chapter 1

Galactoglycerolipid biosynthesis and lipid trafficking between the endoplasmic
reticulum and the plastid in plants

1.1. Introduction

The chloroplast is critical for plants because it is where photosynthesis takes place.
It harbors a unique and also the most extensive membrane system in nature-the thylakoid
membrane. Photosynthetic protein complexes are embedded into the thylakoid membrane
and carry out photosynthesis to provide oxygen in the atmosphere and chemical energy to
heterotrophic organisms in the form of reduced carbon.

The chloroplast has two boundary envelope membranes to separate the interior
from the extraplastidic compartments of the cell. Both membranes consist of lipid
bilayers. However, different from many organisms, such as fungi, animals and bacteria,
whose predominant lipid classes are phosphoglycerolipids, the most abundant lipids in
photosynthetic organisms are non-phosphorous glycoglycerolipids. Among those
glycoglycerolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG) are
predominant in chloroplasts (Ddrmann and Benning 2002).

Glycoglycerolipids are derived from fatty acid precursors, whose synthesis is
conducted in the chloroplasts (Ohlrogge et a1. 1979). Fatty acids can either stay in
chloroplasts and be directly used for glycerolipid synthesis, or they can be exported and
assembled into lipids in the endoplasmic reticulum (ER). Glycerolipids assembled in the
ER serve as building blocks for extraplastidic membranes. Alternatively, lipids could also
return to chloroplasts, where their diacyl glycerol backbones are incorporated into
thylakoid lipids (Roughan and Slack 1982).

Apparently, the assembly of thylakoid membranes requires substantial lipid
exchange between the ER and the chloroplasts, however, most of the lipid trafficking

phenomena still remain poorly understood. In this chapter, I will review studies of

chloroplast lipid biosynthesis, with a focus on the role of lipid trafficking between the ER
and the plastid relevant to chloroplast biogenesis. In addition, I will introduce our current
working model suggesting that the TGD complex is a critical ABC (ATP-binding cassette)
transporter that is involved in phosphatidic acid transfer from the outer chloroplast to

inner chloroplast envelope.

1.2. Galactoglycerolipids are the most abundant lipids in plants

Membranes consisting of lipid bilayers are indispensable structures of all
organisms. In fungi, animals and bacteria, the predominant lipid classes are glycerolipids
that carry a phosphate head group. Likewise, those lipid classes are the major
components of plasma membranes and ER, peroxisomal and mitochondrial membranes in
seed plants. However, in plastids, non-phosphorous glycerolipids carrying one
(monogalactosyldiacylglycerol, MGDG) or two (digalactosyldiacylglycerol, DGDG)
galactose head moieties are the main constituents of the membranes (Ddrmann and
Benning 2002). MGDG and DGDG account for approximately 50% and 25% of the
thylakoid membrane lipids, respectively. Together, they represent the bulk of the
photosynthetic membranes in plant chloroplasts. In addition,
sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (Pthro) represent
about 15% of the thylakoid lipid species (Ddrmann and Benning 2002).

Normally, in MGDG, the galactosyl head group is linked to the diacylglycerol
backbone in B-configuration (Gal [Bl'—>3]-diacylglycerol), whereas the second galactose
of DGDG is bound to the first one in (Jr-configuration (Gal [a1"—’6']- Gal[B1'—>3]-

diacylglycerol) (Figure 1.1 A) (Carter et al. 1956). Unusual oligogalactoglycerolipids

such as trigalactosyldiacylglycerol (TGDG) and tetragalactosyldiacylglycerol (TeGDG)
are also found in some plant species, including Chlorella, pumpkin, rice bran and Adzuki
bean (Benson et al. 1958, Fujino and Miyazawa 1979, Galliard 1969, Ito and Fujino 1975,
Kojima et al. 1990). These lipids have three or four galactosyl head moieties attached to a
diacylglycerol backbone and can be divided into at least three categories based on their
anomeric configuration, as shown in Figure1.1. They can be derived either from the
normal isomer [La-DGDG or isomer B,B-DGDG by galactosylation. It should be noticed
that TGDG detected in the Arabidopsis tgd mutants, which will be described in detail

later, have all [3 configuration (TGDG[B, B, B], Figurel .1 C) (Xu et al. 2003).

Figurel.l. Chemical structures of plant galactoglycerolipids, adapted from
Dfirmann and Benning 2002. A, isomers of MGDG and DGDG. B-D, three categories
of TGDG and TeGDG isomers. The anomeric linkage (a or B) of the glycosidic C1
atoms are indicated in parentheses and the structures. The open hexagons represent the
CngO4 units derived from galactose. Rl-CO- and R2-CO- are acyl groups esterified to

the sn-1 or sn-2 position of glycerol backbone, respectively.

A HO-CH2 l3

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1.3. Three enzyme systems are involved in galactoglycerolipid biosynthesis in plants

Three classes of galactosyltransferases are associated with plastid envelopes and
are responsible for galactoglycerolipid biosynthesis, namely
monogalactosyldiacylglycerol synthase (MGDl, MGD2/3), digalactosyldiacylglycerol
synthase (DGDl, DGD2), and processive galactolipid:galactolipid galactosyl transferase
(GGGT) (Figure 1.2) (Benning and Ohta 2005).

The genes that encode the enzymes of MGDG and DGDG synthesis have been
isolated from different plants, including Arabidopsis (Dormann and Benning 2002,
Jorasch and Heinz 1999, Joyard et al. 1998). MGDG is synthesized in plants by MGDG
synthase (EC 2.4.1.46), which catalyzes the transfer of one galactosyl residue from UDP-
galactose to the sn-3 position of diacylglycerol (DAG) (Benning and Ohta 2005). The
Arabidopsis genome contains three genes encoding functional MGDG synthases, which
could be grouped into the A-type (MGDI) (Figure 1.2 A) and the B-type (MGD2 and
MGD3) (Figure 1.2 B) (Awai et al. 2001, Miege et al. 1999). These different enzymes
have different subcellular localizations, functions, as well as substrate specificities with
regard to the DAG precursor. Though mutant analysis, MGDl was shown to be the major
isoform responsible for the bulk of MGDG synthesis in Arabidopsis. Even partial
inactivation of the gene in the mutant led to a dramatic reduction in MGDG synthase
activity and MGDG level (Awai et al. 2001, Jarvis et al. 2000). MGDl was associated
with the inner chloroplast envelope whereas MGD2 and MGD3 were localized to the
outer chloroplast envelope (see Figure1.2 A and B)(Awai et al. 2001, Miege et al. 1999).

In terms of substrate specificity, MGDl utilizes both 18:2/18z2-DAG and 18:1/1620-DAG

equally, while MGD2 and MGD3 prefer 18:2/ 18:2 DAG over l8:1/16:O-DAG (Awai et
al. 2001).
DGDG synthase catalyzes galactosylation of MGDG to form DGDG. Isolation of

the (1ng mutant in Arabidopsis led to the identification of the gene encoding the enzyme

responsible for the bulk of DGDG synthesis, DGDI (Ddrmann et al. 1995) (Figure 1.2 A).

Disruption of the function of DGDl in the dng mutant results in 90% reduction in
DGDG content (D6rmann et al. 1995). Arabidopsis also has a second DGDG synthase,
DGD2 (Figure 1.2 B), identified by sequence similarity to DGDl (Kelly and Dormann
2002). Both DGDl and DGD2 enzymes are associated with the outer chloroplast
envelope and catalyze the formation of [La-DGDG from UDP-galactose and MGDG
(Kelly et al. 2003, Kelly and Dormann 2002).

A third class of galactosyltransferase, processive galactolipid:galactolipid
galactosyltransferase (GGGT), is capable of synthesizing unusual
oligogalactoglycerolipids such as TGDG and TeGDG (Figure 1.2 C). Its activity has been
observed in chloroplast, however, its function and molecular identity are not yet known

(Heemskerk et al. 1990, Heemskerk et al. 1991).

 

.J

F igurel.2. Three enzyme systems involved in galactoglycerolipid biosynthesis in
plants, adapted from Benning and Ohta 2005. A, MGDl and DGDl are the major
isoforrns of galactosyltransferases to synthesize the bulk of chloroplast
galactoglycerolipids. B, MGD2/3 and DGD2 synthesize galactoglycerolipids in non-
photosynthetic tissues and under phosphate deprivation conditions. C, GGGT is
responsible for the synthesis of oliogalactoglycerlipids. FAS, fatty acid synthase complex;

iE, inner chloroplast envelope; 0E, outer chloroplast envelope; Thy, thylakoids.

 

 

ER

 

   

 

  

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DAG —>

 

 

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10

 

Current evidence suggests that galactoglycerolipids are exclusively synthesized
by these three sets of enzymes associated with the two chloroplast envelopes in plants.
However, galactoglycerolipids could also be present in extraplastidic membranes in non-
photosynthetic tissues or under phosphate-limiting growth conditions (Hartel et al. 2000).
This fact requires substantial transfer of lipid precursors as well as galactoglycerolipids
themselves between different membranes, including ER, chloroplast, thylakoid etc.

However, most of the lipid trafficking processes remain unknown.

1.4. The two-pathway hypothesis of galactoglycerolipids biosynthesis
Galactoglycerolipids could be assembled either de novo in the plastids or from
imported precursors assembled at the ER. This two-parallel-pathway theory was first
proposed by Roughan and his colleagues (Roughan et al. 1980, Roughan and Slack 1982).
The current hypothesis is shown in Figure 1.3. In the plastid (also called prokaryotic)
pathway, fatty acids synthesized in chloroplasts are directly incorporated into chloroplast
lipid species catalyzed by plastid acyl-acyl carrier protein (ACP): glycerol 3-phosphate
acyltransferase encoded by ATS] (Kunst et al. 1988, Xu et al. 2006) and plastid acyl-
ACleyso—phosphatidic acid (PtdOH) acyltransferase encoded by ATS2 (Kim and Huang
2004, Yu and Benning 2003). The PtdOH generated is then dephosphorylated by a
phosphatase to diacylglycerol (DAG). A candidate PtdOH phosphatase (PAP) was
recently reported (Nakamura et al. 2007), however, the definitive proof that this enzyme
is involved is still lacking. The produced DAG together with UDP-galactose serves as

substrate for MGDl to form MGDG (Awai et al. 2001, Jarvis et al. 2000). DGDl then

11

catalyzes the next step of adding one more galactose to MGDG to form DGDG
(Donnann et al. 1999, Kelly et al. 2003).

In the ER (also called eukaryotic) pathway, those plastid-derived fatty acids are
transported to the ER where they serve as building blocks for extraplastidic membrane
lipids by ER-specific acyltransferases. Recent pulse-chase labeling data suggested that
newly exported fatty acids first appear in phosphatidylcholine (PtdCho) by a very active
acyl editing mechanism (Bates et al. 2007, Williams et al. 2000). Alternatively, DAG
moieties of PtdCho can be transported back to the plastid and then enter the
galactoglycerolipids biosynthesis as mentioned above. Therefore, only DAG origins for
galactolipid biosynthesis differ in the two pathways, the final assembly into
galactoglycerolipids by MGDl and DGDI are the same in either pathway.

Two-pathways of galactolipid biosynthesis are present in many plant species,
including the well-studied genetic model plant Arabidopsis. In Arabidopsis, galactolipid
species coming from both pathways are approximately equal in proportion (Browse et a1.
1986). Lipid species derived from different pathways can be distinguished because they
have different fatty acids in the diacylglycerol backbone, a fact due to distinct substrate
specificities of the acyltransferases or acyl exchange enzymes in the ER or the plastid
outer envelope. Lipids derived from the plastid pathway often have a 16-carbon acyl
group at the sn-2 position of the DAG backbone whereas lipids derived from the ER
pathway often have an 18-carbon acyl group at this position (Heinz and Roughan 1983).
Plants that harbor the prokaryotic pathway such as Arabidopsis and spinach are referred

to as ’16:3 plants’; plant species that lost the prokaryotic pathway and thus depend solely

12

on the eukaryotic pathway for glycolipid biosynthesis such as Pea are called ’18:3
plants’(Heinz and Roughan 1983).

Two types of Arabidopsis mutants that affect lipid trafficking in either
galactolipid biosynthesis pathway are available. The act] (atsI) mutant is deficient in the
plastid glycerol 3-phosphate acyltransferase, and thus most of the galactoglycerolipids in
this mutant. are of ER origin (Bates et al. 2007, Kunst et al. 1988, Xu et al. 2006). (Figure
1.3). In other words, the atsI mutation turned Arabidopsis from a ’16:3 plant’ into
an ’1823 plant’. Another type of mutants deficient in the ER pathway was obtained
through a suppressor screen in the dng mutant background (Xu et al. 2003).
Galactoglycerolipids in these so called tgd mutants are derived primarily from the plastid
pathway (Awai et al. 2006, Lu et a1. 2007, Xu et al. 2003, Xu et al. 2005) (Figure 1.3).
The identification and characterization of these tgd mutants and resulting protein
products are the main subject of this dissertation and will be described in detail below

(see chapter 1.6).

13

 

 

 

 

 

Figure 1.3. Two-pathway hypothesis of galactoglycerolipid biosynthesis in plants,
adapted from Benning 2008. ER, endoplasmic reticulum; 0E, outer chloroplast envelope;
iE, inner chloroplast envelope; Thy, thylakoids; FAS, fatty acid synthase complex; AT S1 ,
plastid acyl-acyl carrier protein (ACP): glycerol 3-phosphate acyltransferase; PAP,
plastid phosphatidic acid phosphatase; T GD1,2, 3, components of the proposed ATP-
binding cassette (ABC) transporter for phosphatidic acid import through chloroplast inner
envelope; T 004, a TGD protein associated with the ER; FA, fatty acid; PA, phosphatidic
acid; PC, phosphatidylcholine; DA G, diacylglycerol. Other designations are as in Figure

1.2. Arrows indicate movement of lipids.

14

1.5. Basic polar lipid transfer mechanisms involving plastids

Glycerolipid biosynthesis occurs mainly in plastid envelope and ER membranes.
Since most lipids are not confined in the membranes where they are synthesized,
extensive glycerolipid trafficking within plant cells is required to accomplish membrane
specific lipid distribution. Among all possible aspects of lipid trafficking in plant cells,
those lipid trafficking phenomena that involve the plastid are of our interest and will be
discussed in this chapter.

Plastids not only export newly synthesized fatty acids, but also import ER-derived
glycerolipids whose diacylglycerol backbones are incorporated into thylakoid lipids.
Since plastids originate from ancestral endosymbiotic Gram-negative photosynthetic
bacteria (Reyes-Prieto et al. 2007), similar bacterial lipid transfer mechanisms might still
be present in plastids. In fact, recent studies support this hypothesis by showing that
many bacterial and cyanobacterial components of cell membranes have orthologs in
plastids (Reumann et al. 2005, Vothknecht and 8011 2007). Therefore, the well
characterized lipid trafficking in bacteria and yeast provides us new insights into the less
well studied and more complex plant phenomena. Polar lipid transfer in plants can be
divided into three basic mechanisms: (1) flip-flop movements, (2) intermembrane
transport and transfer by membrane contact sites and (3) vesicular lipid transfer

mechanisms (see Figure 1.4. and below).

1.5.1. F lip-flop movement

Bilayer structures of biological membranes generally consist of two leaflets of

polar glycerolipids. The polar head groups normally face the aqueous environment and

15

the acyl chain groups face the center. Galactoglycerolipids synthesized by DGDl, an
enzyme associated with the cytosolic face of the outer chloroplast envelope membrane
(Figure 1.2) have to be exchanged with the leaflet facing the intermembrane space
(Figure 1.4, process 1). Similarly, the bulk synthesis of MGDG on the outside of the
inner chloroplast envelope membrane by MGD] (Figure 1.2) requires lipid exchange

with the stroma side of the inner envelope membrane (Figure 1.4, process 2).

16

 

Figure 1.4. Postulated lipid transfer processes involved in ER-to-plastid lipid
trafficking in plants, adapted from Benning 2008. Membrane designations are the
same as in Figure 1.3. Bilayer membranes with their two leaflets are drawn. Arrows
indicate different lipid transfer processes: (1) polar lipid flipping across the outer
envelope membrane; (2) polar lipid flipping across the inner envelope membrane; (3)
transmembrane movement of polar lipids through the proposed TGD123 complex; (4)
transfer of lipids between the two envelope membranes; (5) lipid transfer mediated by
lipid transfer proteins (LTPs); (6) lipid transfer through ER-outer envelope membrane
contact zones (PLAMs); and (7) vesicle formation at the inner envelope membrane

mediated by a vesicle inducing protein in plastids (V IPPl).

h; .14.}

The spontaneous movement across the bilayer is dependent on the physical and
chemical properties of lipids and membranes and usually is very slow. For example, the
spontaneous flip-flop movement through a lipid bilayer takes less than Is for DAG but
several hours for lipids with larger polar heads (Bai and Pagano 1997, Jouhet et al. 2007,
Reyes-Prieto et al. 2007). Therefore, the majority of the flipping of polar lipids across a
bilayer membrane does not occur spontaneously, but is catalyzed by lipid transporters or
flippases, either in an energy-independent or energy-dependent way (Daleke 2007,
Pomorski and Menon 2006). Energy-independent flippases catalyze an ATP-independent
movement, in which lipids move along the polar surface created by these proteins (Kol et
al. 2002a, Kol et al. 2002b). They play a role in asymmetric lipid synthesis by facilitating
the equilibration of polar lipids between the two membrane leaflets. Energy-dependent
translocases can assist the movement of specific lipid classes against equilibrated
gradients and thus are involved in the maintenance of membrane asymmetry (Seigneuret
and Devaux 1984).

To date, no energy-independent flippase has been characterized in plants cells
although some gene sequences reveal similarities to flippase sequences from other
organisms. For instance, the first flippase studied in eukaryotic cells are the RFTl protein,
a yeast reticulum pyrophosphoryloligosaccharide-dolichol flippase (Helenius et al. 2002)
and the scramblase, a red blood cell plasma membrane calcium-dependent flippase (Zhou
et al. 1997). RFTl transfers dolichol from the reticulum cytosolic leaflet to the luminal
leaftlet (Helenius et al. 2002). Scramblase is activated by increasing calcium influx and
catalyzes bidirectional transfer of phospholipids between the two plasma membrane

leaflets (Zhou et al. 1997). In Arabidopsis, one RFTl (At5g07630) and one scramblase

l8

(At2g04940) homologue are identified based on sequence similarity, but functional
characterizations are still pending (J ouhet et al. 2007).

ATP-requiring lipid transporters or flippases can be divided into two families: (a)
aminophospholipid translocases that belong to the P-type ATPase superfarnily and (b)
ATP-binding cassette (ABC) transporters that comprise transporters for a variety of
organic and inorganic compounds. In Arabidopsis, 11 genes belong to the P-type ATPase
superfarnily (ALAl to ALA] 1, see Jouhet et al. 2007). Among them, ALAl is found to
be involved in cold tolerance and might affect plasma membrane lipid asymmetry
(Gomes et al. 2000). However, ALAl localization and function remain unknown.

ABC transporters, which are found in all organisms, are one of the largest and
most conserved protein families (Moody et al. 2002, Pohl et al. 2005). These proteins
transport a wide range of compounds including lipids, peptides and sugars through
biological membranes. ABC transporters are often multicomponent protein complexes
that typically consist of two transmembrane domains (TMDs) and two nucleotide-binding
domains (NBDs). In most eukaryotes, all four domains are encoded by one gene to
generate a single polypeptide chain, while the TMDs and NBDs of prokaryotes are
encoded by separate genes (Davidson and Chen 2004, Moody et al. 2002, Pohl et al.
2005). Many prokaryotic ABC transporters require a fifth subunit that functions as a
substrate binding protein (SBP). Nucleotide binding proteins of ABC transporters are
highly conserved among all organisms and share three well conserved motifs required for
nucleotide binding and hydrolysis: the Walker A site (GX4GK(S/T), where X=any
residue), the Walker B site (RX6-3(I)4D, where <I>=hydrophobic residue), and the LSGGQ

signature sequence (Moody et al. 2002).

19

ABC transporters have been extensively studied. In humans, mutations of
members of the ABC protein families cause defects in lipid transport and metabolic
disorders, such as Tangier disease. Fibroblasts from patients with Tangier disease have a
defect in the export of cellular cholesterol and phospholipids from the plasma membrane
to high density lipoproteins (Francis et al. 1995, Remaley et al. 1997). Additionally, the
role of ABC proteins in lipid transport is being studied in non-human eukaryotes such as
Arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and
Lactococcus lactis (Pohl et al. 2005). A good example is the MsbA protein of E. coli,
which has been implicated in lipid transfer from the inner cell membrane to the outer
membrane; its crystal structure has already been solved and is very similar to other
reported ABC transporters (Chang and Roth 2001, Doerrler et al. 2001).

The Arabidopsis genome contains 129 open reading frames (ORFs) belonging to
ABC protein families (Sanchez-Femandez et al. 2001). They play a key role in a variety
of processes including chlorophyll biosynthesis, stomata movement, and ionic fluxes
(Martinoia et al. 2002). Some ABC transporters involved in the export of precursors for
cuticle or wax biosynthesis in the plant epidermis are also reported (Bird et al. 2007, Luo
et al. 2007, Pighin et al. 2004, Ukitsu et al. 2007). A potential ABC transporter identified
as At1g54350 in the Arabidopsis genome is proposed to mediate free fatty acids transfer
from the inner plastid envelope to the outer plastid envelope, where fatty acids are
reactivated to acyl-CoA and enter numerous metabolic pathways (Koo et al. 2004). A
well studied ABC transporter in Arabidopsis, COMATOSE (CTS), PEROXISOMAL
ABC TRANSPORTER1(PXA1) or PEROXISOME DEFECTIVE3 (PED3), is localized

in peroxisomes to import fatty acids or acyl-CoAs for B-oxidation (Footitt et al. 2002,

20

Hayashi et al. 2002, Zolman et al. 2001). Recent labeling experiments on pxaI/cts/ped3
mutant seedlings suggested that this ABC transporter could also mediate the import of
acetate into glyoxysomes, specialized peroxisomes that play a role in seed germination,
when fatty acids are converted to carbohydrates (Hooks et al. 2007). All the above
mentioned ABC transporters are homodimers encoded by a single peptide with a multi-
membrane-spanning permease domain and an ATP-binding cassette (ABC) domain. To
date, the only Arabidopsis ABC transporter involved in ER-to-plastid lipid exchange
identified is the TGDl, 2, 3 complex. These three proteins constitute a bacterial-type
multipartite ABC transporter (Awai et al. 2006, Lu et al. 2007, Xu et al. 2003, Xu et al.
2005). The transporter is localized to the inner chloroplast envelope and proposed to
firnction in PtdOH import through this membrane as will be discussed in detail below

(Figure 1.4, process 3).

1.5.2. Intermembrane transport and transfer by membrane contact sites

As mentioned above, most plant species have two sources of thylakoid lipid
precursors, the ER and plastids. This fact requires substantial lipid exchange between
these two organelles (Figure1.4, processS). In addition, lipids delivered to the outer
chloroplast envelope have to be exchanged with lipids synthesized in the inner
chloroplast envelope to keep the specific lipid distribution and lipid homeostasis (Figure
1.4, process 4). These intermembrane lipid transport events are often carried out by lipid
transfer proteins (LTPs) or membrane contact sites.

LTPs have to target both donor and acceptor membranes to mediate trafficking of

particular lipids between organelles (Holthuis and Levine 2005, Kader 1996). Some LTPs

21

are localized at membrane contact sites where two membranes are in close proximity,
usually less than IOnm (Holthuis and Levine 2005, Loewen et al. 2003a, Loewen et al.
2003b). Some LTPs have two targeting domains for both the lipid donor membrane and
the receptor membranes thus they can either move from one membrane to another or bind
to both membranes simultaneously. This simultaneous binding to both membranes could
potentially stabilize membrane contact sites and promote lipid transport (Holthuis and
Levine 2005). There are many LTPs identified in plants, however, most of the LTPs
characterized so far are generally found to be involved in a variety of cellular processes
such as cell death (Brodersen et al. 2002). Moreover, there is no information concerning
their possible localizations to specific membranes or membrane contact sites. This
questions the actual role of LTPs in ER-to-plastid lipid transfer.

Another type of lipid transfer involves membrane contact between two organelles
(Voelker 2000). MAMs (mitochondria associated membranes) are present in the contact
sites between ER and mitochondria. They are well studied in mammals and yeast
(Achleitner et al. 1999, Vance 1990). MAMs are proposed to be involved in
mitochondrial phosphatidylethanolamine (PtdEtn) biosynthesis, which occurs by
decarboxylation of phosphatidylserine (PtdSer) catalyzed by an isoform of PtdSer
decarboxylase localized to the mitochondria (Rontein et al. 2003). PtdEtn biosynthesis is
dependent on PtdSer supply from ER due to spatial localization of PtdSer synthase in the
ER membrane (Voelker 2000). PtdSer is transported to the mitochondrial inner
membrane by an unknown mechanism, maybe going through contact sites between the
inner and outer membranes of mitochondria (Reichert and Neupert 2002). PtdSer is

subsequently decarboxylated in the mitochondria inner membrane to form PtdEtn before

22

a retro-transfer of PtdEtn to ER probably going also through the MAM (Voelker 2000).
Genetic screens using mutant strains selectively inactivated in the PtdSer decarboxylase
of the mitochondria led to the isolation of enthanolamine auxotrophs that are impaired in
the regulation of arninoglycerophospholipid biosynthesis (Storey et al. 2001 , Trotter et al.
1998, Wu et al. 2000). In addition, MAMs are also possibly involved in the transfer of
other mitochondrial phospholipids synthesized in ER such as PtdCho (Voelker 2000).

Based on yeast studies, it was proposed that PtdEtn is transferred by a similar
mechanism involving plasma membrane associated membrane (PAM) from ER to plasma
membranes (Holthuis and Levine 2005). Membrane contact sites are also observed
between ER and vacuoles. These contact sites possibly participate in the transfer of
PtdEtn, PtdCho and PtdIns with the involvement of previously described LTPs (Staehelin
1997)

Finally, lipid transfer by membrane contact sites between plastid envelope and
other organelles was also reported. Sandelius and coworkers were the first to discover
direct contact sites between the ER and the outer chloroplast envelope membrane (Figure
1.4, process 6) that can be isolated in the form of PLAMs (plastid associated membranes).
During pea chloroplast isolation, some ER-fractions were found to remain associated
with the plastid envelopes (Kjellberg et al. 2000). Moreover, in vivo optical manipulation
reveals strong attracting forces at membrane contact sites between the ER and the plastids
(Andersson et al. 2007). It should be noted that the existence of PLAMs by itself does not
provide a mechanism of ER-to-plastid lipid exchange. It is still not clear what the
physical arrangements of polar lipids in such contact sites are and whether or not proteins

are required to form these contact sites. However, the well studied MAM in yeast system

23

suggests the possibilities of also applying genetic approaches to address lipid trafficking
in plants. Recently, a genetic screen carried out in Arabidopsis has led to the isolation of
mutants disrupted in the transfer of lipids from the ER to the plastids. Mapping of the
mutant genes might provide information about components participating in the formation
of PLAMs in the near future. For instance, the possibility that the
TRIGALACTOSYLGLYCEROL 4 (TGD4) protein functions in PLAMs and lipid

trafficking between the ER and the plastid is emerging (Xu et al. 2008b).

1.5.3 Vesicular lipid transfer

All the lipid transfer mechanisms discussed so far are non-vesicular. On the other
hand, lipid transfer could be realized through a vesicular mechanism, in which vesicles
bud from a donor membrane and fuse with an acceptor membrane. The general
mechanism of vesicular transfer includes several steps with a series of protein factors
(Jiirgens 2004, Nebenfuhr 2002). Briefly, protein complexes are recruited and anchored
to the donor membrane by small type Ras GTPases, coupled to GTP exchange factors.
Then, vesicles bud from the donor membrane through a not-yet-clear mechanism and
move away using the actin cytoskeleton and myosin. A large GTPase called dynamin is
involved in the scission of nascent vesicles from the donor membrane. Finally, SNARE
(soluble N—ethylmaleimide-sensitive factor attachment protein receptors) proteins, located
both on the vesicles (v-SNARE) and on the target membrane (t—SNARE), together with
small GTPases help anchoring and fusion to the acceptor membrane.

Vesicular transfer mechanisms have been extensively studied and a lot of protein

factors have been identified. The process was suggested to mediate numerous transfer

24

events such as ER and Golgi exchanges (Brandizzi et al. 2002, Hawes 2005), lipid
transfer to the plasma membrane (Ikonen 2001, Keller et al. 2001), and lipid transfer to
the vacuoles (Matsuoka and Bednarek 1998). No evidence for the involvement of
vesicular transfer mechanism in ER-to-plastid lipid trafficking has appeared yet.
However, some studies suggest a possibility that a vesicular lipid transfer system might
exist in the transfer of galactoglycerolipids from the inner chloroplast envelope to the
thylakoid membrane, contributing to the biogenesis of the thylakoids (Figure 1.4, process
7). First, vesicle budding from the inner chloroplast envelope membrane was directly
observed by electron microscopy (Carde et al. 1982, Morré et al. 1991). Later, an
Arabidopsis chloroplast-localized THYLAKOID FORMATION 1 (THFl) protein was
shown to control a step required for the organization of vesicles derived from the inner
envelope membrane into mature thylakoid stacks. Deletion of THFl in Arabidopsis leads
to deficient thylakoid formation and variegated leaves (Wang et al. 2004).

The plastid vesicular pathway is dependent on ATP and stromal proteins, among
which a NSF (for N-ethylmaleimide sensitive factor) homolog protein (Hugueney et al.
1995), a dynamin-like protein (Park et al. 1998) and a vesicle inducing protein in plastids
(V IPPl) (Aseeva et al. 2007, Kroll et al. 2001) are identified. VIPPl is essential for basic
thylakoid membrane formation so that thylakoid membrane formation and chloroplast
vesicle transport are abolished in Arabidopsis VIPPl depleted vippI mutant (Aseeva et al.
2007, Kroll et al. 2001). Some data also demonstrate that VIPPl forms a high molecular
weight complex closely associated with the inner chloroplast envelope membrane and
that the C-terminus of the protein protrudes from the complex into the stroma of

chloroplasts possibly for interaction with other proteins (Aseeva et al. 2004).

25

Accordingly, interactions between VIPPl and the chaperon proteins HSP7OB-CDJZ-
CGEl are observed in the green alga Chlamydomonas. This tight association might play
a role in assembly and disassembly of VIPPl oligomers to recycle the system for another
round of vesicle formation (Liu et al. 2005, Liu et al. 2007). Taken together, current
evidence demonstrates that VIPPl is a crucial component of a complex potentially
required for vesicular lipid trafficking between the inner plastid envelope and the
thylakoids. Bioinforrnatics studies suggest that some other vesicular pathway components
are present in plastids (Andersson and Sandelius 2004), yet firrther characterization by

genetic and biochemical approaches is still pending.

1.6. Current progress in the study of lipid trafficking in Arabidopsis

Arabidopsis is a well developed genetic model that has been used to study lipid
transfer phenomena. Two types of Arabidopsis mutants that affect lipid trafficking
between the ER and the plastid have been identified. The act] (atsI) mutant is deficient
in the plastid glycerol 3-phosphate acyltransferase, and thus most of the
galactoglycerolipids in this mutant are derived from the ER pathway (Bates et al. 2007,
Kunst et al. 1988, Xu et al. 2006). All known mutant alleles of atsI are leaky and further
repression of the activity of the mutant gene leads to a severe growth phenotype.
Therefore, the leaky atsI-I allele has become an important tool in double-mutant studies
focusing on lipid trafficking in plants, as mutations in genes encoding components of the
eukaryotic pathway in an atsI -1 background severely impair chloroplast biogenesis and
cause embryo lethality (Dbrmann et al. 1999, Xu et al. 2003, Xu et al. 2005, Xu et al.

2008b).

26

Previous studies in the Benning laboratory have identified another type of mutants
deficient in the ER-derived galactolipid biosynthesis pathway (Awai et al. 2006, Lu et al.
2007, Xu et al. 2003, Xu et al. 2005). Galactoglycerolipids in these mutants are derived
primarily from the plastid pathway. These mutants were given the name
trigalactosyldiacylglycerol (tgd) for the diagnostic accumulation of
oligogalactoglycerolipids (TGDG), which is typically absent in wild-type Arabidopsis
plants.

The tgd mutants were isolated during a suppressor screen in the (1ng mutant
background (Xu et al. 2003), which is deficient in DGDl activity and thus lacks 90% of
DGDG content (Dormann et al. 1995, Dormann et al. 1999). The primary goal of the
screen was to identify mutants that bypass the major galactoglycerolipid biosynthetic
pathway involving MGDl and DGDl (Awai et al. 2001, Harte] et al. 2000, Kelly and
Dormann 2002). Two group of mutants were isolated, with one meeting the original
criteria for the disruption of a regulator of the alternative galactolipid biosynthesis
pathway, which was affected in a mitochondrial outer membrane protein (Xu et al.
2008b). The second group of mutants was the tgd mutants that accumulated novel
oligogalactoglycerolipids TGDG and restored the wild-type level of DGDG content
compared to dgd] parent plants. TGDG accumulated in the tgd mutants have all [3
glycosidic linkages of their head group galactoses, indicating that their production is
independent of MGDl and DGDl galactosyltransferases (See Figurel.1C, Figure 1.2C).
Probably, a processive galactolipid:galactolipid galactosyltransferase (GGGT) is involved

in the synthesis of TGDG in the mutants.

27

The tgd] mutant was the first and best analyzed tgd mutant so far (Xu et al. 2003,
Xu et al. 2005). It accumulates both TGDG and triacylglycerol (TAG) in leaves. The
PtdOH level in the mutant is greatly increased. Pulse-chase labeling experiment with
acetate and oleic acid indicates that the transfer of lipid molecular species from the ER to
the plastid is impaired in the tgd] mutant. Isolated chloroplasts from tgd] plants exhibit a
decreased rate of conversion of labeled PtdOH into galactoglycerolipids. Loss of function
of TGDl is embryo lethal. The TGD] protein is localized in the inner chloroplast
envelope membrane and is similar to the permease component of bacterial ABC
transporters.

Detailed studies of the other two tgd mutants, tgd2 and tgd3, are the major
components of this dissertation and will be introduced chapter by chapter. Chapter 2 and
chapter 4 will be focused on the identification and biochemical study of the TGD3
protein and TGD2 protein, respectively. Characterization of Arabidopsis mutants
disrupted in TGD3 function will be described in chapter 3.

Briefly, both tgd2 and tgd3 mutants have the same phenotype as tgd]. The TGD2
protein is similar to the substrate binding subunit of bacterial ABC transporters and
specifically binds to PtdOH in vitro (Awai et a1. 2001). Recently, I identified a 25-amino-
acid minimal domain that is sufficient for specific PtdOH binding when fused with
Discosoma sp. red fluorescent protein (DsRed) (Lu and Benning 2009). Presumably,
TGD2 is anchored with its N-terminus into the inner chloroplast envelope membrane.
With its C-terminus facing the intermembrane space, it could extract PtdOH from the
outer envelope membrane (Awai et al. 2006, Lu and Benning 2009). The TGD3 protein

was identified through a reverse genetics approach, and contains sequence similarity with

28

non-intrinsic ATPase found adjacent in operons to bacterial orthologs of TGDl and
TGD2 (Lu et al. 2007). Recombinant TGD3 protein fused with maltose binding protein
shows basal ATPase activity and is localized inside the chloroplast beyond the inner
chloroplast envelope membrane (Lu et al. 2007). It could potentially associate with
TGDl on the inside of the inner envelope membrane.

Taken together, it was proposed that TGD], TGD2 and TGD3 form a bacterial
type ABC transporter complex that transports PtdOH through the inner chloroplast
envelope for synthesis of galactoglycerolipids (See Figure 1.3 and Figure 1.4, process 3)
(Benning 2008). The tgd1,2,3 mutants are disrupted in this transporter and thus most of
the galactoglycerolipids in these mutants are derived from the plastid pathway (Awai et
al. 2006, Lu et al. 2007, Xu et al. 2003, Xu et al. 2005). At this stage, how PtdOH would
be formed at the outer envelope remains unclear. However, the recent identification of
the TGD4 protein involved in ER-to-plastid lipid trafficking, which is the first TGD
protein associated with the ER (See Figure 1.3), provides new avenues towards an
understanding of PtdOH formation from ER-derived precursors in predicted contact

zones between the ER and the outer plastid envelope membrane (Xu et al. 2008a).

29

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38

Chapter 2

Identification and characterization of TGD31

 

1This work has been published: Lu, B., Xu, C., Awai, K., Jones, A. D., and Benning, C.
(2007) A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid
import. J. Biol. Chem. 282, 35945-35953. I contributed to all the figures except Figure 2.4,
Figure 2.6 and Figure 2.8.

39

2.1. Abstract

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid
assembly are often distinct from final destination membranes. A striking example is the
biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid
biosynthetic enzymes at the endoplasmic reticulum (ER) and the inner and outer plastid
envelope membranes are involved. This compartrnentalization requires extensive lipid
trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of
endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two genes affected
in two of these mutants, trigalactosyldiacylglycero] 1 (T GDI ) and T GD2, encode the
permease and substrate binding component, respectively, of a proposed lipid translocator
at the inner chloroplast envelope membrane. Here we describe a third gene of
Arabidopsis, T GD3 , encodes a small ATPase proposed to be part of this translocator. As
in the tgd] and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3
mutant carrying a T-DNA insertion just 5’ of the T GD3 coding region. The TGD3 protein
shows basal ATPase activity when expressed as a maltose-binding protein fusion and is
localized inside the chloroplast beyond the inner chloroplast envelope membrane.
Proteins orthologous to TGDl, 2, and 3 are predicted to be present in Gram—bacteria, and
the respective genes are organized in operons suggesting a common biochemical role for
the gene products. Based on the current analysis, it is hypothesized that TGD3 is the
missing ATPase component of a lipid transporter involving TGD] and TGD2 required

for the biosynthesis of ER derived thylakoid lipids in Arabidopsis.

40

2.2. Introduction

Interorganellar non-vesicular lipid trafficking in plant cells is required for the
biogenesis of the characteristic plant cell organelle, the chloroplast (Benning eta]. 2006).
The predominant polar lipids found in the photosynthetic membranes (thylakoids) of
chloroplasts are the galactoglycerolipids monogalactosyldiacylglycerol (MGDG) and
digalactosyldiacyl glycerol (DGDG)(D6rmann and Benning 2002). Their final assembly
from diacylglycerol (DAG) and UDP-galactose requires two major lipid
galactosyltransferases, which in the model plant Arabidopsis are designated MGDl and
DGD1(Benning and Ohta 2005). Of these two enzymes MGD] is associated with the
outside of the inner chloroplast envelope membrane (Xu eta]. 2005) and is responsible
for the bulk of MGDG formation (Jarvis eta]. 2000), whereas DGDl is located on the
outside of the outer chloroplast envelope membrane (Froehlich et a]. 2001) and is
required for the bulk of DGDG biosynthesis (Dormann et a]. 1999). The diacyl glycerol
moiety of the galactoglycerolipids is either synthesized de novo in the plastid or it is
assembled at the endoplasmic reticulum (ER) and needs to be imported into the plastid
(Roughan and Slack 1982).Understanding this complexity of subcellular organization of
glycerolipid biosynthesis in plants and the underlying lipid trafficking phenomena still
poses some of the biggest challenges of modern plant biochemistry. The recent isolation
and characterization of genetic suppressors of the DGDG-deficient dng mutant of
Arabidopsis has provided the first components, TGDl and TGD2, of a potential lipid
transport complex associated with the inner chloroplast envelope membrane in
Arabidopsis (Awai et al. 2006, Xu et a]. 2003, Xu et a]. 2005). The TGDl protein is

similar to the permease component of bacterial ATP binding cassette (ABC) transporters,

41

and the Arabidopsis tgd] mutant shows a complex lipid phenotype; that is, the
accumulation of Oligogalactolipids and triacylglycerols, a disruption of the assembly of
galactolipids from ER-derived precursors, and the elevation of phosphatidic acid (PtdOH)
levels(Xu et a]. 2003, Xu eta]. 2005). The name of the mutants, tgd

(trigalactosyldiacyl glycerol), refers to the characteristic accumulation of
Oligogalactolipids. Presumably, this is a secondary phenotype due to the activation of a
processive galactolipid:galactolipid galactosyl transferase (GGGT) of unknown function,
for which the gene has not yet been identified. The accumulation of PtdOH and reduced
incorporation of labeled PtdOH into galactoglycerolipids by isolated tgd] chloroplasts led
to the suggestion that TGD1 is involved in the transport of PtdOH through the inner
chloroplast envelope membrane (Xu eta]. 2005).The TGD2 protein is similar to
membrane-tethered substrate-binding proteins associated with bacterial ABC transporters
(Awai et a]. 2006). Although tgd2 was not as extensively characterized as the tng
mutant, the Arabidopsis tgd] and tgd2 mutants have a nearly identical lipid phenotype in
all aspects tested. Moreover, the recombinant TGD2 protein lacking the single
membrane-spanning domain specifically binds PtdOH (Awai et a]. 2006). The nearly
identical phenotypes of the two mutants and the organization of bacterial orthologs of
TGD1 and T GD2 in operons (Figure 2. 1 A) strongly suggest that both are involved in a
common biochemical or cell biological process. Moreover, the specific binding of PtdOH
to TGD2 corroborates the hypothesis that the two proteins are components of a PtdOH
transporter at the inner chloroplast envelope. Unlike other eukaryotic ABC transporters or
the MsbA lipid transporter from Escherichia coli (Doerrler 2006), the TGD1 protein

lacks an ATP binding domain. However, the Arabidopsis genome contains 26 genes

42

encoding putative small ATPases with either one or two nucleotide binding domains
(Sanchez-Femandez eta]. 2001). We hypothesized that one of these could encode a small
ATPase potentially associated with the TGD1 protein. Here we describe the identification
and biochemical characterization of TGD3, the small ATPase protein proposed to be

associated with TGD1 and TGD2.

2.3. Experimental procedures

2.3.1 Plant materials and growth conditions

Arabidopsis thaliana wild type and tgd] and tgd2 mutant plants (Awai et a]. 2006,
Xu et a]. 2003) were of the ecotype Columbia-2 (Col-2). The following T-DNA insertion
lines were made available by the Salk Institute Genome Analysis Laboratory (SIGnAL)
and were obtained from the Arabidopsis Biological Resource Center at Ohio State
University: SALK_027270 At4g33460, SALK_129048 At5g60790, SALK_029696
At5g09930, SALK_113472 At5g64840, SALK_116866 At5g14100, SALK_055996
At1g32500, and SALK_040335 At1g65410. Homozygous mutant lines were screened
and used for all experiments. Seeds were surface-sterilized and grown on 0.8% (w/v)
agar-solidified Murashige-Skoog medium (Murashige and Skoog 1962) supplemented
with 1% (w/v) sucrose for 5 days. Wild-type and mutant seedlings of similar
developmental stage were then transferred to a fresh Murashige-Skoog agar plate with

1% sucrose and grown for three additional weeks before lipid analysis.

43

2.3.2 Genotyping and complementation

The T GD3 open reading frame was isolated by reverse transcriptase-PCR
(standard conditions) from mRNA preparations using RNeasy and Omniscript kits
(Qiagen, Valencia, CA) with the primers 5’-ACGGTACCATGCTTTCGTTATCATGC-
TC-3’ and 5’-CTGGTACCCTAGTATCTGATTGGTCCAT-3’. The PCR products were
ligated into pGEM-Teasy (Promega, Madison, WI) and sequenced. The resulting
plasmids were digested with Kpnl and inserted into pCAMBIA13OO (Xu et a]. 2005)
followed by transformation into Agrobacterium. Plants were transformed by the floral dip
method (Clough and Bent 1998) and screened for resistance to hygromycin B (25 rig/ml)
on agar-solidified Murashige-Skoog medium. For an estimation of T GD3 transcript levels
by semi-quantitative reverse transcriptase-PCR, RNA was isolated from Col-2 wild type
and tgd3 mutant plants, and reverse transcription was done using oligo(dT)12-l 8 primers
(Invitrogen). The TGD3-specific primers mentioned above were used for the PCR
reaction. The abundance of ubiquitin (UBQI 0) was tested for control purposes. The
following UBQI 0-specific primers were used: 5’-TCAATTCTCTCTACCGTGATCAA-
GATGCA-3’ and 5’-GTGTCAGAACTCTCCACCTCAAGAGTA-3’. For genotyping
during the complementation analysis, DNA was prepared from 4-week-old plants, and
the following primers were used: T-DNA left border primer (LB), 5’-GCGTGGACCGC-
TTGCTGCAAC-3 ’; P l , 5 ’-AACTGATTGGGACGAGTATC-3 ’; P2, 5 ’-GCTATGCAA-
CAGCAAGAGAC-3’. The PCR conditions were 94 °C 3min followed by 35 cycles at
94 °C for 0.5 min, 54 °C for 0.5 min, and 72 °C for 0.5 min followed by a final step of 5

min at 72 °C.

44

2.3.3 Lipid assays

Lipids were extracted from 4-week-old Col-2 wild-type and tgd3 mutant seedlings
grown on Murashige-Skoog medium with 1% sucrose. Fatty acid methyl esters were
prepared as previously described (Ddrmann eta]. 1995) and quantified by gas
chromatography according to Rossak eta]. (Rossak et a]. 199 7). Polar lipids were
analyzed on activated ammonium sulfate-impregnated (Dormann et a]. 1995) silica gel
TLC plates (Si250PA; Mallinckrodt Baker) developed with chloroform/methanol/acetic
acid/water (85/25/10/4, v/v). Neutral lipids were separated on untreated TLC plates
developed with petroleum ether/ether/acetic acid (80/20/1, v/v). Lipids were visualized
by brief exposure to iodine vapor or staining with a-naphthol to detect glycolipids
(Benning et a]. 1995). The fatty acid compositions at the sn-2 position of individual lipids
were determined using Rhizopus lipase (Sigma) digestion according to Siebertz and
Heinz (Siebertz and Heinz 1977) with modifications as described in Miquel eta].
(Miquel et a]. 1998). Fatty acid methyl esters were formed from lyso-lipids, and the fatty

acid methyl esters were quantified by gas chromatography.

2.3.4 Mass spectrometry

Lipid extracts were characterized using liquid chromatography/mass spectrometry
(MS) on a Waters LCT Premier time-of-flight mass spectrometer equipped with
Shimadzu LC-20AD pumps and SIL-SOOO autosampler. Extracts were analyzed using V-
mode Operation and electrospray ionization in both positive and negative ion modes
similar to described protocols (Houjou et a]. 2005) except that, in place of MS/MS

spectra, mass spectra were acquired in alternating acquisition functions at low (15 V) and

45

high (75 V) potentials on aperture one to generate spectra with and without in-source
collision induced dissociation. The latter conditions allow for production of characteristic
fragment ions that support structure assignments. High performance liquid
chromatography separations were performed using a Restek Allure C18 column (1 X150
mm) and a ternary gradient based on (a) 10 mM aqueous ammonium acetate, (b)

methanol, and (c) 2-propanol.

2.3.5 Subcellular localization assays

For the generation of a green fluorescent protein fusion construct, the entire
coding region of Atl g65410 was inserted into the Kpnl site of pCAMBIAl 300GFP (Xu
eta]. 2005) using primers 5’-ACGGTACCATGCTTTCGTTATCATGCTC-3’ and 5’-
CTGGTACCGTATCTGATTGGTCCAT-3’. Transient expression of the construct in
tobacco and microscopy of the resulting transgenic leaves was performed as previously
described (Xu et a]. 2005). For the in vitro import experiments using pea chloroplasts the
same conditions and treatments were used as published for the localization of the MGD]

protein (Xu et a]. 2005).

2.3. 6 Recombinant MBP-ATGDS protein production and purification

The coding sequence for TGD3 leading to a truncated protein missing 46 N-
terminal amino acids (designated as ATGD3) was PCR-amplified. As template, the
pGEM-Teasy plasmid derivative mentioned above was used in combination with primers
5’-GGATCCTGCATAGCTCCACCTCAGAAC-3 ’ and 5’-GTCGACTAGTATCTGAT-

TGGTCCATCG-3’. The fragment was inserted into pGEM-Teasy (Promega) and

46

sequenced. This plasmid served as template for F94A mutagenesis by using the
forward/reverse primers and the QuikChange site-directed mutagenesis kit from
Stratagene (La Jolla, CA). The forward and reverse primers for F94A are 5’-GTAGAGA-
TGTCTATAAATCGQCEGGGGAGAAACATATCTTG-3 ’ and 5 ’-CAAGATATGTTT-
CTCCCCC_GQCGATTTATAGACATCTCTAC-3’. The underlined sequences are codons
for the mutated residue (alanine). After digestion with BamHI and Sall, either the wild
type ATGD3 or the mutant AT GD3F 94A fragment was inserted 3 ’ of the E. coli malE
coding sequence into the pMalc2x vector (New England Biolabs, Ipswich, MA) to
generate the maltose-binding protein fusion proteins MBP-ATGD3 or MBP-ATGD3 F94A.
For recombinant protein production, a 500-ml culture was grown at 37 °C, inoculated

with 2 ml of an overnight culture. The protein was induced with 0.1 mM isopropyl-B-D-
thiogalactopyranoside at an A600 of 0.4—0.6 and incubated at 28 °C for 4 h. The cells were

then collected by centrifugation and lysed by sonication in the presence of 1mg/ml
lysozyrne. Crude lysate was centrifirged at 18,000Xg for 20 min, and the soluble fraction
was loaded onto an amylose column (New England Biolabs). Fusion proteins were
purified according to manufacturer’s instructions. Protein concentration was determined
according to Bradford (Bradford 1976) using bovine serum albumin as a standard.
Protein purity was verified by SDS-PAGE. After purification, samples were stored at

4 °C for a few weeks without significant loss of activity.

2. 3. 7 Assay for A T Pase activity

Purified MBP-ATGD3 protein was assayed for ATPase activity at a protein

concentration of ~O.5—8 11M in SO-ul reaction mixtures containing assay buffer (40 mM

47

Tris-HCI, pH 7.6,150 mM NaCl, 2 mM MgClz). The MBP-ATGD3 protein was diluted in
assay buffer to a final volume of 25 ul. To initiate the reaction, 25 ul of ATP in the assay
buffer ranging from O to 2 mM (diluted from a 10 mM stock made in the same assay
buffer) were added, giving a final ATP concentration of 0 to lmM. ATPase reactions
were incubated at 37 °C for the indicated time and stopped by the addition of SOul 12%
SDS. Released Pi was quantified by a colorimetric method, as described by Chifflet et a].
(Chifflet et a]. 1988) using potassium phosphate as the standard. After the addition of
SDS, 100 111 of a solution containing equal volumes of 12% ascorbic acid in 1 M HCl and
2% ammonium molybdate were added, and the samples were incubated at room
temperature for 5 min. This was followed by the addition of 150 pl of a solution
composed of 2% sodium citrate, 2% sodium meta-arsenite, and 2% acetic acid. After 10
min of incubation at 37 °C, absorbance was measured at 850 nm. The assay was linear in
the range of 0—25 nmol of Pi. The activity was corrected for nonenzymatic release of Pi
from ATP and for Pi contamination by subtracting absorbance readings obtained in the
absence of protein and ATP, respectively. A vector expressing only MBP was used to
control for endogenous ATPase activity from 'E. coli. The kinetic constants were
determined by nonlinear least squares fitting to the equation V, = Vmax[S]/(Km + [5]) using

Microcal Origin 7.0 (Microcal Software, Northampton, MA).

48

2.4. Results

2. 4. 1 Identification of T GD3 by genomics and reverse genetics

Bacterial genes involved in the same biochemical process are commonly linked in
operons. This fact has been successfully exploited in genome annotation or the
identification of individual genes (Osterman and Overbeek 2003). Accordingly, the
location of TGD1 and TGD2 encoding homologs in bacterial operons (Figure 2. 1 A)
adjacent to putative ABC proteins suggests that these are the ABC components of the
respective transport complex. Based on comparative genomics, it seemed likely that

Arabidopsis has an ABC protein associated with the TGD1/2 transporter.

49

Figure 2.1. Isolation of the T GD3 gene by reverse genetics. A, linear representation of
the Arabidopsis T GD homologs in different bacterial species. The number designations
(1—3) correspond to open reading frames presumed to encode homologs of Arabidopsis
TGD1, TGD2, and TGD3 proteins, respectively. Letters refer to the bacterial open
reading frame designations in the respective operons. Species and GenBankTM protein
accession numbers for TGD3 orthologs: E. coli (ZP_00728201), Pseudomonas putida
(ZP_01714441), Xylellafastidiosa (NP_779845), Synechococcus sp. J A-2—3B’a
(YP_477326), Arabidopsis thaliana (N P_564850). B, an alignment of the TGD sequence
with various ABC-transporter associated ATPases for which crystal structures are
available: HlyB (PDB code lMTO), MalK (PDB code 1G29), MJ 1267 (PDB code 1G9X),
and HisP (PDB code lBOU). Open boxes mark conserved residues, and black boxes
indicate identical residues. The relative positions of the main functional sites are assigned,
and phenylalanine residue Phe94 of TGD3, which was mutated to alanine, corresponds to

the A-loop conserved aromatic residue as indicated.

50

  

 

llD

 

E 13 C) B At

 

13|1|2| llll

 

13 C D EF

 

 

l3|2|

 

 

 

 

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.5-50bp

100
KS can
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ozdoo
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A Eco/i yrb | 3I1I2I
F
P. putida tth
X. fastidiosa tth I3 I 1 II 2| I
A e c o
Synechococcus TGD
A. thaliana TGD

B 60 '70 80
TGD3 51 PQNLDNDATKFDSLTKSGGGMCKERGLENDSDV
HlyB 1 ............................... DI R
Hall 1 ............................... MA R
M31267 1 ........................... MRDTMRI R
HIIP 1 ............................ MMSEN
TGD3
HlyB
M31!
M31267
HisP
TGD3 .GLIsnanI ........ sondi
HlyB .......... ADPNWLR. .RQV
Mal! ..DPEKGIFVPP ...... XDR I
M312€7 ..NK.EPAEL ........ YHY
HisP VRDKDGQLKVADKNQLRLLRTMQ

 

51

 

 

180
8 P ......
SII SLA .NPG
TVY F ......
TVL IGEINPG
TV 3 ......

 

 

TGD3
HlyB
HAIR
MJ1267
HiaP

TGD3
HlyB
Malx
MJ1267
HisP

TGD3
HlyB
Hal!
MJ1267
HiaP

TGD3
HlyB
MalK
M31267
HiaP

  

 

 

 

 

 

 

   
  
 
 

 

 

190 200 210 220
......LLY sxnsznor anvonx nnan.ps xx
nsvxxvrra LAGA.HDFI an ....GYN GEQ.GA QRQ
......PLK .xvpaozr AELLGLT LNRK.PR QRQ
aspnnsnrr IPKEEE ILEPLKLS DRK.AG our
......xpr LGLSKHDA LAKVGID oexrpv
C-motif
00...... L
I RNMBKI ..
AELKKL .
FNHVLEL .
RIMQQL .
Walker B
270 280 290 300
IRSVHMTDEDAVGKPGK nsn* RA
..... 0.000.000... LSfl N.
00.0.0.000000000 Qv TM
. ........ ........ LDI LNYI
.. .............. e anv
320 330
HT .rrrsrnprvoora. ............................
RH LLSEPES...0. 00000000000000 00.0.0.0..000000000

SP YDKPANTFVAGFIGSPPMNFLDAIVTEDGFVDFGEFRLKLLPDQF
RG ..... O O 0
DP FGNPQSPRLQQFL....... .......... . ..... .........

.00.. ......... 0 ........ 0 ..... 0.000....

Figure 2.1. (cont’d)

52

An extensive and possibly complete inventory of transport ATPases and non-
intrinsic ABC proteins potentially associated with membrane transporters in Arabidopsis
has been published (Sanchez-Femandez et a]. 2001) listing 26 soluble nucleotide binding
domain proteins. Because TGD1 and TGD2 are associated with the inner chloroplast
membrane, which is homologous to the bacterial cell membrane, we expected that the
respective ABC protein was localized on the inside of the inner chloroplast membrane
requiring an N-terminal chloroplast targeting sequence. Nine of the above-mentioned
ABC proteins were predicted to be targeted to the plastid using ChloroP (Emanuelsson et
a]. 1999): At1g32500, At1g65410, At3g10670, At4g04770, At4g33460, At5g09930,
At5g14100, At5g60790, At5g64840. For seven (listed under “Experimental Procedures”)
of these candidate genes, T-DNA inactivation lines (Alonso et a]. 2003) were available at
the time and were selfed and selected for homozygosity. The homozygous mutant lines
were tested for the presence of tri galactosyldiacyl glycerol, which is diagnostic for
mutants with impaired TGD1 or TGD2 function. Only one inactivation line
(SALK_O40335) carrying a T-DNA 5’ of the presumed ATG start codon of Arabidopsis
gene At1g65410 (Figure 2.2A) showed accumulation of the oligogalactolipid (Figures
2.2D and 2.3B). This mutant was clearly leaky as the abundance of the respective mRNA
estimated by semi-quantitative reverse transcriptase-PCR was only partially reduced in
the T-DNA line (Figure 2.2B). A completely disrupted allele was not available (see also
below). To confirm that the mutant phenotype was not due to secondary mutations, a
wild-type cDNA was expressed in the homozygous T-DNA line disrupted in At1g65410.
Two transgenic homozygous T-DNA lines lacking the accumulation of the

oligogalactolipid are shown in Figure 2.2C and D. The restoration of the wild-type

53

phenotype in the homozygous T-DNA lines by the corresponding wild-type cDNA
confirmed that the accumulation of Oligogalactolipids in the T-DNA lines was due to the
disruption of the At1g65410 locus. From here on, the SALK_O40335 line will be referred
to as tgd3 mutant line (carrying the here characterized tgd3-I mutant allele). The
At1g65410-encoded protein was previously designated Non-intrinsic ABC Protein 11

(N AP] 1) (Sanchez-Femandez et a]. 2001) and will be referred to here as
trigalactosyldiacylglycerol 3 (TGD3) fitting more closely with its function. Comparing
the amino acid sequence of TGD3 with proteins encoded by bacterial genomes using
BLAST (Altschul et al. 1997), ABC protein-encoding open reading frames adjacent to
putative T GD1/2 homologs provided some of the best hits, with putative TGD3 homologs
from Cyanobacteria, e. g. Synechococcus (Figure 2.1A), showing 52% identity and the E.

coli homolog 32% identity.

54

Figure 2.2. Identification of the TGD3 locus (At1g65410). A, structure for the tgd3
mutant allele corresponding to Salk_040335 line. The coding region ofAt1g65410 is
shown as black boxes. T-DNA insertion position and primers (P1, P2, and left T-DNA
border primer LB) used for PCR amplification are indicated. B, semiquantitative reverse
transcriptase-PCR analysis of T GD3mRNAlevels in wild-type (Col-2) and tgd3 plants.
The expression of ubiquitin (UBQI 0) in the same samples was used as a control. C,
genotypic analysis at the T GD3 locus. Wild-type (Col-2), tgd3 mutant, and two tgd3
homozygous complementation lines expressing the T GD3 cDNA (cTGD3/tgd3) are
shown. Primers were the same as in A, and diagnostic DNA fragments are shown with
their respective lengths in base pairs. D, lipid phenotype of the different plant lines in C.

A section of the thin-layer chromatogram stained for glycolipids is shown.

55

A5:

 

 

 

 

 

- TGDG

56

2. 4.2 The tgd3 mutant shares a complex lipid phenotype with other tgd mutants

It is important to note that all three tgd mutant alleles (tng -1 , tgd2-1, tgd3-1)
analyzed thus far and discussed here are leaky, leading to attenuated phenotypes. More
severe impairment of the system as previously shown for TGD1 causes embryo-lethality
(Xu et a]. 2005) making fully gene-disrupted mutants currently inaccessible to analysis. If
the TGD3 protein indeed is part of a lipid transport complex along with TGD1 and TGD2,
mutant lipid phenotypes for all three loci should be similar. Therefore, leaf lipid extracts
from the tgd] , tgd2, and the tgd3 lines were compared by thin-layer chromatography of
neutral (Figure 2.3A) and polar lipids (Figure 2.3B). The tgd3 mutant extracts contained a
lipid co-migrating with triacyl glycerol (Figure 2.3A), previously identified in the tng
mutant (Xu eta]. 2005) and shown also to accumulate in tgd2 mutant extracts (Awai et al.
2006). In addition, polar lipid extracts of the tgd3 mutant contained a new lipid staining
with a-naphthol, which is diagnostic for the presence of hexoses (Figure 2.38). This lipid
co-chromatographed with authentic trigalactosyldiacyl glycerol (TGDG) accumulating in

tgd] and tgd2.

57

Col-2
tgd 1
tgd2
tgd3
CD
Col-2
tgd 1
t d2
tgd3

l

MGDG - 0-.-

 

TAG -

 

Figure 2.3. Lipid phenotype of the tgd3 mutant compared with the tgd], tgd2 mutant,
and Col-2 wild type. Total lipids were extracted from 4-weekold seedlings grown on
Murashige-Skoog agar plates containing 1% sucrose and separated by thin-layer
chromatography. A, thin-layer chromatogram of neutral lipids. Lipids were visualized by
exposure to iodine vapor. B, thin-layer chromatogram of polar lipids. Lipids were
visualized by sugar-specific a-naphthol staining. 0, origin; PIG, pigments; TAG,

triacyl glycerol.

58

Further confirmation of the identities of TGDG and triacylglycerol lipids was
provided by liquid chromatography/MS analyses of leaf extracts of Col-2 wild type and
tgd3 mutants (Figure 2.4, A—C). Extracted ion chromatograms generated in negative ion
mode yielded a strong peak at m/z 1129.6 for the tgd3 mutant corresponding to
[M+acetate]' for TGDG 34:6, which was the dominant TGDG lipid detected, but minimal
amounts in the wild-type extracts. The in-source collision-induced dissociation spectra
supported this assignment in the form of a fragment ion at m/z 277 corresponding to the
C1823 fatty acid anion. Triacylglycerols were characterized using positive ion
electrospray ionization, displaying peaks at m/z 892.7 to 902.8 corresponding to
[M+NH4]+ of triacylglycerols with 54 carbons and a total number of double bonds
ranging from 3 to 8 (Figure 2.4F). Extracted ion chromatograms for signals in the range
of m/z 890—905 showed about 5-fold greater signal in the tgd3 mutant than the wild type
(Figure 2.4, D and E). Ion abundances for both TGDG and triacylglycerol lipid peaks
were consistent with quantitative analysis of fatty acid methyl esters by gas

chromatography coupled to flame ionization detection.

59

Figure 2.4. Liquid chromatography/MS analyses of lipid extracts of tgd3 mutant
and Col-2 wild type Arabidopsis leaves. Negative mode electrospray extracted ion
chromatograms (XIC) for m/z 1129.6, the [M+acetate]' ion for TGDG 34:6, for extracts of
tgd3 mutant (A) and Col-2 wild type (B) leaves. The mass spectrum at retention time
21.32 min for the tgd3 mutant (C) shows additional peaks corresponding to [M+Cl] ' and
[M+formate] ' (unlabeled). Positive mode electrospray extracted ion chromatograms over
the range of m/z 890—905 reflect the abundance of triacylglycerols with 54 carbons and
various numbers of double bonds for tgd3 mutant (D) and Col-2 wild type (E) leaves. The
summed mass spectrum over the peak eluting at 27.87 min (F) shows [M+NH4] ' peaks

corresponding to triacylglycerols with 54 carbons and from 3—8 double bonds.

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61

The overall lipid composition of the tgd3 mutant differed from that of the wild
type as shown in Figure 2.5A. Most notably, the relative amounts of the major
chloroplast lipids MGDG and DGDG were decreased, whereas the presumed precursor of
galactolipids derived from the ER pathway, phosphatidylcholine (Roughan and Slack
1982), was more abundant. Analysis of the fatty acid composition of MGDG and DGDG
revealed distinct changes in the fatty acid profiles in tgd3 as compared with the wild type
(Figure 2.5, B and C). Most notably, fatty acids were generally more saturated, and 18:3
fatty acid content was reduced. Moreover, the tgd3 fatty acid profile changes were very
similar to those observed for the tgd] and tgd2 mutants (Awai et a]. 2006). In general, the
tgd mutants impaired in the ER pathway have an increased 16-carbon-to-18-carbon fatty
acid ratio (Awai et a]. 2006) in their galactolipids. This is particularly visible for DGDG,
which is to a large extent derived from the ER pathway (Heinz and Roughan 1983).
Accordingly, the 16-carbon-to-18-carbon ratio for the digalactolipid increased from 0.29
to 0.76 in the tgd3 mutant (Figure 2.5C). This phenomenon is due to the substrate
specificities of the different acyltransferases in the plastid and the ER leading to 18—
carbon fatty acids at the sn-1 position and l6-carbon fatty acids at the sn—2 position of the
diacylglycerol backbone for plastid-derived lipids. Those lipids derived from the ER
pathway carry 18-carbon fatty acids in both positions (Heinz and Roughan 1983).
Positional analysis using Rhizopus lipase (Supplemental figure 2.1) confirmed an
increase in 16-carbon fatty acids in the sn-2 position of MGDG and DGDG of the tgd3
mutant. Overall, tgd3 has a complex lipid phenotype very similar to tgd] and tgd2 that is

consistent with an impairment of the ER pathway of galactolipid biosynthesis. This

62

similarity in phenotypes also suggests that the TGD1, TGD2, and TGD3 proteins are

involved in the same biochemical process.

63

Figure 2.5. Polar lipid composition and fatty acid content in the tgd3 mutant. A, 4-
week-old Col-2 wild type and tgd3 mutant seedlings grown on the same Murashige-
Skoog agar plate were analyzed. Five replicates were averaged, and the SE. are shown. A,
polar lipid composition (relative mol%) determined by quantification of fatty acid methyl
esters derived from individual lipids. Fatty acid composition of MGDG (B) and DGDG
(C). Fatty acids are indicated with the number of carbonsznumber of double bonds. PE,
phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG,

sulfoquinovosyldiacyl glycerol.

64

CI] Col-2

- tgd3
0

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- tgd3

 

 

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Supplemental Figure 2.1. Fatty acid composition of lyso-MGDG (A) and lyso-DGDG
(B) in the tgd3 mutant and C012 wild type lines. Relative abundance (mol%) of 16 and
l8-carbon fatty acids at the glycerol sn-2 position of MGDG and DGDG were
determined from the lyso derivatives after digestion of the lipids with Rhizopus sp. lipase.

Values represent the means of three measurements and the standard error is shown.

66

2. 4.3 The T GD3 protein is imported into the chloroplast

The TGD3 sequence has a predicted chloroplast transit peptide. To verify the
localization of the protein, the cDNA was C-terrninally fused with a green fluorescent
protein and transiently expressed in tobacco. The result shown in Figure 2.6, A—C, shows
the green fluorescent protein fluorescence associated with chloroplasts, which are
identified by their chlorophyll fluorescence. To further narrow the location of TGD3, in
vitro translated labeled TGD3 protein was incubated with isolated pea chloroplasts. Two
proteases were used to determine the sub-chloroplast localization; thermolysin, a large
protease that can only digest cytosol-exposed domains of outer envelope membrane
proteins unless detergent such as Triton-X-IOO is added (Cline et a]. 1984), and trypsin,
which can penetrate the outer envelope membrane but not the inner (Jackson et a]. 1998).
‘ The result of this analysis is shown in Figure 2.6D. The TGD3 preprotein was shortened
upon import consistent with the removal of a transit peptide. Moreover, the mature TGD3
protein was resistant to thermolysin and trypsin treatment but was digested by
thermolysin in the presence of detergent. These results were consistent with import of
TGD3 into the chloroplast beyond the inner chloroplast envelope membrane. Based on
these results, TGD3 would be available for association with the TGD1_TGD2 complex at

the stroma side of the inner envelope membrane.

67

Figure 2.6. Subcellular localization of TGD3. A—C, transient expression of the TGD3-
encoding cDNA fused to the N terminus of the green fluorescent protein coding sequence.
Confocal images are shown with red chlorophyll fluorescence of chloroplasts (A), green
fluorescence specific for the TGD3-green fluorescent protein fusion protein (B) and an
overlay of A and B. The scale bar equals 8 um. D, import of in vitro translated TGD3
protein into isolated pea chloroplasts. An autoradiogram of the protein gel is shown. The
sensitivity of the imported protein to thermolysin (TL), trypsin (TR), and to thermolysin

in the presence of Triton X-l 00 (TX) was tested. The type of plastid treatment is indicated
by +/-. The translation product mixture (T P) containing theTGD3preprotein (pP) is

shown as well. The mature protein (mP) after removal of the transit peptide is indicated.

68

 

-pP

”V's-lia-
- mP

 

69

2. 4.4 Recombinant T GD3 protein has AT Pase activity

The TGD3 protein contains motifs characteristic for other small ATPases
associated with bacterial membrane transporters (Ambudkar et a]. 2006, Davidson and
Chen 2004) designated Walker-A, Walker-B, A-loop, and Q-loop (Figure 2.1B). To test
the biochemical function of TGD3, an N-terminal truncated cDNA missing the coding
region for the first 46 amino acids representing the predicted transit peptide was fused to
the C-terminus—encoding end of the E. coli maltose binding protein open reading frame
and expressed in E. coli. Of several expression systems tested, this was the only
providing soluble TGD3 protein. Purification of the recombinant protein designated
MBP-ATGD3 on an amylose column is shown in Figure 2.7A. The firsion protein was
highly purified (estimated ~90%) and migrated as a 78-kDa protein. The fusion protein in
the E3 fraction was assayed for its ability to hydrolyze ATP. For control purposes,
maltose-binding protein by itself or a mutant cDNA giving rise to an F94A point mutant
of TGD3 fused in the same manner as the truncated wild type cDNA were included
(MBP-ATGD3F94A). It should be noted that the F 94A mutation did not affect protein
expression or protein purification. Replacing the A-loop residue homologous to Phe94 in
TGD3 in bacterial orthologs with a nucleophilic cysteine residue was previously shown
to greatly decrease the ATP-dependent transport activities (Zhao and Chang 2004). Under
standard assay conditions as defined under “Experimental Procedures,” the reaction was
linear for more than 40 min (Figure 2.78). Furthermore, the reaction velocity increased
linearly with MBP-ATGD3 protein concentration in the range tested (Figure 2.7C).
Varying the concentration of ATP, 3 classic Michaelis-Menten hyperbolic substrate

velocity function was observed for the MBP-ATGD3 protein (Figure 2.7D). Estimated

70

kinetic constants based on this experiment were 151.7 uM for the Km and 0.026 umol
min'lmg'l for Vmax. These values are in the same range as previously reported for ATP
hydrolysis by other ABC transporters (Schneider and Hunke 1998). The MBP-
ATGD3F94A mutant protein had less than 20% residual activity insufficient for accurate
determination of the kinetic constants (Figure 2.7D). The maltose-binding protein had no

activity.

71

Figure 2.7. Purification and ATPase activity of recombinant TGD3 protein. The
TGD3 protein is N terminal truncated lacking the transit peptide and fused to the C
terminus of maltose-binding protein designated as MBP-ATGD3. A, purification of MBP-
ATGD3 in E. coli. Analysis of 10 ul of total protein from flow-through (P), washing
fraction (W), and a series of different elution fractions (1—7) by SDS-PAGE is shown.
Proteins were visualized by Coomassie Blue staining. The E3-fraction contained ~5 ug of
protein. B, time-course of ATPase activity. The assay was conducted at a protein
concentration of ~8 uM and final ATP concentration of lmM at 37 °C. Buffer solutions
containing no protein were included as background control. Maltose-binding protein
treated identical to MBP-_TGD3 was included to test for endogenous ATPase activity.
Relative activity is shown as absorbance readings at 850 nm. C, production of inorganic
phosphate as a function of protein concentration at lmM ATP. Reactions were performed
at 37°C with an incubation time of 30 min. Absorbance readings were corrected for ATP
spontaneous hydrolysis. Three replicates were averaged, and the SE. are shown. D,
ATPase activity of MBP-ATGD3 and MBP-ATGD3F 94A. The phenylalanine at position
94 (A-loop) was mutated to an alanine residue by site-directed mutagenesis. ATPase
assays were performed at various ATP concentrations as indicated. Three replicates were

averaged, and the SE. are shown.

72

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73

2.5. Discussion

The previous identification of TGD1 and TGD2 proteins and the characterization
of the respective mutants (Awai eta]. 2006, Xu eta]. 2003, Xu eta]. 2005) provided
reasonable evidence suggesting that these two proteins are involved in PtdOH transfer
possibly from the outer envelope through the inner envelope membrane of chloroplasts in
Arabidopsis. Typically, ABC-type lipid transporters such as the biochemically well
characterized MsbA protein from E. coli (Doerrler et a]. 2001, Doerrler and Raetz 2002)
consist of a dimer of a bifunctional protein, with a membrane-spanning permease domain
and a second ATP binding domain. In the case of the TGD1/TGD2 transporter, TGD1
represents the permease domain only, and TGD2 is similar to a membrane-tethered
substrate binding protein like those associated with other bacterial ABC transporters. The
TGD3 (N AP] 1) protein of Arabidopsis described here appears to be the missing ATP
binding component that would be required for a functional TGD1_TGD2_TGD3
complex as proposed in Figure 2.8. Several lines of indirect evidence are consistent with
the current hypothesis for the composition of the PtdOH transporter of the inner
chloroplast envelope membrane and the involvement of the TGD3 (N API 1) protein; 1)
most Gram-bacteria and mycobacteria contain potential orthologs of the three
Arabidopsis TGD proteins including the newly described TGD3 protein. Their respective
genes are typically linked in operons consistent with their function in the same
biochemical process (Figure 2.1A). 2) Of the 26 genes of Arabidopsis encoding small
ATP-binding proteins without membrane-spanning domains (Sanchez-Femandez et al.
2001), 9 were predicted to be targeted to the chloroplasts. Of the seven genes tested, only

the tgd3 mutant corresponding to the gene encoding TGD3 (N AP] 1) showed a phenotype

74

identical to the previously characterized tgd] and tgd2 mutants. 3) The TGD3 (N AP] 1)
protein was localized to the stroma of chloroplasts and showed basic ATPase activity in
vitro when expressed as a maltose-binding protein fusion. With the presumed ATPase
component of the TGD transport complex identified, reconstitution and direct proof of
biochemical transport activity should in principle be possible. However, our current
efforts have been hampered by the fact that the production of detergent-soluble,
functional full-length TGD1 and TGD2 proteins in E. coli has not yet been feasible. Even
the TGD3 protein described here could only be functionally produced fused to maltose-
binding protein. The difficulties in demonstrating lipid transport by integral membrane
complexes are also evident from the fact that even for the well studied MsbA protein no
direct evidence of lipid transfer across the membrane is available. Indirect evidence for
lipid transport by MsbA is currently based on the respective mutant phenotype (Doerrler
et a]. 2001) and the observation that specific lipids stimulate the ATPase activity of
MsbA in vitro (Doerrler and Raetz 2002). Testing the possible stimulation of ATPase
activity of TGD3 was not considered a viable approach, because unlike the MsbA protein,
the TGD3 protein lacks the permease domain that would directly interact with the lipid
substrates. As for the TGD complex of Arabidopsis, the precise function of MsbA still
remains under debate, especially since a mutant of Neisseria meningitis lacking the
MsbA ortholog remains capable of exporting phospholipid building blocks of the outer
membrane (Tefsen eta]. 2005). Unfortunately, technical barriers currently prevent us
from directly and unambiguously demonstrating the lipid transfer activities of MsbA-like
or TGD complex-like lipid transporters. Despite these difficulties, we are now reasonably

certain about the localization of the TGD complex in the inner chloroplast membrane.

75

Because the localization data for TGD1 or TGD2 left open questions, the clear result for
TGD3 presented here provides crucial evidence. For example, TGD1 was partially
thermolysin-sensitive in chloroplast import experiments (Xu eta]. 2003, Xu eta]. 2005),
and therefore, it seemed possible that at least subpopulations of TGD1 might be localized
in the inner and outer chloroplast envelope membranes. Alternatively, the TGD1 protein
could be present in a fusion zone between the two membranes. Likewise, TGD2 wild-
type protein was resistant to trypsin, but the TGD2-green fluorescent protein fusion
protein was not (Awai eta]. 2006). This result could be interpreted as the presence of
TGD2 in localized domains between the inner and the outer envelope membrane, which
might be inaccessible to trypsin. On the other hand, the evidence for TGD3 presented
here is unambiguous; TGD3 is imported into the chloroplast beyond the inner envelope
membrane and processed during in vitro import experiments (Figure 2.6B). The only way
TGD3 could interact with TGD1 and TGD2 as suggested by the identical phenotypes for
the respective mutants and the organization of predicted bacterial orthologs is if TGD1
and TGD2 are intrinsic proteins of the inner chloroplast envelope membrane as shown in
Figure 2.8, thereby confirming previous conclusions drawn about TGD1 and TGD2
localization. Demonstrating a direct interaction of the three TGD proteins in the inner
chloroplast envelope has proven difficult in our hands using available in vivo techniques.
It will require either the isolation of the native complex or its reconstitution from
recombinant proteins, both of which have not yet been accomplished. However, our
current data clearly suggest that TGD3 is the missing ATPase subunit of this transporter,
bringing us a step closer to the actual reconstitution of a core transporter possibly

consisting of the TGD1 permease and the TGD3 ATP-binding protein.

76

oE

Figure 2.8. Model of the proposed TGD lipid transporter complex. A dimeric
composition is shown with numbers corresponding to the TGD1, TGD2, and TGD3

proteins. Outer (0E) and inner chloroplast envelope membranes (iE) are indicated.

77

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Benning, C. and Ohta, H. (2005) Three Enzyme Systems for Galactoglycerolipid
Biosynthesis Are Coordinately Regulated in Plants. J. Biol. Chem. 280, 2397-2400.

Benning, C., Xu, C., and Awai, K. (2006) Non-vesicular and vesicular lipid trafficking
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Davidson, A. L. and Chen, J. (2004) ATP-binding cassette transporters in bacteria.
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Doerrler, W. T. (2006) Lipid trafficking to the outer membrane of Gram-negative
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Doerrler, W. T. and Raetz, C. R. (2002) ATPase activity of the MsbA lipid flippase of
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Doerrler, W. T., Reedy, M. C., and Raetz, C. R. (2001) An Escherichia coli mutant
defective in lipid export. J. Biol. Chem. 276, 11461-11464.

Diirmann, P., Balbo, 1., and Benning, C. (1999) Arabidopsis galactolipid biosynthesis
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D6rmann, P. and Benning, C. (2002) Galactolipids rule in seed plants. Trends Plant Sci.
7, 112-118.

Diirmann, P., Hoffmann—Benning, S., Balbo, I., and Benning, C. (1995) Isolation and
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Emanuelsson, 0., Nielsen, H., and von Heijne, G. (1999) ChloroP, a neural network-
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Froehlich, J. E., Benning, C., and Diirmann, P. (2001) The di galactosyldiacyl glycerol
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a manner independent of the general import pathway and does not depend on direct
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Heinz, E. and Roughan, G. (1983) Similarities and differences in lipid metabolism of
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Jackson, D. T., Froehlich, J. E., and Keegstra, K. (1998) The hydrophilic domain of
Tic110, an inner envelope membrane component of the chloroplastic protein
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Jarvis, P., Diirmann, P., Peto, C. A., Lutes, J ., Benning, C., and Chory, J. (2000)
Galactolipid-deficiency and abnormal chloroplast development in the Arabidopsis MGD
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Miquel, M., Cassagne, C., and Browse, J. (1998) A new class of Arabidopsis mutants
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Rossak, M., Schfifer, A., Xu, N., Gage, D. A., and Benning, C. (1997) Accumulation of
sulfoquinovosyl-l-O-dihydroxyacetone in a sulfolipid- deficient mutant of Rhodobacter
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Roughan, P. G. and Slack, C. R. (1982) Cellular-Organization of Glycerolipid
Metabolism. Ann. Rev. Plant Physiol. 33, 97-132.

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Arabidopsis thaliana ABC protein superfamily, a complete inventory. J. Biol. Chem. 276,
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Schneider, E. and Hunke, S. (1998) ATP-binding—cassette (ABC) transport systems:
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Siebertz, H. P. and Heinz, E. (1977) Labeling Experiments on Origin of
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35961-35966.

Xu, C., Fan, J., F roehlich, J. E., Awai, K., and Benning, C. (2005) Mutation of the
TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.
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80

Chapter 3

Germination, growth and lipid analysis of Arabidopsis plants disrupted in T GD3

81

3.1. Abstract

Galactoglycerolipids are predominant in the chloroplasts of seed plants. They can
either be synthesized de novo in the plastids or transported to endoplasmic reticulum and
later return to the plastids and enter the galactolipid biosynthesis pathway. Arabidopsis
mutants that are disrupted in the incorporation of endoplasmic reticulum-derived lipid
precursors into thylakoid lipids are available. Three genes affected in these mutants,
trigalactosyldiacylglycero] 1 (TGD1), T GD2, and T GD3, encode the permease, substrate
binding component and ATP binding domain, respectively, of a proposed phosphatidic
acid (PtdOH) translocator at the inner chloroplast envelope membrane. The TGD3
protein shows basal ATPase activity when expressed as a maltose-binding protein fusion
and is localized inside the chloroplast beyond the inner chloroplast envelope membrane.
Similar to the tgd] and tgd2 mutants, a tgd3 mutant carrying a T-DNA insertion just 5’ of
the TGD3 coding region (tgd3-1) also accumulates triacylglycerols and trigalactolipids.
However, the T GD3 gene expression level is only slightly reduced in this tgd3-1 mutant.
Mutants fully disrupted in T GD3 were unavailable to analysis at the time of the study. By
utilizing RNA silencing technique, we generated Arabidopsis mutant plants with more
severely disrupted TGD3 function. The phenotype of these RNAi mutant plants are
nearly identical to tgd3-1, and do not show the expected severe growth defect and
embryo lethality seen in other tgd mutants, raising the possibility of another ATPase

associated with the proposed TGD complex.

82

3.2. Introduction

The chloroplast harbors the most extensive membrane system in nature- the
thylakoid. Different from most organisms and organelles, the major lipid classes in the
chloroplasts are galactoglycerolipids with one (monogalactosyldiacylglycerol, MGDG) or
two (digalactosyldiacylglycerol, DGDG) galactose moieties (Dormann and Benning
2002). MGDl and DGDl are the two major galactosyltransferases required for
galactoglycerol lipids final assembly from diacylglycerol (DAG) and UDP-galactose
(Benning and Ohta 2005). MGDl is located at the outside of the inner chloroplast
envelope membrane (Xu et a]. 2005) and responsible for the bulk of MGDG synthesis
(Jarvis eta]. 2000), whereas DGDl is located at the outside of the outer chloroplast
envelope membrane (Froehlich et a]. 2001) and plays a major role in the bulk of DGDG
synthesis (Dormann et al. 1999).

The original location of lipid biosynthesis and final destinations of the lipids are
often different, a fact which requires substantial interorganellar lipid trafficking within
the plant cells (Benning et al. 2006). For example, the diacylglycerol moiety of the
galactoglycerolipids is either synthesized de novo in the plastid or is assembled at the
endoplasmic reticulum (ER) and imported into the plastid (Roughan and Slack 1982).
Previous studies identified a group of tgd mutants in Arabidopsis that are disrupted in the
ER-pathway so that most of the galactolipids in these mutants are of plastid origin (Awai
et a]. 2006, Lu eta]. 2007, Xu eta]. 2003, Xu eta]. 2005). These mutants have nearly
identical lipid phenotypes in all aspects tested such as the accumulation of
oligogalactolipids and triacylglycerols (Awai eta]. 2006, Lu et a]. 2007, Xu et al. 2003,

Xu eta]. 2005). Four TGD genes have been identified so far. Their bacterial orthologs are

83

often organized in operons (Lu et a]. 2007). Taken together, these facts strongly suggest
that the T GD gene products are involved in a common biochemical or cell biological
process. Our current hypothesis is that the T GD] , T GD2 and T GD3 gene encodes a
protein similar to the permease component, membrane-tethered substrate-binding protein
and the ATP binding domain of a bacterial ATP binding cassette (ABC) transporter (Lu
eta]. 2007), respectively. The specific binding of phosphatidic acid (PtdOH) to TGD2
suggests that PtdOH is the substrate transported by this TGD1-3 transporter complex
located at the inner chloroplast envelope (Awai eta]. 2006).

In chapter 2, I detailed the identification and biochemical characterization of
TGD3, the small ATPase protein proposed to be associated with TGD1 and TGD2 (Lu et
a]. 2007). The TGD3 protein shows basal ATPase activity when expressed as a maltose-
binding protein fusion and is localized inside the chloroplast beyond the inner chloroplast
envelope membrane (Lu et a]. 2007). It is important to notice that all three tgd mutant
alleles (tng -1 , tgd2-1, tgd3-1) analyzed thus far are leaky, leading to attenuated
phenotypes. More severe impairment of the system as previously shown for TGD1 causes
embryo-lethality (Xu eta]. 2005), making fully gene-disrupted mutants currently
unavailable to analysis. An Arabidopsis mutant carrying a T-DNA insertion in the
promoter region of T GD3 (tgd3-1) does not have obvious growth defects as compared to
wild-type Col-2, possibly due to the leakiness of T GD3 gene expression. Therefore, I
utilized RNA interference (RNAi) technique to silence T GD3 expression and generate
transgenic Arabidopsis plants disrupted more severely in the TGD3 function. Here, I
performed experiments to systematically characterize germination, root growth and lipid

phenotypes of these tgd3 mutants.

84

3.3. Experimental Procedures

3. 3. I . Plant materials and growth conditions

Arabidopsis thaliana wild type plants were of the ecotype Columbia-2 (Col-2).
The tgd3-1 T-DNA insertion lines (SALK_040335, At1g65410) were obtained from the
Arabidopsis Biological Resource Center at Ohio State University. The T GD3 transgenic
lines were generated as described below. Homozygous mutant lines were screened and
used for all experiments. All seeds were surface-sterilized with 20% bleach, 0.05%
TritonX-IOO for 10 min and were rinsed 5 times in sterile water before grown on 0.8%
(w/v) agar-solidified Murashige-Skoog medium (Murashige and Skoog 1962)
supplemented with 0 or 1% (w/v) sucrose when appropriate.

For germination and seedling growth assay, wild-type and mutant seeds were first

stratified at 4°C in the dark for 3 days prior to being transferred into an incubator. The

incubator has a photon flux density of 60—80 umol m—2 sec—l, a light period of 16 h
(22°C), and a dark period of 8 h (18°C). After 10 days, seedlings were either transferred
to soil or to fresh agar plates. Soil grown plants were put into a 16h photoperiod with a
day temperature of 22°C and a night temperature of 20°C at a photon flux density of
100—120 umol m_2 sec—l. Wild-type and mutant seedlings of similar developmental

stage were used for lipid analysis and growth measurement.

3.3.2. Lipid and fatty acid analysis
Total lipids were extracted by vigorously shaking leaf tissue for 5 min in 300 uL

of methanol/chloroform/formic acid (2:1:0.1, v/v). Then 150 uL of 1 M KC1, 0.2 M

85

H3PO4 was added and the tubes were vortexed before centrifuged at 16,000 g for 5 min.

The organic phase (upper phase) was loaded onto a treated (soaked in 0.15 M (N H4)2SO4

and dried, then baked at 120°C for 2 hrs) silica-60 TLC plate (Si250PA; Mallinckrodt

Baker, Phillipsburg, NJ). For lipid quantification, a solvent system of
acetone/toluene/water (91 :30:7, v/v) was used and lipids were visualized with iodine
vapor. Distinct bands were then scraped off the plate and lipid composition was
determined by fatty acid methyl ester (FAME) analysis and gas chromatography as

described previously (Dorrnann eta]. 1995, Rossak et a]. 1997)

3.3.3. Root elongation assays

All seeds used were produced from plants grown in identical conditions and were
of the same age. When appropriate, 0 or 1% sucrose was added to the medium prior to
being autoclaved. Seeds were sown in a straight line and the agar plates were arranged
vertically. Plant growth were recorded by a SPOT CCD microsc0pe digital camera
(Diagnostic instruments, Sterling Heights, MI), and root length was measured by
software Spot Advanced (version 4.0.2) every 24 h. The same set of agar plates was used
throughout the experiment. Ten individual plants were measured and values were

averaged.

3.3.4. Generation and analysis of T GD3 RNAi plants
For the generation of a TGD3 RNA interference (RNAi) line, 5’UTR and the first

exon region of TGD3 gene sequence (~375bp) were amplified by PCR using the primers

5’- CCGCTCGAGAAAAATGGCAATGTGAC -3’ and 5’—CGCAAGCTTATCGAATT-

86

TGGTGGCATC-3’ (XhoI and HindIII; for amplification of sense fragment) and 5’-CG-
GACTAGTAAAAATGGCAATGTGACTCAC-3 ’ and 5’-ATTGGATCCATCGAATTT-
GGTGGCATC-3’( Spel and BamHI; for amplification of antisense fragment). The
underlined sequences indicate the restriction enzyme sites. The resulting fragment was
cloned into the intermediate vector pSK-int (Guo et a]. 2003) in sense and antisense
orientations separated by an actin intron using the corresponding restriction enzyme sites
on the vector. Subsequently, the entire sense-intron-antisense cassette was cloned into the
hormone-inducible expression vector pER8 (Zuo eta]. 2000). Alternatively, the pSK-int
vector harboring TGD3 RNAi cassette ( pSK-TGD3i) was cut by Xhol and Sacl and
inserted into the SalI and Sacl sites of pCambial 300MCS (Xu et a]. 2005) followed by
transformation into Agrobacterium. The details of construction of these plasmids are
illustrated in Figure 3.1. Plant transformation was achieved by the floral dip method
(Clough and Bent 1998). Transgenic plants were selected by MS medium containing 25
ug/ml of Hygromycin B. For an estimation of T GD3 transcript levels by semi-
quantitative reverse transcriptase-PCR, RNA was isolated from Col-2 wild-type and
T GD3 RNAi mutant plants, and reverse transcription was done using oligo(dT);2-13
primers (Invitrogen). The T GD3 specific primers used for the PCR reaction were: 5’—AC-
GGTACCATGCT’I‘TCGTTATCATGCTC-3 ’ and 5 ’-AGGTTTGTGTCACAAGCTCA-
3’. Meanwhile, the abundance of actin (A C T 1 ) was tested for control purposes. The
following AC T 1 specific primers were used: 5’-CCTCTCGATTTCAGGTAGAAGAAA-
ATG-3 ’ and 5 ’-CCATCTCCAGAGTCGAGCACAATA-3 ’.

To evaluate the function of the inducible T GD3i-pER8 system, T2 seeds from

independent transgenic lines were germinated on the regular MS medium containing

87

Hygromycin B. After two weeks, resistant plants were transferred into a liquid MS
solution containing inducer B-estradiol. Samples were collected and subject to TLC

analysis afler 6 days induction.

88

Figure 3.1. Generation of TGD3i constructs.A, linear representation of the T GD3 gene
(At1g65410). The coding region is shown as black boxes. B, nucleic tide sequences from
the T GD3 gene 5’-UTR and the first exon. Exon was shown as black box.PCR primers
with restriction enzyme sites used to amplify and clone sense and antisense fragments are
indicated. C, schematic diagrams of the inducible and constitutive T GD3i constructs.
pSK-int, an intermediate vector contains actin 11 intron (ATU27981, nt 1957-2111) with
multiple cloning sites on both arms of the intron. DNA fragments encoding sense and
antisense RNA (from B) were cloned into pSK-int at 5’ and 3’ arms of the intron in
appropriate restriction sites indicated by solid lines, resulting pSK-TGD3i. pER8-TGD3i,
the inducible pER8-TGD3i construct contains a chimeric transactivator with the regulator
domain of an estrogen receptor (see Zuo et al., 2000 for details of pER8 vector). T GD3i,
T GD3 DNA sequences encoding the intron-containing inverted-repeats.
pCAMBIA1300MCS-TGD3i, a constitutive T GD3i construct with a fragment of T GD3i

cloned into the binary vector pCAMBlA1300MCS.

89

51
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3.3.5. Dominant-negative mutation analysis

Coding region of T GD3 was amplified by reverse transcriptase-PCR (standard
conditions) from mRNA preparations using Plant RNeasy kit (Qiagen, Valencia, CA)
with the primers 5’-ACGGTACCATGCTTTCGTTATCATGCTC-3’ and 5’-CTGG-
TACCCTAGTATCTGATTGGTCCAT-3’. The PCR products were ligated into pGEM-
Teasy (Promega, Madison, WI) and sequenced. The resulting plasmids served as template
to create F94A mutation by site-directed mutagenesis strategy. The following
mutagenesis primers were used: 5’-GTAGAGATGTCTATAAATCG@GGGGAGA-
AACATATCTTG-3 ’ and 5’-CAAGATATGTTTCTCCCC_(&(_3CGATT'TATAGACAT-
CTCTAC-3’. The underlined sequences are codons for the mutated residue (Alanine).
After digestion with Sall, the mutant T GD3F 94A fragment was inserted into
pCambia1300MCS (Xu et a]. 2005) followed by transformation into Agrobacterium.
Plant transformation was achieved by the floral dip method (Clough and Bent 1998).
Transgenic plants were selected by MS medium containing 25 ug/ml of Hygromycin B
and analyzed for lipid phenotype as described above.

2 For semi-quantitative PCR of TGD3 and T GD3F 94A (tgd3) transcripts, the
following primers were used: T GD3, P1: 5’-CCGCTCGAGAAAAATGGCAATGTGA-
C-3’ and P2: 5’-CGCAAGCTTATCGAATTTGGTGGCATC-3’; T GD3 and tgd3, P5: 5’-
CCGGTCGACATGCTTTCGTTATCATG- 3’ and P6: 5’-AGGTTTGTGTCACAAGCT-
CA—3’; ACT I , 5’-CCTCTCGATTTCAGGTAGAAGAAAATG-3’ and 5’-CCATCTCC-
AGAGTCGAGCACAATA-3’. Primers are shown by arrows in Figure 3.1. Isolation of
RNA and reverse transcription were done as described above. Standard PCR conditions

were used.

91

3.3.6. Chlorophyll quantification
Chlorophyll was extracted from leaves and seedlings using 100 volumes (pL/mg)

of 80% acetone and was quantified as previously described (Lichtenthaler 1987).

3.4. Results

3.4. 1. Oligogalactolipids accumulate in T GD3i leaves

The T GD3 gene was identified via a reverse-genetics approach. Previously, a T-
DNA insertion line (SALK_040335, tgd3-1) carrying a T-DNA 5’ of the presumed ATG
start codon of the T GD3 gene showed accumulation of oligogalactolipids (Lu eta]. 2007).
The grth of the tgd3-1 mutant line was only slightly reduced, possibly due to leakiness
of the allele.

Because no knockout allele of T GD3 was readily available, we used an RNA
interference (RNAi) approach to enhance the mutant phenotype of tgd3-1 and thereby
demonstrate the leakiness of the tgd3-I allele. Two representative RNAi lines are shown
in Figure 3.2. The abundance of the T GD3 mRNA was greatly reduced in these lines
(Figure 3.2C), resulting in slightly diminished growth (Figure 3.2A) and obvious

accumulation of the oligogalactolipids-TGDG (Figure 3.28).

92

 

0
41°
46° 49

A .
B IIMGDG
DGDG
* “r '“ ,reoe

C3

 

Figure 3.2. Oligogalactolipids accumulate in T GD3-RNAi plants. A, morphology of 6-
week-old plants grown on soil. Wild-type (Col-2), tgd3—1 T-DNA mutant, and two
constitutively expressed TGD3-RNAi homozygous lines (3 5S:TGD3i/Col-2) are shown. B,
lipid phenotype of the different plant lines in A. A section of the thin-layer chromatogram
stained for glycolipids is shown. C, semi-quantitative reverse transcriptase-PCR analysis
of TGD3 mRNAlevels in wild-type (Col-2) and TGD3-RNAi plants. The expression of

actin (AC T1 ) in the same samples was used as a control.

93

3.4.2. The phenotype of T GD3i and tgd3-1 are very similar

The overall lipid composition of the tgd3-1 mutant differed from that of the wild
type (Lu eta]. 2007). Most notably, the relative amounts of the major chloroplast lipids
MGDG and DGDG were decreased, whereas the presumed precursor of galactolipids
derived from the ER pathway (Roughan and Slack 1982), phosphatidylcholine, was more
abundant.

Two independent homozygous 35S: T GD3i/Col-2 lines were screened and
analyzed for their lipid phenotype (Figure 3.3A). The overall lipid compositions of these
two lines are very similar to each other and the tgd3-1 mutant, with the exception of
phosphatidylglycerol (PG) and digalactosyldiacylglycerol (DGDG). A decrease in
DGDG and increase in PG were observed in the tgd3-1 mutant, however, an opposite
trend was found in TGD3 i plants. In both lines, PG was decreased about 50-60% while
DGDG was increased 10-20%.

Analysis of the fatty acid composition of MGDG and DGDG also revealed
distinct changes in the fatty acid profiles in 35S: T GD3i/Col-2 plants as compared with
the wild type (Figure 3.3 B and C). Similar to tgd3-1 plants, fatty acids were generally
more saturated, and 18:3 fatty acid content was reduced. In general, the tgd mutants
impaired in the ER pathway have an increased 16-carbon-to-18-carbon fatty acid ratio in
their galactolipids (Awai eta]. 2006). This is particularly visible for DGDG, which is to a
large extent derived from the ER pathway (Browse eta]. 1986). Accordingly, the 16-
carbon-to-18-carbon ratio for the digalactolipid increased 2.6 fold in the tgd3-1 mutant
and 1.8 and 3 fold for the two 35S: T GD3i/Col-2 lines, respectively (Figure 3.3 C). This

phenomenon is due to the substrate specificities of the different acyltransferases in the

94

 

plastid and the ER leading to l8-carbon fatty acids at the sn-1 position and 16-carbon 1"
fatty acids at the sn-2 position of the diacyl glycerol backbone for plastid-derived lipids.

Those lipids derived from the ER pathway carry 18-carbon fatty acids in both positions

(Heinz and Roughan 1983). Overall, 358: T GD3i/Col-2 plants have a complex fatty acid

composition very similar to tgd3-1 that is consistent with an impairment of the ER

pathway of galactolipid biosynthesis.

95

 

Figure 3.3. Polar lipid composition and fatty acid content in the tgd3 mutant. 4-
week-old seedlings grown on the Murashige-Skoog agar plates from Col-2 wild type,
tgd3-1 T-DNA mutant and two independent homozygous 35S:TGD3i/Col-2 lines were
analyzed. Five replicates were averaged, and the SE. was shown. A, polar lipid
composition (relative mol%) determined by quantification of fatty acid methyl esters
derived from individual lipids. B, fatty acid composition of MGDG. C, fatty acid
composition of DGDG. Fatty acids are indicated with the number of carbonsznumber of
double bonds. MGDG, monogalactosyldiacylglycerol; PG, phosphatidyl glycerol; DGDG,
digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacyl glycerol; PE,

phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine.

96

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97

3.4.3. Impaired growth and development of T GD3i

Chapter 2 describes identification of the tgd3-I T-DNA mutant allele and includes
some data about lipid phenotype and fatty acid composition. It must be pointed out that
during routine growth of the tgd3-1 plants, no obvious growth defect was observed
except that their leaves are slightly pale-green and growth is slightly retarded both in
leaves and roots compared to the wild type. After obtaining homozygous 358: T GD3i
plants, systematic analyses of the growth phenotype of these transgenic plants defective
in TGD3 function were conducted, as described in this chapter. Root elongation assays
were performed. As shown in Figure 3.4, tgd3-I plants grow almost the same as wild
type Col-2, but 358: T GD3i plants grow slower than wild type. In general, T 0031' plants
grow more slowly on MS medium supplemented with 1% (w/v) sucrose compared to
Col-2, and this growth delay is more obvious when plants are grown on MS medium
containing no sucrose. The result also indicated that the initiation of root and shoot
growth maybe delayed in T GD3i mutants as at 2 days after sowing (DAS) mutants are
notably smaller and pale with shorter roots compared with Col-2. One possibility is that

the germination itself is inhibited in these seedlings.

98

 

 

Figure 3.4. Delayed root growth in tgd3 mutants. Morphology of 2-day-old (A) or 8-
day-old (B) Col-2 wild type and tgd3 mutant seedlings gown on MS medium contains no
sucrose or 1% (w/v) sucrose. White arrows indicate un—gerrninated seeds. Root length
were measured and shown as a fianction of days-after-sowing (DAS) for Col-2 wild type,
and tgd3 mutants grown on MS medium contains no sucrose (C) or 1% sucrose (D). 10

seedlings from each line were measured, values were averaged and SE was shown.

99

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Days after sowmg Days after sowing

100

 

Germination tests were subsequently conducted to explore the possibility that the
tgd3 mutants germinate later than wild type plants. Figure 3.5 shows the germination
rates of wild type, tgd3-1, and two RNAi lines using seeds of the same age. As early as 1
DAS, wild type seeds have completely germinated (defined by radicle emergence from
the seed coat) and the roots are already elongating on either 0% or 1% sucrose (Figire
3.5). However, there are only about 90% of the seeds germinated for tgd3-1 and
3SS:TGD3i linel sown on MS medium containing no or 1% sucrose and they reach a
maximum germination of ~98% after 4DAS. Moreover, only 80% of the 358: TGD3i line
2 seeds germinated and reached its maximum of 95% at 8 DAS, suggesting that this line

carries a stronger allele of T GD3 mutation.

 

 

 

 

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Days after sowing

Days after sowing

Figure 3.5. Delayed germination in tgd3 mutants. Col-2 wild type and tgd3 mutant

seeds of the same age were first stratified at 4°C in the dark for 3 days prior to being

transferred into an incubator. Germination rates were recorded for seeds sown on 0% (A)

or 1% (B) sucrose containing MS medium.

Another noticeable morphology of tgd3 mutants is slight chlorosis, namely, pale-
green leaves, and early flowering (Figure 3.6, A and B). When grown on agar plates, total
chlorophyll content is reduced by 15-30% (Figure 3.6D). Shoot fresh weight was also

reduced by ~30% in tgd3 mutants compared to the Col-2 wild type (Figure 3.6C).

  

   

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Figure 3.6. Impaired growth and slight chlorosis phenotype of tgd3 mutants. Pictures
were taken for 4-weeks-old seedlings grown on 0% (A) or 1% (B) sucrose containing MS
medium. Shoot fresh weight and chlorophyll content were measured. Values are

represented as Mean a: SE from 10 seedlings.

102

 

3. 4. 4. Overexpression of T GD3F 94A cDNA in wild type causes a dominant negative
effect

The similarities of tgd], tgd2, tgd3 mutant phenotypes and the fact that bacterial
orthologs of these Arabidopsis genes are organized in operons suggested that these
proteins might form a large complex and act together in the same cellular process.
However, due to technical difficulties, we still lack definitive evidence to support this
hypothesis. Previous studies on TGD2 demonstrated that expression of the tgd2-1 cDNA
in the wild type recapitulates the tgd2-1 phenotype (Awai et al. 2006).

There are no tgd3 point mutant alleles available except the T-DNA insertion line
at the time of the study, so we set out an experiment to generate a mutant version by site-
directed mutagenesis. In Chapter 2, we have reported that mutation of Phenylalanine 94
to Alanine greatly decreased TGD3 ATPase activity in vitro when expressed as a MBP-
fusion protein in E. coli (Lu et al. 2007). We introduced this TGD3F94A mutant protein
in wild type plants by expressing the mutant cDNA under the control of the 3SS-CMV
promoter in the wild type background.

Initially, two independent T1 transgenic lines were obtained and further analyzed
(Figure 3.7). It is interesting that grth of 355: T GD3F 94A/Col-2 line 2 was obviously
different than wild type and the other 355: T GD3F 94A/Col-2 line (Figure 3.7A). One'
possible explanation is that overexpression of the mutant T GD3F 94A cDNA also
randomly inactivates another gene involved in flowering so that a late flowering
phenotype was observed. This should be an exception. Further isolation of other
independent transgenic lines proved that most of the overexpression lines behave similar

to wild-type and tgd3-I in growth (data not shown).

103

Constitutive overexpression of mutant T GD3F 94A cDNA led to the accumulation
of a lipid cochromatographing with the trigalactolipid accumulating in the tgd3-1 mutant,
namely TGDG (Figure 3.7B). Semi-quantitative RT-PCR confirmed that this dominant
negative effect was not due to cosuppression of the genomic wild type TGD3 gene and
the tgd3 (T GD3F 94A) cDNA expression construct, since mRNA derived from both genes
was abundant in the transgenic lines (Figure 3.7C). One explanation of this dominant
negative effect is that the TGDF94A mutant protein is impaired in its ATPase activity but
can still physically be part of its native protein complex, thereby disrupting overall
fimction of the process involving the complex. This result provided indirect proof that
TGD3 is part of a large lipid transfer complex that involves TGD1 and TGD2. An
antibody against native TGD3 protein would eventually be required to explore the

expression level and function of TGD3 proteins in vivo.

104

 

 

Figure 3.7. Dominant negative effect caused by expression of TGD3F 94A mutant
cDNA in Col-2 wild type. A, morphology of 6-week-old plants grown on soil. Wild-type
(Col-2), tgd3-l T-DNA mutant, and two constitutively expressed T GD3F 94A T1 lines
(35$:TGD3F94A/Col-2) are shown. B, lipid phenotype of the different plant lines in A. A
section of the thin-layer chromatogram stained for glycolipids is shown. C, semi-
quantitative RT-PCR analysis of both TGD3 and T GD3F 94A (tgd3) mRNA levels in
wild-type (Col-2) and T GD3F 94A plants. The expression of actin (A CT 1) in the same

samples was used as a control.

105

 

 

 

3.4. 5. Inducible silencing of T GD3 in Arabidopsis

Potentially, the most effective silencing mediated by intron-containing double-
strand RNAi could produce phenotypes resembling those of the null alleles of the target
genes. However, if the target gene is required for basic cell function or development,
constitutive silencing of the gene may produce detrimental effects or even cause plant
lethality resulting in no recovery of transgenic plants for further investigation. To
circumvent this problem, an inducible gene silencing approach was recently developed
and considered as a effective substitute for constitutive silencing (Guo et al. 2003).

In the study of T GD3 function, parallel to the constitutive overexpression of
T GD3RNAi, a chemical inducible TGD3RNAi system was also constructed and evaluated.
The construct T GD3RNA i-pER8 was introduced into Arabidopsis wild-type Col-2
background by Agrobacterium-mediated transformation (Xu et al. 2005). T2 seeds from
independent transformation lines were germinated on the regular MS medium containing
Hygromycin B. After two weeks, resistant plants were transferred into a liquid MS
solution containing B-estradiol. Samples were collected and subject to TLC analysis after
6 days. As shown in Figure 3.8A, 3 out of the 6 lines tested have accumulated TGDG
after B-estradiol induction for 6 days at a concentration as low as 2uM. Similarly, 2 out of
the 12 lines tested showed accumulation of triacylglyerols (TAG) (see Figure 3.88).
However, in the control wild-type Col-2 plant treated the same way, a faint band
migrated at the same position as TGDG was also observed, suggesting that the presence
of oligogalalctolipids might be due to stress condition, .e. g. hypoxia (seedlings immersed
under liquid solution for 6 days). Other ways of applying the inducer such as spraying the

surface of leaves or direct germination of seeds on inducer-containing MS medium did

106

 

F113...

not lead to positive results. Therefore, the actual effectiveness of this inducible RNAi
system is still under question. Compared with 358 promoter driven constitutive T 0031' ,
which leads to clear and unambiguous results, I conclude that in the case of T GD3,

constitutive RNAi works better.

107

TGD3 RNAi- -pER8
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Figure 3.8. Inducible silencing of TGD3 in Arabidopsis. The construct T GD3RNAi-
pER8 was introduced into Arabidopsis wild-type Col-2 background by A grobacterium-
mediated transformation. T2 seeds from independent transgenic lines were germinated on
the regular MS medium containing Hygromycin B. After two weeks, resistant plants were
transferred into a liquid MS solution containing 2 or SM B-estradiol. A, TGDG
phenotype of the seedlings after induction. B, TAG phenotype of the seedlings after

induction with ZuM B-estradiol.

108

 

11..

3.5. Discussion

The previously identified tgd3-1 T-DNA mutant is a much weaker allele than all
other tgd mutants in all aspects tested. The T GD3 mRNA level is only partially reduced
in the T-DNA line. Therefore, a dsRNA mediated gene silencing approach was used to
generate transgenic plants with more severely disrupted T GD3 gene function. Inverted
repeats of T GD3 fragment was introduced into wild-type Arabidopsis plants mediated by
Agrobacterium infiltration, driven by either a constitutive promoter or a chemical
inducible promoter. The results demonstrated that constitutive expression system works
more effectively than the chemical inducible system. In general, the tgd3-1 mutant
phenotype was further enhanced possibly due to dramatically decreased mRNA
expression. The transgenic TGD3RNAi plants have more heavily impaired root growth
and slower germination rate, similar fatty acid composition as compared to tgd3-I, all of
which are as expected.

However, there are also some results that are uninterpretable at this stage. For
example, TGD3RNAi plants have an opposite effect on DGDG and PG levels as
compared to the tgd3-1 mutant. DGDG content in these RNAi plants is slightly increased
whereas the PG content is greatly decreased (Figure 3.3). This might be due to low
efiiciency or specificity of RNA silencing mediated by dsRNA that possibly affects other
genes (see below).

Moreover, the growth phenotype of tgd3 plants also differs from tgd] and tgd2.
Previous studies showed that ~50% of the seeds from the homozygous tgd] -1 allele are
aborted during seed development. This abortion ratio can be further increased to ~90% in

the tgd] -I / T GD] -RNAi lines with the reduced tng-l mRNA level, suggesting that

109

hi. .__._J

 

TGD1 has an essential role in seed development (Xu et al. 2005). Similar to the tgd] -1
mutant, the tgd2-1 mutant also produced ~ 43% of aborted seeds (Awai et al. 2006).
However, both tgd3-1 and TGD3RNAi seeds are developed normally. No abortion of
seeds was observed.

Taken together, there are two possible explanations:

1) The mRNA level was still only partially reduced by dsRNA-mediated silencing.
Although semi-quantitative RT-PCR revealed almost complete disrupt of T GD3 (Figure
3.2C), it can only give an estimate. More accurate quantitative assays such as real-time
quantitative PCR or northern blot are required to demonstrate the actual abundance of
TGD3 mRNA. Therefore, it is possible that T GD3 expression is still leaky in T GD3i
transgenic plants and thus lead to weak phenotypes.

2) There is probably an additional ATPase associated with TGD1. The T GD3
gene was identified via a reverse genetics approach (Lu et al. 2007). The Arabidopsis
genome has 26 genes encoding small ATPases. Among those 26 genes, only nine have
chloroplast transit peptide as predicted by ChloroP (Emanuelsson et a1. 1999). We
analyzed seven T-DNA insertion lines (Alonso et al. 2003) available at the time for those
nine genes and identified Non-intrinsic ABC Protein 11 (NAPl 1) as the missing ATPase
associated with the TGD1/2 transporter. Later, NAP 11 was renamed T GD3 because the
mutant disrupted in NAP 11 accumulates TGDG.

However, recently STRING (Search Tool for the Retrieval of Interacting
Genes/Proteins) database is being developed for known and predicted protein interactions
(Jensen et al. 2009). In addition to TGD3, another ATPase NAP3 (Atl g67940) was found

to be associated with TGD1. The predicted interaction of NAP3 with TGD1 has a bit

110

score of 0.671, which follows that of TGD3 (NAPl 1) (a score of 0.868), indicating strong
confidence of this prediction.

NAP3 and NAPll proteins share a sequence similarity of ~34%. NAP3 does not
have a chloroplast targeting peptide, as predicted by ChloroP, a primary reason why it
was ruled out of initial consideration. However, based on the information from
SUBA( the Arabidopsis Subcellular Database), several other programs predict its
targeting to cytosol or Golgi apparatus whereas MS/MS data shows its localization to
plasma membrane or vacuole (Heazlewood et al. 2007). This indicates that no definite
localization of the NAP3 gene product is known so that the possibility of its association
with TGD1, which is a chloroplast targeted protein, is emerging. Unfortunately, no T-
DNA insertion lines are available which are disrupted in the NAP3 gene. In order to
demonstrate whether or not NAP3 could be another ATPase associated with TGD1/2
transporter, either antisense or RNA silencing approach would be required to generate
mutant alleles disrupted in the target genes and used for testing the TGDG lipid
phenotype. Hopefully, the study of this NAP3 protein would help us explain some of the

currently uninterpretable results for the 3SSzTGD3RNAi mutant plants.

11]

“‘1-.-"- "-1

 

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Guo, H. S., Fei, J. F., Xie, Q., and Chua, N. H. (2003) A chemical-regulated inducible ‘
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Heazlewood, J. L., Verboom, R. E., Tonti-Filippini, J., Small, 1., and Millar, A. H.
(2007) SUBA: The Arabidopsis subcellular database. Nucleic Acids Res. 35, D213-D218.

Heinz, E. and Roughan, P. G. (1983) Similarities and Differences in Lipid-Metabolism
of Chloroplasts Isolated from 18-3 and 16-3 Plants. Plant Physio]. 72, 273-279.

Jarvis, P., Dormann, P., Peto, C. A., Lutes, J., Benning, C., and Chory, J. (2000)
Galactolipid-deficiency and abnormal chloroplast development in the Arabidopsis MGD
Synthase 1 mutant. Proc. Natl. Acad. Sci. U. S. A. 97, 8175-8179.

Jensen, L. J ., Kuhn, M., Stark, M., Chaffron, S., Creevey, C., Muller, J., Doerks, T.,
Julien, P., Roth, A., Simonovic, M., Bork, P., and von Mering, C. (2009) STRING 8-a

global view on proteins and their functional interactions in 630 organisms. Nucleic Acids
Res. 37, D412-D416.

Lichtenthaler, H. K. (1987) Chlorophyll and carotenoids: pigments of photosynthetic
biomembranes. Meth. Enzynol. 148, 350-382.

Lu, B., Xu, C., Awai, K., Jones, A. D., and Benning, C. (2007) A small ATPase protein
of Arabidopsis, TGD3, involved in chloroplast lipid import. J. Biol. Chem. 282, 35945-
35953.

Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bio assays
with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

Rossak, M., Schiifer, A., Xu, N., Gage, D. A., and Benning, C. (1997) Accumulation of
sulfoquinovosyl-l-O-dihydroxyacetone in a sulfolipid- deficient mutant of Rhodobacter
sphaeroides inactivated in squ. Arch. Biochem. Biophys. 340, 219-230.

Roughan, P. G. and Slack, C. R. (1982) Cellular-Organization of Glycerolipid
Metabolism. Annu. Rev. Plant Physio]. Plant Mol. Bio]. 33, 97-132.

Xu, C., Fan, J., Froehlich, J. E., Awai, K., and Benning, C. (2005) Mutation of the
TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.
Plant Cell 17, 3094-3110.

Xu, C., Fan, J., Riekhof, W., Froehlich, J. E., and Benning, C. (2003) A permease-like
protein involved in ER to thylakoid lipid transfer in Arabidopsis. EMBO J. 22, 23 70-2379.

Zuo, J. R., Niu, Q. W., and Chua, N. H. (2000) An estrogen receptor-based
transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant
J. 24, 265-273.

113

 

Chapter 4

Biochemical analysis of the phosphatidic acid binding site in Arabidopsis TGD2
. 2
protein

 

2This work has been published: Lu, B. and Benning, C. (2009) A 25-amino acid
sequence of the Arabidopsis TGD2 protein is sufficient for specific binding of ‘
phosphatidic acid. J. Biol. Chem. 284(26): 17420-17427. Iperformed all the experiments
and wrote the first draft of the paper.

114

4.1. Abstract

Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the
biosynthesis of endoplasmic reticulum (ER) derived thylakoid lipids. TGD2 is proposed
to be the substrate binding protein of a presumed lipid transporter consisting of the TGD1
(permease) and TGD3 (ATPase) proteins. The TGD1, 2, and 3 proteins are localized in
the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-
terrninal membrane-spanning domain into the inner envelope membrane while the C-
terminal domain faces the inter-membrane space. It was previously shown that the C-
terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate. the PtdOH
binding site of TGD2 in detail, the C-terrninal domain of the TGD2 sequence lacking the
transit peptide and trans-membrane sequences was fused to the C-terminus of the
Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the
resulting DR-TGD2C fusion protein following production in E. coli. The DR-TGDZC
protein bound PtdOH with high specificity as demonstrated by membrane lipid-protein
overlay and liposome association assays. Internal deletion and truncation mutagenesis
identified a previously undescribed minimal 25 amino acid fragment in the C-terminal
domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-
mer were different from that of TGD2C suggesting that additional sequences of TGD2

providing the proper context for this 25-mer are needed for wild-type like PtdOH binding.

115

 

4.2. Introduction

Many plants including Arabidopsis have two parallel pathways of thylakoid lipid
biosynthesis involving enzymes at the inner plastid envelope or the endoplasmic
reticulum (ER), respectively (Roughan et al. 1980, Roughan and Slack 1982). In
Arabidopsis the two pathways contribute nearly equally to the bulk of
galactoglycerolipids (Browse et a]. 1986), which are the predominant lipids in thylakoid
membranes. Synthesis by the ER pathway involves the assembly of phosphatidic acid
(PtdOH) and phosphatidylcholine (PtdCho) at the ER from fatty acids synthesized in the
chloroplast. The current hypothesis suggests that PtdCho returns to the outer plastid
envelope, where it is converted to PtdOH by a phospolipase D (Benning 2008). The
TGD1, 2, and 3 proteins are postulated to be involved in the transfer of PtdOH from the
outer envelope membrane to the inside of the inner envelope membrane (Awai et a]. 2006,
Lu et a]. 2007, Xu et a]. 2003, Xu et a]. 2005), where PtdOH is dephosphorylated by a
PtdOH phosphatase to diacylglycerol, which is the precursor of galactoglycerolipid
biosynthesis at the inner chloroplast envelope membrane. Alternatively, nascent fatty
acids are assembled de novo at the chloroplast inner envelope into PtdOH, which then
enters galactoglycerolipid biosynthesis as described above. In either pathway, PtdOH is a
key intermediate for thylakoid lipid biosynthesis.

The TGD1, 2, and 3 proteins resemble the permease, substrate binding, and
ATPase subunits of a multipartite ABC transporter which is localized in the inner
chloroplast envelope (Awai et a]. 2006, Lu et al. 2007, Xu et a]. 2003, Xu et a]. 2005) .
Mutants with reduced function of these proteins have complex lipid phenotypes. They

accumulate diagnostic oligogalactolipids such as trigalactosyldiacylglycerol giving rise to

116

their name, and triacylglycerols in their leaves. Moreover, galactolipid molecular species
derived from the ER-pathway are underrepresented and pulse-chase-labeling experiments
indicate a reduced flux through the ER-pathway in the mutants.

TGD2 appears anchored with its N-terminal domain into the inner envelope
membrane such that the C-terminus faces the inter-membrane space (Awai et al. 2006) .
The C-terminus of TGD2 was shown to specifically bind PtdOH. The TGD2 protein does
not share sequence similarity with known PtdOH-binding proteins or domains. However,
it does contain a mycobactrial cell entry domain (MCE) required for mycobacterial entry
into host cells (Chitale eta]. 2001).

For the few cellular protein targets of PtdOH described thus far, no uniform
PtdOH binding motif has emerged making the reliable prediction of protein-PtdOH
interactions or PtdOH binding domains difficult. In mammalian cells, protein kinase Raf-
] (Ghosh et a]. 1996, Ghosh et a]. 2003), protein phosphatases SHP-l (Frank et a]. 1999)
and PPl (Jones and Hannun 2002), and protein kinase Cs (Lopez-Andreo eta]. 2003) are
among the best studied PtdOH binding proteins. In yeast, the SNARE protein Sp020p
(Nakanishi et a]. 2004) and the inositol-regulated transcriptional repressor Opilp
(Loewen et a]. 2004) are recognized as PtdOH binding proteins.

At this time, even fewer PtdOH-binding proteins have been identified in plants.
The PtdOH binding proteins ABIl (ABA insensitive 1) (Zhang et al. 2004) and PDKI
(phosphoinositide-dependent kinase 1) (Anthony et al. 2004) were only recently reported.
ABIl is a protein phosphatase 2C that negatively regulates signaling of the growth
regulator abscisic acid. Arabidopsis PDKl is a protein kinase that binds both PtdOH and

phosphoinositides (Deak et al. 1999), but its kinase is activated only by PtdOH (Anthony

117

 

et al. 2004). Additional PtdOH targets were isolated from Arabidopsis cell lysates
employing a PtdOH-analogue-affinity matrix followed by mass spectrometry analysis
(Testerink et a]. 2004). Phosphoenolpyruvate carboxylase was identified and shown to
bind preferentially PtdOH over other phospholipids (Testerink et a]. 2004). The TGD2
protein contains a new PtdOH binding site which we characterized and delineated as

described below.

4.3. Experimental procedures

4. 3. 1. Expression and purification of DR-TGD2 fusion proteins

All TGD2 truncated proteins used in this study were derived from the TGD2-
dTMD-pQE3l plasmid (also known as TGD2C-pQE31) using a PCR based
strategy(Awai et a]. 2006). Following digestion with Nco I and Xho I, the PCR fragment
was ligated into the DsRed-plel-His plasmid (provided by Dr. Michael Garavito,
Michigan State University, East Lansing) to produce Discosoma sp. red fluorescent
protein (DR)-TGD2C fusion proteins. Internal deletion mutants and/or point mutants
were generated by site-directed mutagenesis applied to TGD2C-DsRed-plw01-His using
a PCR-based protocol, with the primers and mutation sites listed in Supplemental table
4.1. All fusion proteins were expressed in the Escherichia coli strain, BL21 (DE3)
(Novagen, Madison, WI). An overnight pre-culture of LB medium (5 mL) was used to

start a 200 mL culture in LB medium. The protein was induced with 50 11M IPTG
(isopropyl-B-D-thiogalactopyranoside) at OD600 0.6-0.8, 16°C and growth was continued

overnight. Cultures were cooled to 4°C, washed twice and resuspended in lysis buffer (50

118

mM Tris-HCl, pH7.5, 300 mM NaCl, 10 mM imidazole). The cells were lysed by
sonication, followed by centrifugation at 18,000Xg. The resulting supernatant was applied
to a Ni-NTA agarose column (Qiagen, Valencia, CA). Proteins binding non-specifically
were washed off the column using lysis buffer containing 20 mM imidazole. The His-
tagged protein was then eluted with lysis buffer containing 250 mM imidazole. Samples
were concentrated and dialyzed into assay buffer (10 mM KHZPO4, pH7.4), using
Amicon centrifugal filter devices (Millipore, Billerica, MA). Protein concentration was
determined according to Bradford (Bradford 1976) using bovine serum albumin as a
standard. The fusion proteins were analyzed for purity by SDS-PAGE followed by

Coomassie Brilliant Blue staining (Laemmli 1970) and stored at 4°C for several weeks

without significant degradation or loss of lipid binding activity. The fusion proteins were
soluble and, typically, their purity was greater than 90%. The different DR-TGD2 fusion

proteins used in this study are listed in Supplemental table 4.1.

119

 

 

 

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4. 3. 2. Phylogenetic analysis of T GDZ

Full-length TGD2 amino acid sequences were searched against the National
Center for Biological Information (NCBI) non-redundant protein database using the
BLAST tool (Altschul et al. 1997) and the resulting sequences with high similarities and
identities were aligned using ClustalX software (version 2.0.10) (Larkin et al. 2007).
Generation of the bootstrapped phylogenetic tree was performed using the PHYLIP

software package as previously described (Mamedov et al. 2005).

4. 3. 3. Protein-lipid overlay assay

Membrane lipid strips were purchased from Echelon Biosciences (Salt Lake City,
UT). The strips were first blocked with 3% bovine serum albumin in TBST (lOmM Tris-
HCl, pH 8.0, 150 mM NaCl, and 0.25% Tween-20) for two hours and incubated in 0.5
ug/mL DR-TGD2 fusion protein solution in the blocking buffer at 4°C overnight. The
strips were washed 3 times, 10 min each with TBST and soaked in 3% bovine serum
albumin in TBST with a penta-His mouse monoclonal antibody (Sigma-Aldrich, St.
Louis, M0) at 1:2,000 dilution at 4°C overnight. The strips were washed twice with
TBST and soaked in 3% bovine serum albumin in TBST with horse radish peroxidase-
conjugated anti mouse antibody (Bio-Rad, Hercules, CA) at 1:20,000 dilution for an hour
at room temperature. Following washing with TBST for 1 hour, the protein was detected

by using a chemiluminescence detection system (Sigma-Aldrich, St. Louis, MO).

122

 

4. 3. 4. Liposome-association assay

The liposome association assay was performed according to (Sano et al. 1998).
Lipids (dioleoyl-phosphatidylcholine, or dioleoyl-phosphatidic acid) were dried under a
stream of nitrogen and incubated in TBS (50 mM Tris-HCl, pH 7, 0.1 M NaCl) at 37°C
for an hour followed by vigorous vortexing for 5 min. The liposomes were precipitated at
20,000Xg and washed twice with ice-cold TBS. Liposomes (250 ug) were mixed with

purified DR-TGDZ fission protein and TBS to make a final 100 uL solution. The mixture

was incubated at 30°C for 30 min and washed twice with ice-cold TBS by centrifugation

at 20,000X g at 4°C. The liposome pellet mixed with sample buffer was analyzed by SDS-

PAGE (Laemmli 1970). Immuno-detection of the His-tagged protein was accomplished
using the above mentioned penta-His antibody at 1:10,000 and the anti mouse antibody at
l:50,000 dilution. For visualization and quantification a chemiluminescence detection kit

from Sigma was used.

4. 3. 5. Quantitative analysis ofimmunoblots

Quantification and detection of signals on protein immunoblots were done by two
methods: 1) by film-based autoradiography followed by densitometry scanning. Distinct
protein bands were quantified using the computer software Multi Gauge V3.0 (Fujifilm
USA, Valhalla, NY), and 2) using a camera-based gel documentation system (a LAS-
3000, Fuji Film Life Science USA, Stamford, CT). The linear range for detection
extended over 4 orders of magnitude for the camera based system as compared to 1.5
orders of magnitude for the film-based system. Linearity of the response of the respective

detection system was established using a serial dilution of a test protein. Under the

123

conditions used, both detection systems were in the linear range of detection and gave
similar results during quantification of signals and were used interchangeably as
indicated. The data obtained by both methods were fit to a Hill equation modified for
ligand binding data (Ghosh et al. 1996, Orr and Newton 1992) using OriginPro8 (Origin
lab corporation, Northampton, MA). Relevant variables calculated by this method were k
indicating the PtdOH lipid fraction at half maximal binding and the Hill coefficient n

indicating cooperativity.

4.4. Results

4. 4. I. TGD2 orthologs in plants and C yanobacteria

The TGD2 gene encodes a 381 amino acid protein with a calculated molecular
mass of 41.6 kDa as summarized for At3g20320 at The Arabidopsis Information
Resource (http://www.arabidopsis.org). The TGD2 protein contains a mycobacterial cell
entry domain (MCE, amino acids 127-204), which is the only known conserved domain
in TGD2 and is found in surface proteins of pathogenic mycobacteria. These proteins are
essential virulence factors proposed to facilitate the bacterial entry into mammalian host
cells (Chitale et al. 2001). The molecular or biochemical functions of this domain, which
was strictly defined by conservation in different proteins, are not known. At the outset of
this study it was hypothesized that the MCE domain might play a role in mediating
PtdOH binding to TGD2. A trans-membrane-spanning domain (amino acids 96-118) and
a chloroplast targeting peptide (amino acids 1-45) in TGD2 were also predicted as shown

in Figure 4.1A. Putative TGD2 orthologs in plants, green algae and cyanobacteria (Figure

124

 

4.1, B and C) have conserved regions beyond the MCE domain (Figure 4.1B). None of
the conserved amino acid residues in the primary sequence resembled previously
described PtdOH binding motifs. Therefore, a systematic mutagenesis approach was

necessary to identify amino acids in TGD2 relevant to PtdOH binding.

125

 

Figure 4.1. Sequence analysis of TGD2. A, primary structure of TGD2 indicating a
predicted transit peptide (TP), transmembrane domain (TMD) and a conservative
mycobacterial cell entry (MCE) domain. B, an alignment of the TGD2 amino acid
sequence with its orthologs in plants and green algae is shown. The predicted TGD2
secondary structure is indicated above the sequence. Open boxes mark conserved
residues, and black boxes indicate identical residues. C, an unrooted tree showing the
relatedness of predicted TGD2 orthologs in plants, green algae and Cyanobacteria is
shown. Boot strapping values >950 are marked by +, those between 500 and 950 are
marked with a solid circle, and those under 500 are marked by open square. The gene
bank accession numbers for the included sequences in B and C are: Arabidopsis thaliana,
NP_566659.1; Vitis vinifera, CAN71395.1; Oryza sativa, EAY77419.1; Physcomitrella
patens, XP_001778862.1; Ostreococcus tauri, CAL53419.1; Chlamydomonas reinhardtii,
XP_001699315.1; Prochlorococcus marinus str. NA TLZA, YP_292846.1;
Prochlorococcus marinus str. MIT 9301, YP_00109053 7.1; Synechococcus sp. WH 5 701,
ZP__01083418.1; Synechococcus sp. CC9902, YP_376253.1; Synechococcus sp. JA-2-
3B'a(2-13), YP_477327.1; Anabaena variabilis, YP_323182.1; Nodularia spumigena,
ZP_01630545.1; Crocosphaera watsonii, ZP_00516249.1; Cyanothece sp. PCC 8801,
ZP_02940544.1; Microcystis aeruginosa, CAO90615.1; Acaryochloris marina,

YP_001516641.1; Thermosynechococcus elongatus, NP_683197.1.

126

 

 

    

 
 
  

 
 

 

 

 

 

 

 

 

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4. 4. 2. Specific binding of the DR-TGDZC WT fusion protein to PtdOH

A His-tagged version of the C-terminal domain of TGD2, TGD2C-His, was found
to specifically bind PtdOH(Awai et al. 2006). This truncated TGD2 protein was difficult
to solubilize and mutant forms were completely insoluble when the respective constructs
were expressed in E. coli. To overcome this problem, we fused the C-terminal TGD2
domain to the C—terminus of an engineered monomeric Discosoma sp. red fluorescent
protein (DR) (Bevis and Glick 2002). Contrary to maltose binding protein and
glutathione-S—transferase that were initially considered as fiJSiOIl partners for TGD2C, DR
did not show lipid binding by itself in the protein-lipid overlay or liposome lipid binding
assays that were employed in this study (see supplemental Figure 4.1 and Figure 4.2).
However, the soluble DR-TGDZC fusion protein specifically bound to PtdOH as
demonstrated in the protein-lipid overlay assay shown in Supplemental Figure 4.1,
confirming previous results on the TGD2C-His-tagged protein(Awai et a1. 2006).
Moreover, deletion and point mutant variants of the DR-TGDZC protein encoding

constructs generally expressed well in E. coli and the resulting proteins were soluble.

130

 

DR DR-TGDZC * "

LPtdOH— ’7 7 " ‘-S1P
LPtdCho— —Ptdlns(3,4)P2
PtdIns— —Ptd|ns(3,5)P2
PtdIns(3)P— ' —Ptdlns(4,5)F:2

PtdIns(4)P— ' -Ptd|r15(3,4.5)F§3

PtdIns(5)P— O -—PtdOH
PtdEtn_ r-PtdSer
PtdCho _. J _ rB'ank

5

Supplemental figure 4.1. Binding of DsRed-TGDZC WT fusion protein to PtdOH.

The TGD2 protein is N-terminally truncated lacking the TMD and C-terminally fused to

the Discosoma sp. red fluorescent protein (DsRed, DR) open reading frame. Fusion

protein was expressed and protein-lipid overlay assay was conducted with commercial

phospholipid-containing membrane strip. Lipids: LPtdOH, lysophosphatidic acid;

LPtdCho, lysophosphatidylcholine; PtdIns, phosphatidylinositol; PtdIns(3)P,

phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-

phosphate;Ptdlns(5)P, phosphatidylinositol S-phosphate; PtdEtn,

 

"JJiciiiauuiauliuc, PtdCho, phosphatidylcholine; SIP, Sphingosine l-phosphate;

PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,5)P2, phosphatidylinositol

3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3,

phosphatidylinositol 3,4,5-bisphosphate; PtdOH, phosphatidic acid; PtdSer,

phosphatidylserine.

131

Figure 4.2. Binding of the DR-TGDZC WT fusion protein to PtdOH as a function of
weight percent of PtdOH in a PtdOH/PtdCho mixture. A, qualitative analysis of DR-
TGDZC wild-type protein and DR (DsRed) binding to liposomes. Liposomes were
prepared from a mixture of dioleoyl-PtdOH and dioleoyl-PtdCho in which the weight
percent of PtdOH was varied from 0-100% (w/w), maintaining total lipid at 250 ug and
total protein at lug. Westem-blots detected by chemiluminescent kit are shown. B,
quantitative analysis of the association of DR-TGDZC wild-type protein to
PtdOH/PtdCho liposomes determined by direct fluorometry using a digital camera based
system (left). The values are plotted as a function of PtdOH concentration in the
liposomes (right). The data were fit to a modified Hill equation for receptor-ligand

binding.

132

 

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133

 

To obtain corroborating and more quantitative evidence for PtdOH binding, a 41 ~—
liposome binding assay was used. Purified recombinant proteins were incubated as
described under Experimental Procedures with liposomes of different lipid composition.
Following centrifugation, proteins bound to the liposomes were associated with the lipid
pellet, whereas non-binding proteins remained in the supernatant (Figure 4.2A). Because
lipids are not-soluble in water, PtdOH was mixed at different weight-per-weight ratios
with phosphatidylcholine (PtdCho) to determine the weight% fraction of PtdOH required
for binding to DR-TGDZC. This fraction provided an indication for the affinity of the
DR-TGD2C wild-type protein to PtdOH and allowed a quantitative comparison to mutant
versions of the TGD2C protein. The DR-TGD2C wild-type fusion protein was found to
bind liposomes consisting of PtdCho, PtdOH lipid mixtures depending on the fraction of
PtdOH (Figure 4.2A, left panels). On the contrary, DR alone was exclusively present in
the supernatant and did not bind to the liposomes (Figure 4.2A, right panels). At the
protein concentration tested (1 pg total protein), a significant increase in binding between
30-40% PtdOH was observed. In Figure 4.23, the blot was analyzed using a digital
camera system which shows linear responses over four orders of magnitude. The
individual signals were quantified, and the resulting data were plotted and fit to the Hill
equation modified for receptor-ligand binding (Ghosh et al. 1996, Orr and Newton 1992).
Because we established that the detection system itself was not saturated under the
conditions used (see Experimental Procedures), we interpreted the observed saturation as
true saturation of binding of the protein to the liposomes. When the data were fit to the
modified Hill plot, half maximal binding at 39.6% of PtdOH was apparent. Using the

film-based detection methods, a similar value was obtained (see below). This value is

134

comparable to the results obtained for RafC-PtdOH association (20 mol% PtdOH)(Ghosh
et al. 1996). From the binding plot, a Hill number of 5.7 was calculated, suggesting
positive cooperativity (Figure 4.2B). Again, this value is similar to that obtained for
RafC-PtdOH interaction (Hill number between 3.3 and 6.2) (Ghosh et al. 1996). It seems
likely that TGD2 is forming a homo-multimer showing cooperativity of PtdOH binding

among the involved C-terminal TGD2 domains.

4. 4. 3. Identification of T GDZC amino acid sequences necessary for PtdOH binding
Because most previously described amino acid sequences involved in PtdOH-
binding lack recognizable similarities at the primary sequence level (Munnik and
Testerink 2005), a deletion and truncation approach was used to systematically identify
sequences necessary for PtdOH binding by TGD2C. The described liposome binding
assay was sufficiently quantitative to compare PtdOH binding to different versions of
DR-TGDZC. Pure PtdOH liposomes were used which gave maximal binding of DR-
TGD2C (ref. Figure 4.2B). In parallel, a negative control was included using pure PtdCho
liposomes. To test for specificity of binding, liposomes containing 50% (w/w) PtdOH and
PtdCho were included as well. All DR-TGDZC mutant proteins described here were
soluble and more than 90% pure based on Coomassie Brilliant Blue protein gel staining
(see Supplemental figure 4.2). Schematic representations of the mutant proteins and the
respective binding data are shown in Figure 4.3. In an initial set, five truncated mutant
versions ranging in lengths from l30-to-180 amino acids across the length of the TGD2C
sequence (Figure 4.3, B and C) were tested for binding. All of the truncated proteins

(DR-TGD2C T1 -T5, Figure 4.3, B and C) showed significant PtdOH binding to pure

135

__F_

 

 

 

PtdOH liposomes, suggesting that a fragment from residues 221-to-250 common to these U
fragments might be involved in PtdOH binding.
In a second set of experiments, two internal deletion mutants probing the 221 -to-
250 sequence were generated (DR-TGD2C D1 and D2; Figure 4.3C) and tested for
PtdOH binding. Surprisingly, deleting the entire segment (residues 221-250, DR-TGDZC
D1; Figure 4.3C) did not affect binding, while deleting a smaller fragment within this
segment (residues 221-225, DR-TGDZC D2; Figure 4.3C) diminished binding of the
fusion protein to PtdOH. It seems possible that DR-TGD2C D1 with the larger deletion
folds just right to still reconstitute a nearby site, whereas the smaller deletion in DR-

TGD2C D2 does not allow reconstitution.

136

Supplemental figure 4.2. SDS—PAGE of the proteins used for liposome binding assay. lug of
total proteins were loaded each lane and stained with Coomassie Blue R-250. A, same
proteins as in Figure 4.3 B. B, same proteins as included in Figure 4.3C. C, same proteins
as in Figure 4.4A. D, same proteins as in Figure 4.4C. E, same proteins as in Figure 468.
Protein molecular weight markers are indicated where appropriate. All the proteins
including DsRed control show an additional weaker band underneath the band which is
identical to calculated mass, most likely due to alternative translation starting site or N-

terrninal truncation, since both bands can be detected by anti-His antibody.

137

 

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138

Figure 4.3. Identification of a PtdOH binding domain in TGD2C. A, primary
structure of TGD2 indicating a predicted transit peptide (TP), transmembrane domain
(TMD) and a conservative mycobacterial cell entry (MCE) domain. B,C series of deletion
and truncation mutants were generated for TGD2C and fused to the C-terminus of the DR
open reading frame. The black oval represents the DR (DsRed) carrier protein, grey bars
represent deletions. Liposome-association assays were performed to assess binding of
various mutants to PtdCho, PtdOH/PtdCho mixtures or PtdOH liposomes. Westem—blots
detected by chemiluminescent kit are shown. PtdOH-specific binding data are
summarized on the right based on visually examined signal intensity (+++++, ++++, +++,

++, +, indicate decreasing signal intensity, — no binding).

139

 

A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

145 96—118 127-204 381
E] |TMD| MCE |
DsRed (DR) . '
DR-TGDZCWT(119-381) .: I +++++
DR-TGDZC T1(119—250) ++
DR-TGDZC T2 (171—300) . I +++
DR-TGDZC T3 (221—350) F fl ++
DR-TGDZC T4 (201-381) . fl ++++
100%PtdCho 50%PtdCho+50%PtdOH 100%PtdOH
DR wr T1 T2 T3 T4 DRWT T1 T2 T3 T4 DR WT T1 T2 T3 T4 kDa
’ -64
-
-37
—26
DR-TGDZC T5(119-300) . l +++++
DR-TGDZCD1(A221-250)I. Stiff: J *H
DR-TGDZC 02 (4221-225). 5 J +
100%PtdCho 50%PtdCho+50%PtdOH 100%PtdOH
DR WTT5 D1D2 DRWT T5 D1D2 DRWT T5 01 oz
. 3 kDa
‘_64
- - - - ---.- _49
' —37
—-26

140

 

4. 4. 4. A TGDZ minimal domain sufficient for PtdOH binding

Coarse mapping described above suggested peptide sequences overlapping or
surrounding residues 221-225 as a major contributing factor to PtdOH binding by TGD2.
Fine mapping was initiated from residue 225 in both directions starting with the fragment
containing residues 119-225 which fused to DR (DR-TGD2C T6; Figure 4.4A). This
fragment tested positive for PtdOH binding with liposomes. Truncations were made from
the N-terminus of DR-TGD2C T6 to narrow down this PtdOH binding site in the TGD2
protein. The two truncation clones DR-TGD2C T7 and T8 still showed binding to
PtdOH (Figure 4.4A). Apparently, the clones DR-TGD2C T6-T8 have a 25 amino acid
peptide (residues 201—225) in common that is sufficient to mediate binding to PtdOH.
Specificity of PtdOH binding by the minimal fragment DR-TGDZC T8 was confirmed
using the protein-lipid overlay assay applied to a membrane containing multiple lipids
(Figure 4.48).

Probing from TGD2 amino acid 221 towards the C-terminus, a fragment
consisting of residues 221-250 (DR-TGD2C T9; Figure 4.4A) did not show binding of
PtdOH when fused to DR, indicating that this sequence might not play a direct role in
PtdOH binding. This result was consistent with the previous observation that DR-TGD2C

D1 lacking this specific fragment still binds PtdOH (Figure 4.3C).

141

 

Figure 4.4. Fine mapping of a TGD2 minimal PtdOH binding sequence. A, truncation
mutants fused to DR (DsRed) focusing on the PtdOH binding domain. PtdOH binding
activity was assessed by liposome-association assay. B, specificity of the minimal PtdOH
binding domain fused to DR was verified by protein-lipid overlay assay using a
phospholipid-containing membrane strip. Lipids: LPtdOH, lysophosphatidic acid;
LPtdCho, lysophosphatidylcholine; PtdIns, phosphatidylinositol; PtdIns(3)P,
phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-
phosphate;Ptdlns(5)P, phosphatidylinositol S-phosphate; PtdEtn,
phosphatidylethanolamine; PtdCho, phosphatidylcholine; SIP, Sphingosine l-phosphate;
PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,5)P2, phosphatidylinositol
3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3,
phosphatidylinositol 3,4,5-bisphosphate; PtdOH, phosphatidic acid; PtdSer,
phosphatidylserine. C, PtdOH binding of point mutations of the minimal PtdOH binding
domain fused to DR shown by liposome-association assay with 100% PtdOH liposomes.
A section (residues 201-225) of the same sequence alignment as in Figure 4.1 was shown.
Point mutations in the sequence are indicated by arrows. P1, DR-TGDZC T8 R204A; P2,
DR-TGDZC T8 N205A; P3, DR-TGD2C T8 E209A; P4, DR-TGD2C T8 H216A; P5,
DR-TGDZC T8 E218A; P6, DR-TGD2C T8 C219A; P7, DR-TGD2C T8 K221A; P8,

DR-TGDZC T8 E222A.

142

 

A

DR-TGDZC T6 (119-225) .
DR-TGDZC T7 (171-225)

J
DR-TGDZC T8 (201-225) I .2

DR-TGDZC T9 (221-250)

100% PtdCho 50% PtdCho+50%PtdOH 1 00% Ptd OH
DR WT T6 T7 T8 T9 DR WTT6 T7 T8 T9 DRWTT6 T7 T8 T9

 

 

 

 
   

   
  

kDa
I. -64
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—26
DR-TGDZC T8
. ‘ 4
LPtdOH— /"' —S1P
LPtdCho- .—Ptdlns(3,4)P2
PtdIns- —Ptdlns(3.5)P2
Ptd|n5(3)P-' , —Ptd|ns(4.5)P2
PtdIns(4)P-. -Ptd|ns(3.4.5)P3
PtdIns(5)P- . . . —PtdOH
Ptd Etn — - PtdSer
PtdCho- 1._ ’B'ank
C P1 P2 P3 P4 p5 P6 p7 P8
219 229
TGD2 IMPRNPPPIEPTSVGLHPE G EIGL
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143

 

In the few cases in which PtdOH binding sites have been analyzed in detail, basic
amino acids or tryptophan were found to be critical for PtdOH binding (Munnik and
Testerink 2005). In particular, a recent study suggested that electrostatic interactions of
PtdOH with basic amino acids (i.e. lysine and arginine), along with hydrogen bond
interactions, form the basis of the specific binding of PtdOH to its cognate targets
(Kooijman et al. 2007). Similarly, we set out to map residues critical to PtdOH binding
within the identified minimal PtdOH binding region of TGD2C (residues 201-225).
Based on sequence similarity of TGD2 to its most closely related orthologs in plants and

green algae, several charged or conserved amino acids were targeted for mutational

analysis. In every case these amino acids were changed to alanine to simplify the analysis.

Point mutant versions of DR-TGD2C T8 are shown in Figure 4.4C and were tested for
PtdOH binding using pure PtdOH liposomes. Of all the point mutants analyzed only the
substitution in DR-TGDZC T8 K221A drastically reduced PtdOH binding. No detectable
binding to pure PtdCho liposomes was observed for any of the point mutants (data not
shown). Taken together, these data suggest that TGD2 residues 201-225 represent a not
previously described PtdOH binding domain, which is localized near but not in the MCE
domain. Lysine 221, a positively charge amino acid at physiological conditions seems to
be a critical residue within this PtdOH binding domain that might be required for

electrostatic interactions with the negatively charged phosphoryl group of PtdOH.

4. 4. 5. PtdOH binding properties of the minimal PtdOH binding domain
Positive cooperativity of ligand binding to its target as observed for PtdOH

binding by DR-TGD2C (Figure 4.28) usually requires the interaction of multiple protein

144

subunits or protein domains. On the other hand, the minimal 25-amino acid PtdOH
binding domain represented by DR-TGDZC T8 (residues 201-225) was hardly expected
to show this cooperativity. To test this hypothesis, we measured PtdOH binding to DR-
TGD2C T8 (see Figure 4.5, A and C) using PtdCho/PtdOH mixed liposomes and pure
lipid liposomes. For consistency, we analyzed DR-TGD2C in parallel as well (Figure
4.5B). In this experiment we used film-based detection and quantification of the Western
signal as described under Experimental Procedures and obtained very similar results for
DR-TGD2C (Figure 4.5, A and C; k=3 7.7%, n =2.5) to results observed for camera—based
detection (Figure 4.28; k =39.6%, n =5.7). Binding of DR-TGD2C T8 to PtdOH was
much weaker compared to DR-TGD2C to the extent that interpretations regarding

cooperativity were inconclusive (Figure 4.5C) and a fit to a Hill plot was not attempted.

145

 

Figure 4.5. Reduced affinity of the minimal PtdOH binding domain for PtdOH. A,
PtdOH binding for DR-TGDZC (wild type). B, PtdOH binding for DR-TGD2C T8
carrying the 201-to-225 residue minimal domain. C, quantification of relative binding of
PtdOH for DR-TGDZC and DR-TGDZC T8. D, PtdOH binding for DR-TGDZC T8. E,
PtdOH binding for DR-TGDZC D3 (DR-TGD2C with deletion of minimal PtdOH
binding domain residues 201-225). F, quantification of relative binding of PtdOH for
DR-TGDZC T8 and DR-TGD2C D3. Liposome association assays were performed. Data
shown in A, B, and C were obtain in the same representative experiment, likewise data

obtained in D, E, and F were obtained in the same representative experiment.

146

 

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147

We also constructed an additional DR-TGD2C D3 deletion mutant lacking only
residues 201-225 and compared it directly to DR-TGD2C T8 carrying only residues 201-
225 (Figure 4.5, D-F). The deletion mutant DR-TGDZC D3 still had residual binding
activity but less than observed for DR-TGDZC T8 carrying the identified 25 amino acid
minimal PtdOH binding domain. When the data were fit to a Hill plot, positive
cooperativity was indicated (Figure 4.5F; k = 53%, n = 8.2). Again, the analysis of DR-
TGD2C T8 gave data too noisy to draw conclusion regarding cooperativity (Figure 4.5F).
Taken together, these data suggest that TGD2C residues 201-225 are important to PtdOH
binding and sufficient by themselves. They might represent the core of a primary PtdOH
binding site, but in its absence a secondary weaker PtdOH binding site may be present, or
possibly additional residues participate in the formation of a genuine PtdOH binding site
in the native TGD2 protein.

To test the possibility for secondary PtdOH binding sites or residues participating
in PtdOH binding by TGD2, an additional series of mutants with truncated sequences
starting either from the C-terminus (residue 381) or from the center of the protein
(residue 204) were generated, fused to DR and tested in pure PtdOH liposome binding
assays as shown in Figure 4.6. Comparing the PtdOH binding activity of the different
truncation clones, three regions were identified between residues 161 and 204, 251 and
300, or 291 and 340 that could contribute to binding of PtdOH to TGD2 outside of the

core segment represented by residues 201-225.

148

 

 

 

A

145 96-118 127-204 381
[TP] ITMD“ MCE I I

B

DR-TGDZC T10 (225-381) . l

 

 

DR-TGDZC T11(225-350) l

 

DR-TGDZC T12 (225300) J

oR-Toozc T13 (225250)

DR-TGDZC T14 (119204) 1

DR-TGDZC T15 (119-160)

DR-TGDZC T16 (291-381) r l

DR-TGDZC T17 (341-381) .:I

T10 T11 T12 T13 T14 T15 T16 T17

 

 

" - "" W”

Figure 4.6. Additional TGD2 sequences contributing to PtdOH binding. A, primary
structure of TGD2 indicating a predicted transit peptide (TP), transmembrane domain
(TMD) and a conserved mycobacterial cell entry (MCE) domain. B, internal deletion and
truncation mutants of TGD2C fused to the C-terminus of the DsRed protein (black oval).

Liposome-association assays were performed to assess binding of various mutants to

PtdOH liposomes.

149

 

4.5. Discussion

A crystal structure for TGD2 binding to its ligand PtdOH will ultimately be
required to map the three-dimensional relationships between TGD2 amino acid residues
and the ligand. In the absence of such a structure, systematic functional dissection of the
primary amino acid sequence offers a valuable opportunity and the next logical step to
learn more about this interaction. Previous genetic evidence has provided a solid in vivo
framework for the function of TGD2 as the possible substrate binding protein of an ABC-
type transporter hypothesized to be involved in ER-to-plastid lipid trafficking and
required for chloroplast development(Awai et al. 2006). Other proteins involved in this
process constitute the permease (TGD1) and ATPase (TGD3) of this presumed lipid
ABC-type transporter in the inner plastid envelope (Benning 2008). The evidence to date
that this system transports PtdOH is still indirect and is based on the observations that the
tgd] mutant of Arabidopsis accumulates PtdOH and that isolated tgd] plastids show
reduced incorporation of PtdOH into plastid lipids (Xu et al. 2005), and most
convincingly that TGD2 specifically binds PtdOH (Awai et al. 2006). TGD2 appears
anchored with a transmembrane domain into the inner envelope membrane, but a major
C—terminal domain presumably faces the intermembrane space (Awai et al. 2006). The
fact that the TGD2 wild-type protein is resistant to the protease trypsin, which penetrates
the outer envelope but not the inner envelope membrane of chloroplasts, but that a mutant
TGD2 protein and a green fluorescent protein fused to TGD2 are sensitive to the protease,
suggests that the TGD2 protein is in a trypsin-inaccessible location, possibly in a junction
between the inner and outer envelope membrane. Taken together, these observations let

us hypothesize that TGD2 might provide the substrate PtdOH to the TGD1-TGD3

150

complex in the inner envelope membrane by extracting PtdOH from the intermembrane
leaflet of the outer plastid envelope membrane. While this hypothesis is difficult to prove
at this time, one step towards a better understanding of TGD2 function is a delineation of
its actual PtdOH binding site and its mode of PtdOH binding. Important questions are
whether the PtdOH binding site of TGD2 is localized in the intermembrane space,
whether it is close to the predicted transmembrane domain and, therefore, close to the
inner plastid envelope membrane, or whether it is synonymous to the MCE domain, for
which a molecular function is not yet known, but which appears to be involved in
membrane penetration by mycobactrial pathogens (Chitale et al. 2001).

The initially encountered protein solubility issue was solved by fusing the
TGD2C protein lacking the predicted N-terminal membrane-spanning domain to an
engineered highly soluble monomeric DR carrier protein (Bevis and Glick 2002). It
should be noted that neither glutathione-S-transferase, nor maltose-binding protein were
useful for this purpose, because in our hands both carrier proteins showed significant
lipid binding above background on their own. However, DR does not bind lipids by itself.
We generally tested binding of PtdOH to the fusion proteins using two assays, a protein-
lipid overlay assay providing a measure for the specificity of binding and a liposome
binding assay providing a semi-quantitative assessment of the affinity of TGD2 or its
mutant derivatives for PtdOH. To avoid misinterpretation of the binding data, care was
taken to stay within the linear range of the assays. Baring the technical limitations of the
available approaches in mind, we were able to delineate a 25 amino acid stretch in the
TGD2 protein that was sufficient for PtdOH binding, even though with reduced affinity.

Upon generation of a point mutant (K221A) within the minimal domain, PtdOH binding

151

 

was diminished, identifying lysine 221 as a critical residue involved in this process. This
finding is consistent with other work proposing basic amino acids and/or tryptophan as
critical residues for PtdOH binding (Kooijman et al. 2007, Munnik and Testerink 2005).

In the reverse experiment, deleting residues 201-225 from TGD2C did not fully
abolish PtdOH binding activity (Figure 4.5, E and F) suggesting the presence of other,
secondary amino acids involved in PtdOH binding. The presence of six proline residues
in the identified primary binding site represented by TGD2 residues 201-225 suggests
that this minimal region forms a loop-strand fold lacking helical or B-strand structure as
indicated by secondary structure prediction (Figure 4.13). Additional elements are clearly
required to hold this loop in place and enable the TGD2 protein to bind PtdOH in a
cooperative manner as suggested by the DR-TGDZC binding data.

In general, PtdOH binding sites of different proteins lack conservation in their primary
structures (Munnik and Testerink 2005). Likewise, the observed TGD2 PtdOH binding
site does not follow an established pattern. Presumed orthologs of TGD2 (Figure 4.1)
show more similarity in sequences outside the identified minimal PtdOH binding site
than within it. N-terminally adjacent to the minimal PtdOH binding is the MCE domain, a
highly conserved feature initially found in mycobacterial cell surface proteins required
for entry of the pathogen into the host cell (Chitale et al. 2001). This study clearly shows
that the MCE domain is not required for PtdOH binding and must have other
functionalities that nevertheless could lead to cell membrane fusion or penetration as
would be necessary for mycobacterial entry into mammalian host cells. Whether the close
proximity of the two sequence features in TGD2 is necessary for the function of the

protein can only be speculated at this time. The central location of the identified binding

152

 

domain approximately ~100 residues C-tenninal of the predicted membrane-spanning
domain does not rule out the possibility that TGD2 could extract PtdOH out of the outer
envelope membrane assuming that it is anchored with its N-terminus into the inner
envelope membrane. How PtdOH would be formed at the outer envelope remains unclear.
However, the recent identification of the TGD4 protein involved in ER-to-plastid lipid
trafficking, which is the first TGD protein associated with the ER(Xu et al. 2008),
provides new avenues towards an understanding of PtdOH formation from ER-derived
precursors in predicted contact zones between the ER and the outer plastid envelope

membrane.

153

 

 

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Munnik, T. and Testerink, C. (2005) Phosphotidic acid: a multifunctional stress
signaling lipid in plants. Trends Plant Sci. 10, 368-375.

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of a SNARE protein by control of intracellular localization. Mol. Biol. Cell 15, 1802-
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Roughan, P. G. and Slack, C. R. (1982) Cellular organization of glycerolipid
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Sano, H., Kuroki, Y., Honma, T., Ogasawara, Y., Sohma, H., Voelker, D. R., and
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Testerink, C., Dekker, H. L., Lim, Z. Y., Johns, M. K., Holmes, A. B., Koster, C. G.,
Ktistakis, N. T., and Munnik, T. (2004) Isolation and identification of phosphatidic acid
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Xu, C., Fan, J., Cornish, A. J., and Benning, C. (2008) Lipid trafficking between the
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TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.
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Chapter 5

Conclusions and perspectives

157

 

 

5.1. Major Conclusions

This dissertation focused on the study of TGD3 and TGD2 protein, the ATPase
component and the substrate binding protein of a proposed bacterial type ABC
transporter, respectively. The TGD3 gene was isolated by a reverse genetics approach.
Arabidopsis plants with a T-DNA inserted in the 5’-UTR region have reduced TGD3
mRNA expression levels and phenotypes similar to that of tgd] -1 (Xu et al. 2003, Xu et
al. 2005), including retarded growth, accumulation of trigalactosyldiaeylglycerol (TGDG)
and triacylglycerol (TAG) (Lu et al. 2007). By using a double stranded RNA mediated
RNA silencing technique, the TGD3 mRNA level was further reduced and the phenotype
was enhanced (see chapter 3). However, the fact that the resulting TGD3i plants still have
normal seed development in combination with bioinformatics data led to the speculation
that there might be a second ATPase associated with TGD1 that could partially substitute
for TGD3. Further experiments to test this hypothesis are still pending.

When fused with MBP, the TGD3 protein has basal ATPase activity in vitro.
Upon mutating the conserved phenylalanine to alanine, the ATPase activity was almost
abolished. Overexpression of this F94A mutant cDNA caused a dominant-negative effect
in the Col-2 wild-type background, supporting the hypothesis that TGD3 is involved in a
complex. One explanation is that the TGD3 F94A mutant protein is nonfunctional but
could still be part of its native complex thereby disrupting the overall function of the
complex.

The TGD2 protein binds specifically to phosphatidic acid (PtdOH) (Awai et al.
2006). In this study, the binding domain of TGD2 was narrowed down to a 25 amino acid

segment, which by itself is sufficient to mediate specific PtdOH binding of TGD2. The

158

l C -‘AW‘Z-J
. .-

 

 

binding property of this 25-mer is different from that of WT, suggesting that there might
be other sequences involved to fulfill complete and complex WT-like binding. A crystal
structure of TGD2 is ultimately required to demonstrate the three dimensional

relationship between TGD2 and its ligand.

5.2. Remaining questions and future directions

Lipid transfer into and out of plastids is extensive as tissues expand. The
discovery of the TGD proteins provides new insights into lipid—trafficking phenomena
involving plastids, particularly the import of lipid precursors from the ER during
chloroplast biogenesis. However, at this time there are still some important issues that
need to be addressed. For example, no direct interaction among the three TGD proteins
has been demonstrated, the transporter has not been reconstituted in vitro, and its
substrate specificity has not yet been determined.

Meanwhile, additional components involved in the ER that facilitate the ER-
chloroplast outer envelope membrane contact sites and lipid import into the plastid
remain to be discovered, as some evidence suggests that such proteins must exist (Xu et
al. 2008). Hopefully, the availability of the extensive genomic resources for plants and
photosynthetic bacteria will accelerate the discovery process in the near future.

I will discuss below remaining questions in the study of TGD proteins and

propose some future directions as well.

159

5. 2. 1 Demonstration of direct interaction of the three TGD proteins

The TGD proteins are hypothesized to form a bacterial type ABC transporter.
This is based on the nearly identical lipid phenotypes of the respective Arabidopsis
mutants with disrupted TGD protein function and the fact that their bacterial orthologs
are often organized in an operon. However, direct proof that the proteins interact with
each other is lacking.

Polyclonal antibodies against individual TGD proteins were generated. Further
purification and characterization of the antibodies are needed to test their efficiency and
specificity. Upon availability of specific antibodies, co-immunoprecipitation experiments
should be conducted according to the methods described in ref (Pandey and Assmann
2004). Total proteins will have to be extracted from HA-tagged TGD1 plants. The TGD1
protein will be pulled down by anti—HA antibodies and protein A-Sepharose. TGD1
and/or the putative complex will be extracted and solubilized by nonionic detergents such
as Triton X-100, Nonidet P-40, and CHAPS. Protein immunoprecipitated with anti-HA
antibodies will be probed with anti-TGD2 (Awai et al. 2006) or anti-TGD3 antibodies
(Lu et al., unpublished) by immunoblotting. TGD2 and/or TGD3 proteins are expected in
the complex, confirming that TGD1 is indeed associated with the TGD3 and/or TGD2
proteins.

Additionally, the purified complex could be submitted to the Mass spectrometry
facility of MSU and explore all the components of the complex. This will help unveil
additional components involved in lipid transport between the ER and the chloroplasts
that might be associated with the currently known TGD proteins. The potential sites of

protein-protein interactions between TGD1 protein and other proteins can be explored by

160

 

using homobifunctional amino group-specific chemical cross-linkers such as
dimethyladipimidate (DMA), dimethylsuberimidate (DMS) and bis (sulfosuccinimidyl)
suberate (BS). The reactive residues will be identified by matrix-assisted laser
desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic
peptides. Such an approach has been successfully used to map protein—protein
interactions between MutL and MutH (components of an E. coli DNA repair system)
(Giron-Monzon et al. 2004) and interactions between UreD, UreF and Urease from

Klebsiella aerogenes (Chang et al. 2004).

5. 2. 2. Investigation of the function of MCE domain in TGD2

The amino acid sequence of the TGD2 protein between residues 127-204 has
similarity to the MCE (mammalian cell entry) related proteins (Marchler-Bauer et al.
2009). These proteins are from Mycobacterium tuberculosis and found to be necessary
for colonization of, and survival within macrophages (Arruda et al. 1993, Flesselles et al.
1999)

Previously, the MCE gene in the Mycobacterium tuberculosis genome was cloned
and the protein product promoted uptake into Hela cells of polystyrene latex
microspheres coated with the protein (Chitale et al. 2001). Although the exact function of
the MCE protein is still unknown, it might serve as a membrane interacting domain on
the surface of Mycobacterium tuberculosis that is capable of eliciting plasma membrane
perturbations in non-phagocytic mammalian cells.

In-depth biochemical analysis revealed that the MCE domain in TGD2 appears to

be not critical for PtdOH binding (Lu and Benning 2009). However, the identified

161

 

 

minimal PtdOH binding domain (residue 201-225) of TGD2 is just outside of the MCE
domain and even overlaps partially with it (see Chapter 4, Figure 4.4). Furthermore,
residuesl61-204, which are part of the predicted MCE domain, still might be involved in
PtdOH binding (see Chapter 4, Figure 4.6). Therefore, PtdOH binding by the MCE
domain itself needs to be tested. For that purpose, the MCE domain of TGD2 should be
cloned and expressed as a DsRed fusion protein and tested for specific PtdOH binding
similar as described in Chapter 4.

In addition, even if the MCE domain turns out to be not directly involved PtdOH
binding, there are still possibilities that the MCE domain has other functions such as
membrane fusion or penetration as that would be required by bacterial cell entry into
mammalian host cells. To test that, an uptake and internalization experiment using HeLa
cells and latex beads coated with DsRed-TGDZMCE domains according to previously
reported methods (El-Shazly et al. 2007) should be conducted.

Briefly, latex beads coated with purified recombinant DsRed-TGDZMCE protein
will be incubated with HeLa cell monolayer for varying lengths of time at 37°C. Cell
suspension will be collected and observed under fluorescence microscope with an
excitation at 556 nm and emission at 586 nm for DsRed-monomer red fluorescence.
Transmission electron microscopy will also be used as a second method to confirm the
uptake of beads coated with DsRed-TGDZMCE protein by HeLa cells. I expect that the
DsRed-TGDZMCE protein could also facilitate the uptake and internalization of latex
beads by HeLa cells, similar to McelA, Mce4A, and LprM/Mce3E (Chitale et al. 2001,
El-Shazly et al. 2007, Saini et al. 2008). The expected result would suggest a possible

function of TGD2 MCE domain in membrane perturbations and invaginations, which

162

 

 

might be required for the transport of lipids or other substrates across chloroplast L

envelopes in plants.

5.2.3 Reconstitution of T GD transporter and in vitro uptake assay

Previously, we hypothesized that TGD2 binds a lipid and contributes to the
substrate specificity of the transporter. This is based on the facts that TGD2 is similar to
the substrate binding protein of bacterial ABC transporters and that the Arabidopsis tgd2
mutant phenotype is consistent with an impairment in ER-to-plastid lipid trafficking.
Later, TGD2 was shown to bind PtdOH specifically, and the incorporation of PtdOH into
galactoglycerolipids was reduced by isolated tgd] chloroplasts, which is consistent with
PtdOH as the substrate for the proposed transporter complex (Awai et al. 2006, Xu et al.
2005).

However, PtdOH is usually maintained at very low levels and often serves as
precursor. It is also rapidly converted into other lipids in plants under normal conditions.
Therefore, we cannot rule out the possibilities that there are other lipids that are
transported by the proposed TGD complex. For example, recent rapid pulse-chase
labeling experiments with pea or rape seed leaves have shown that newly exported fatty
acids are first incorporated into PtdCho instead of PtdOH, presumably due to a very
active acyl editing mechanism (Bates et al. 2007, Williams et al. 2000). These results
suggest that the first glycerolipid formed outside the plastid from newly synthesized fatty
acids is PtdCho.

The recent discovery of TGD4, a novel Arabidopsis TGD protein, suggests that

TGD4 protein is possibly associated with the ER. It has been speculated that TGD4 is

163

directly involved in the formation of plastid associated microsomes (PLAMs) and is

 

required for the transfer of PtdCho to the outer plastid envelope (Xu et al. 2008).
Recombinant TGD4 protein has some PtdCho binding activity when expressed as a
maltose binding protein fusion (Xu et al., unpublished). Therefore, it is reasonable to
hypothesize that the TGD transporter system might be required for the transfer of PtdOH
formed at the outer chloroplast envelope from PtdCho to the inside of the inner envelope
membrane where the PtdOH phosphatase resides (Malherbe et al. 1992). This process
could produce diacyl glycerol for subsequent galactoglycerolipid biosynthesis (See
Chapter 1, Figure 1.3). For this hypothesis, one would expect a phospholipase D involved
in the conversion of PtdCho to PtdOH at the outside of the outer chloroplast envelope
membrane, for which the gene has not been identified.

It is extremely difficult to determine the substrates and substrate specificity of
lipid transporters because no reliable in vitro assays are available. Also, a functional
reconstitution of these membrane proteins involved is generally very challenging. The
latter issue poses an even greater hurdle for the analysis of multi-component transporters
assembled from different peptides, including the proposed TGD transporter. Recently, we
successfirlly generated a recombinant maltose binding protein-TGD1-TGD3 fusion
protein and purified it to homogeneity, which provides hope for functional reconstitution
of the transporter in vitro. Preliminary data suggested that this single MBP-TGD1-TGD3
fusion protein potentially has basal ATP hydrolysis activity (Z.Wang, B.Lu, C.Benning,
unpublished work), similar to the TGD3 protein alone. Experiments need to be conducted

in the near future to confirm the result and test whether the ATPase activity could be

164

 

stimulated in the presence of PtdOH, the proposed substrate of the transporter, or
inhibited by vanadate and other compounds.

In addition, purified fusion protein could be functionally reconstituted into
proteoliposomes and tested for its ability to translocate or transport fluorescent lipids.
This could be done by using a dithionite reduction assay in the presence or absence of
DsRed-TGD2 protein. Such a system has been successfully used to demonstrate the
function of the E. coli MsbA protein in a reconstituted system in vitro (Eckford and
Sharom 2008). MsbA functions as an ATP-dependent lipid translocase that transports
lipid A from the inner to the outer leaflet of the cytoplasmic membrane. By using a
fluorescence quenching assay, MsbA was shown to have ATP-dependent flippase activity

for several fluorescent NBD-lipids (Eckford and Sharom 2008)

5.3. Summary

Only a few PtdOH targets have so far been identified in plants. Moreover, most of
the PtdOH targets studied are involved in PtdOH signaling, including the first PtdOH
target ABIl(ABA insensitive 1) (Zhang et al. 2004) and PDKl (phosphoinositide-
dependent kinase 1) (Anthony et al. 2004). However, PtdOH can also serve as an
important metabolite involved in phospholipids biosynthesis and membrane remodeling.
For instance, in the model plant Arabidopsis thaliana, there are two parallel pathways
that contribute to the bulk synthesis of the predominant galactolipids in thylakoid
membrane (Roughan et al. 1980, Roughan and Slack 1982). In either pathway, PtdOH is

an important intermediate for the bulk of thylakoid lipid biosynthesis.

165

 

 

The TGD2 protein of Arabidopsis has been identified as a novel PtdOH target in
this described dissertation (See Chapter 4). However, together with TGD1 and TGD3,
these Arabidopsis proteins, which are involved in lipid transfer from the outer to the inner
chloroplast envelope membrane, need to be reconstituted and tested for transport activity
as well as substrate specificity.

How the TGD transport complex works still remains unclear. Our current
speculation is that the TGD2 protein seems critical to trigger the first step of import by
binding to the lipid substrates and thus modulates the activity of the TGD1, 3 core
transporter. With its N-terminus tethered into the inner chloroplast envelope, the TGD2
C-terminus could face the intermembrane space and even touch the outer envelope.
Therefore, TGD2 could extract PtdOH formed at the outer envelope and deliver it to
TGD1, which then acts as a flippase to mediate transport of PtdOH through the
membrane with the energy supplied by ATP hydrolysis catalyzed by TGD3. Two pieces
of evidence supported TGD2 localization in two-membrane contact zones: 1) TGD2
contains an MCE domain typically found in cell surface proteins that are needed by
mycobacteria cell entry into mammalian host cells (Chitale et al. 2001). Functional study
of the TGD2 MCE domain is currently under way. 2). It was observed that both the
TGD2-OPP fusion and the tgd2-1 mutant protein are sensitive to Trypsin treatment,
whereas the wild type TGD2 protein by itself was resistant (Awai et al. 2006). One
possible interpretation is that TGD2 is in a complex or a membrane contact domain
inaccessible to Trypsin.

In summary, whether or not this TGD complex is located in membrane contact

zones between the outer and inner envelopes and whether or not this complex transports

166

 

PtdOH from the outer to the inner envelope still remains an interesting and challenging
hypothesis, which will require substantial amount of work to test. If transport of PtdOH
by the TGD complex can be confirmed by future efforts, this will be the first PtdOH
transporter identified and will greatly advance our understanding of lipid transport and
chloroplast biogenesis.

On the other hand, the proposed TGD complex belongs to ABC transporters, one
of the largest and best conserved protein families found in all organisms (Moody et al.
2002, Pohl et al. 2005). Some ABC transporters involved in the export of precursors for
cuticle or wax biosynthesis in the plant epidermis are reported (Bird et al. 2007, Luo et al.
2007, Pighin et al. 2004, Ukitsu et al. 2007). Another well studied ABC transporter in
Arabidopsis, COMATOSE (CTS), PEROXISOMAL ABC TRANSPORTER1(PXA1) or
PEROXISOME DEFECTIVE3 (PED3), is localized in peroxisomes to import fatty acids
or acyl-CoAs for B-oxidation (Footitt et al. 2002, Hayashi et al. 2002, Zolman et al.
2001). However, all the currently identified ABC transporters in plants are a homodimer
encoded by a single peptide with a multi-membrane-spanning permease domain and an
ATP-binding cassette (ABC) domain. To date, the TGD1, 2, 3 complex is the only
Arabidopsis ABC transporter involved in ER-to-plastid lipid exchange that resembles a
bacterial-type multipartite ABC transporter (Awai et al. 2006, Lu et al. 2007, Xu et al.
2003, Xu et al. 2005).

Many bacteria have orthologs of the Arabidopsis TGD1, 2, 3 genes organized in
operons. For example, the TGD orthologs in Pseudomonas putida were proposed to form
a toluene efflux pump and thus required for toluene tolerance (Kieboom et al. 1998b,

Kieboom et al. 1998a, Kim et al. 1998). However, toluene has not yet been confirmed as

167

 

 

a substrate. It is possible that the bacterial orthologs are involved in lipid remodeling of - -

i
I:

the cell membrane in response to solvent stress. Hopefully, in-depth functional studies of
the TGD transporter will lead to a better overall understanding of ABC lipid transporters

and provide new insights into their bacterial orthologs.

168

 

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