A NOVEL TIP30 COMPLEX REGULATES ENDOCYTIC TRAFFICKING OF SIGNALING
RECEPTORS BY FACILITATING THE TRANSPORT OF VACULOLAR (H+)-ATPASES TO
EARLY ENDOSOMES
By
Chengliang Zhang

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Genetics
2010

ABSTRACT
A NOVEL TIP30 COMPLEX REGULATES ENDOCYTIC TRAFFICKING OF SIGNALING
RECEPTORS BY FACILITATING THE TRANSPORT OF VACULOLAR (H+)-ATPASES TO
EARLY ENDOSOMES
By
Chengliang Zhang
TIP30, a 30-kDa HIV-1 Tat-interacting protein, is a tumor suppressor whose expression is
altered in a variety of human cancers. Tip30-deficient mice spontaneously develop hepatocellular
carcinomas and other tumors as well as mammary preneoplasic lesions. However, the molecular
mechanism of TIP30 function remains largely unraveled.
In this study, we show that a novel protein complex consisting of TIP30, Endophilin B1
(Endo B1), acyl-CoA synthetase long-chain family member 4 (ACSL4) interacts with Rab5a,
and facilitates Rab5a recruitment to early endosomes by promoting efficient fusion between
Rab5a vesicles and endocytic vesicles. Rab5a vesicles are EEA1-negative vesicles carrying
vacuolar (H+)-ATPases (V-ATPases), an endosome acidification enzyme that causes ligandreceptor dissociation. Inhibition of TIP30, ACSL4 or Endo B1 impairs Rab5a vesicles loading on
early endosomes and causes the mislocalization of V-ATPases, leading to delayed EGF-EGFR
dissociation and prolonged EGFR signaling. Furthermore, we show that both arachidonic acid
and coenzyme A are essential for the fusion of Rab5a vesicles with endocytic vesicles in vitro.
TIP30, ACSL4 and Endo B1can promote vesicle fusion in the presence of arachidonic acid and
coenzyme A and can transfer the arachidonyl group to endosomal phosphatidic acid to produce
triacylglycerol, which induces membrane tethering and stacking. Together, these results identify
a novel function for Rab5a in endocytic trafficking and suggest a mechanism, in which addition

of the hydrophobic arachidonyl group to phosphatidic acid by the TIP30 protein complex may
bring membranes into close contact to allow for membrane fusion.
Supporting the above findings is the observation that Tip30 deletion dramatically accelerated
the onset of mammary tumors in the MMTV-Neu transgenic mouse model of breast cancer,
which overexpresses another EGFR family member, HER2/Neu. Similar to liver cells, deletion
of Tip30 in mouse mammary cells also caused the trapping of EGF-EGFR complexes in early
endosomes, thereby leading to delayed EGFR destruction and sustained EGFR signaling in
response to EGF treatment.
We further found that unlike tumors developing in MMTV-Neu mice, almost all of which are
estrogen receptor-negative and progesterone receptor-negative (ER-/PR-), tumors arising in
Tip30-/-/MMTV-Neu

mice

are

almost

exclusively

ER+/PR-

mammary

tumors.

Immunofluorescence studies showed that Tip30 is predominantly expressed in ER+ mammary
epithelial cells (MECs) and its deletion leads to an increase in the number of phospho-ER
positive cells in mammary glands and accelerated activation of Akt in MMTV-Neu mice.

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my mentor, Dr. Hua Xiao, for the continuous
support of my Ph.D. study and research. He has been motivating, encouraging, and enlightening.
I have been amazingly fortunate to have an advisor who gave me the freedom to explore on my
own and at the same time the guidance necessary for me to proceed through the doctorial
program and complete my dissertation.
I am very grateful to my guidance committee, Dr. Karen Friderici, Dr. Hans Cheng, Dr. Jerry
Dodgson and Dr. Richard Schwartz for their encouragement, insightful comments and helpful
suggestions concerning my research as well as my future career.
I am also thankful to all former and current members of the Xiao lab for their help and
support: George Chen, Dr. Isum Hoshino, Dr. Mikito Mori, Dr. David Ankrapp, Aimin Li, and
Shenglan Gao.
I want to thank Dr. Melinda Frame for her help with confocal microscopy, Dr. Alicia Pastor
for her help with Electron Microscopy, and Douglas Whitten in the Proteomics core facility for
his excellent work in protein identification.
I would like to acknowledge members in the Histopathology Lab: Amy S. Porter, Kathy A.
Joseph and Rick A. Rosebury.
Special thanks to my friends, Dr. Jianjun Luo, Dr. Bill Underwood, Dr. Yanfeng Zhang, Dr.
Xiaoli Gao, Dr. Young Nam Lee, for helping me get through the difficult times, and for their
help with my experiments.

iv

Thanks to Genetics Program and Plant Research Laboratory for offering me the opportunity
to study at Michigan State University and for the initial financial support. I thank all people who
work in Genetics Program and Department of Biomedical and Integrative Physiology for their
help and support throughout my study at MSU. I am especially grateful to Dr. Barbara Sears for
her various forms of support.
This thesis is dedicated to my parents and my wife for their never-ending love, concern,
support and strength all these years, which motivates me to work hard and do my best.

v

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................................... ix 
LIST OF FIGURES .........................................................................................................................x 
LIST OF ABBREVIATIONS ....................................................................................................... xii 
Chapter 1 Introduction ...................................................................................................................1 
1.1 Receptor-mediated endocytosis ...........................................................................................2 
1.1.1 Endosomes as a platform for receptor signaling .........................................................2 
1.1.2 Dissociation of receptor-ligand complexes .................................................................4 
1.1.3 Vacuolar (H+)-ATPases (V-ATPases) and the termination of receptor
endosomal signaling....................................................................................................5 
1.2 Vesicle membrane fusion .....................................................................................................6 
1.2.1 Fatty acids and vesicle membrane fusion ...................................................................6 
1.2.2 Role of SNARE and SM (Sec1/Munc18-like) proteins in membrane fusion .............9 
1.2.3 Endophilins and membrane fusion............................................................................11 
1.3 Rab GTPases in vesicle transport ......................................................................................11 
1.3.1 Rabs proteins are critical for vesicle transport ..........................................................12 
1.3.2 Rab5 is required for endocytic trafficking ................................................................14 
1.4 TIP30 is a tumor suppressor ..............................................................................................14 
1.5 Objectives ..........................................................................................................................17 
REFERENCES ........................................................................................................................18 
Chapter 2 Rab5a vesicles transport vacuolar (H+)-ATPases to early endosomes via
membrane fusion ..........................................................................................................31 
Abstract ....................................................................................................................................32 
Introduction ..............................................................................................................................33 
Results ......................................................................................................................................35 
TIP30 interacts with Rab5a, Endo B1 and ACSL4............................................................35 
Inhibition of TIP30, ACSL4 or Endo B1 leads to delayed EGFR degradation and
sustained EGFR signaling .........................................................................................46 
TIP30, ACSL4 and Endo B1 are involved in endocytic trafficking of EGF-EGFR..........47 
TIP30, ACSL4, and Endo B1 promote the recruitment of Rab5a to early endosomes .....53 
Rab5a vesicles transport V-ATPases to early endosomes .................................................54 
TIP30 complex and arachidonic acid promote fusion between endocytic and Rab5a
vesicles in vitro .........................................................................................................58 
Vesicle tethering and stacking induced by acylation of phosphatidic acid .......................63 
Discussion ................................................................................................................................68 
Function of TIP30, ACSL4 and Endo B1 in endocytic trafficking ...................................68 
A role for Rab5a in transport of endosomal targeting proteins .........................................69 
Lipids and membrane fusion ..............................................................................................70 
Materials and methods .............................................................................................................72 
vi

EGFR internalization and immunofluorescence ................................................................72 
Purification of endocytic and Rab5a vesicles ....................................................................73 
In vitro vesicle fusion assay ...............................................................................................73 
Lipid acylation ...................................................................................................................74 
Statistical analysis ..............................................................................................................74 
Acknowledgments....................................................................................................................74 
Supplemental materials and methods.......................................................................................89 
Cell culture, DNA constructs and shRNAs........................................................................89 
Immunoprecipitation ..........................................................................................................89 
Purification of recombinant proteins .................................................................................90 
Mouse hepatocyte isolation................................................................................................90 
MS/MS spectrometry analysis ...........................................................................................91 
REFERENCES ........................................................................................................................92 
Chapter 3 Tip30 Deletion in MMTV-Neu Mice Leads to the Development of Estrogen
Receptor-Positive and Progesterone Receptor-Negative Mammary Tumors via
Enhancing EGFR Signaling .........................................................................................99 
Abstract ..................................................................................................................................100 
Introduction ............................................................................................................................101 
Results ....................................................................................................................................103 
Tip30 deletion significantly accelerates mammary tumorigenesis in MMTV-Neu
mice. ........................................................................................................................103 
Deletion of Tip30 results in a shift from development of ER-/PR- tumors to ER+/PRtumors in the MMTV-Neu mouse model................................................................108 
Estrogen and progesterone promote growth of Neu+/Tip30-/- mammary tumors. ..........112 
Deletion of Tip30 leads to a progressively increased numbers of p-ER and p-Akt
positive cells in the mammary gland from MMTV-Neu mice. ..............................113 
Tip30 deletion leads to delayed EGFR degradation and sustained EGFR signaling. ......122 
Discussion ..............................................................................................................................123 
Materials and Methods ...........................................................................................................126 
Mice, primary MECs and tumor cells ..............................................................................126 
Immunofluorescence and immunohistochemistry ...........................................................127 
EGFR internalization assay..............................................................................................128 
Statistical Analysis ...........................................................................................................128 
Disclosure of Potential Conflicts of Interest ....................................................................128 
Acknowledgments..................................................................................................................128 
REFERENCES ......................................................................................................................130 
Chapter 4 Conclusions, Discussions and Future Directions .......................................................135 
4.1 EGF dissociates from EGFR and exits the early endosomes in membrane-bound
vesicles .............................................................................................................................136 
4.2 Rab5a vesicles deliver endosomal targeting proteins via membrane fusion ...................138 
4.3 TIP30, ACSL4 and Endo B1 form a protein complex that interacts with Rab5a and
mediates the fusion of Rab5a vesicles with endocytic vesicles .......................................140 
4.4 Arachidonic acid and the synthesis of arachidonyl-coA by ACSL4 is essential for
membrane fusion ..............................................................................................................141 
vii

4.5 Tip30 deletion in MMTV-Neu transgenic mice leads to the development of ER+/PRtumors ..............................................................................................................................142 
REFERENCES ......................................................................................................................144 

viii

LIST OF TABLES
Table 1.1 A summary of known functions of Rab proteins .......................................................... 13
Table 3.1 ER and PR staining in mammary tumors ................................................................ 106

ix

LIST OF FIGURES

Figure 1.1 The classic view of the endocytic pathway. ...................................................................3
Figure 1.2 Proposed mechanism of SNARE protein function in membrane fusion. .......................9
Figure 1.3 TIP30 localizes in the cytosol and inhibits the AKT signaling pathway. ....................16
Figure 2.1 TIP30 interacts with Rab5a, ACSL4, and Endo B1. ...................................................36
Figure 2.2 TIP30, Rab5a, ACSL4, and Endo B1 promote the endocytic downregulation of
EGFR. ..........................................................................................................................40
Figure 2.3 TIP30 and Rab5a depletion delays the exit of EGF from early endosomes. ...............48
Figure 2.4 TIP30, ACSL4, and Endo B1 are required for the localization of Rab5a to early
endosomes. ...................................................................................................................51
Figure 2.5 Rab5a vesicles transport V-ATPase to early endosomes. ............................................55
Figure 2.6 The TIP30 protein complex, arachidonic acid and coenzyme A promote the fusion
between endocytic and Rab5a vesicles. .......................................................................59
Figure 2.7 Acylation of phosphatidic acid promotes vesicle tethering and stacking.....................65
Supplemental Figure 2.8 Related to Figure 2.1, TIP30, ACSL4 and Endo B1forms a protein
complex that may function on the EGFR endocytic pathway. ....................................76
Supplemental Figure 2.9 Related to Figure 2.2, Knockdown of TIP30, Rab5a, ACSL4, and
Endo B1 in PLC/PRF/5 cells. ......................................................................................78
Supplemental Figure 2.10 Related to Figure 2.3 and 2.4, Deletion of TIP30, ACSL4, or Endo
B1 Delays EGF-EGFR dissociation and inhibits the recruitment of Rab5a to
endocytic vesicles ........................................................................................................79
Supplemental Figure 2.11 Related to Figure 2.5, Rab5a vesicles carry V-ATPases. ....................81
Supplemental Figure 2.12 Related to Figure 2.6, Examination of vesicle fusion using TEM
and the purification of recombinant proteins. ..............................................................83
Supplemental Figure 2.13 Related to Figure 2.6, Spectrometric analysis of PA derivatives. .......85

x

Figure 3.1 Tip30 deletion significantly accelerates the onset of mammary tumors in MMTVNeu mice. ...................................................................................................................104
Figure 3.2 Mammary tumors arising in Neu+/Tip30-/- mice are exclusively ER+/PR-...............106
Figure 3.3 Growth of ER+/PR- tumors arising in Neu+/Tip30-/- mice depends upon estrogen
and progesterone. .......................................................................................................109
Figure 3.4 Representative immunohistochemical staining of p-ERα in mammary glands and
mammary tumors from Neu+/Tip30-/- and Neu+/Tip30+/+ mice. ..............................114
Figure 3.5 Representative immunohistochemical staining for p-Akt in mammary tumors and
mammary glands from Neu+/Tip30-/- and Neu+/Tip30+/+ mice. ...............................116
Figure 3.6 Deletion of Tip30 in MECs leads to delayed EGFR degradation. .............................119
Figure 4.1 Function of the TIP30 complex ..................................................................................137
Figure 4.2 Phase separation of Rab5a in Triton X-114 ...............................................................139

xi

LIST OF ABBREVIATIONS

Abbreviation Description
ACSL4 Acyl-CoA synthetase long-chain family member 4
APPL Amyloid precursor protein-like
BSA Bovine serum albumin
C-terminus Carboxyl terminus
EDTA Ethylenediaminetetraacetic acid
EEA1 Early endosomal antigen 1
EGF Epidermal growth factor
EGFR EGF receptor
Endo B1 Endophilin B1
ER Endoplasmic reticulum
ER Estrogen receptor
EYFP Enhance yellow fluorescent protein
g gram
GDI GDP dissociation inhibitor
GTP Gunanosine 5’triphosphate
HA Hemagglutinin
HCC Hepatocellular carcinomas
HIV Human immunodeficiency virus
kDa Kilo Dalton
kDa Kilodalton

xii

Lamp Lysosomal-associated membrane protein
LDL Low-density lipoprotein
LDLR Low-density lipoprotein receptor
ml milli-liter
M Mol
MAPK Mitogen-activated protein kinase
MVB Multivesicular bodies
NSF NEM sensitive factor
N-terminus Amino-terminus
PA Phosphatidic acid
PBS Phosphate based buffer solution
pH potential Hydroxyl
PI Phosphoinositide
PI(4)P Phosphatidylinositide-4-phosphate
PLD Phospholipase D
Rab Ras like from brain
SCLC Small cell lung carcinoma
SDS Sodium dodecyl sulfate
shRNA Shot hairpin RNA
siRNA small interference RNA
SM Sec1/Munc18-like
SNAP Soluble NSF acceptor protein
SNAP-25 Synaptosome associated protein-25

xiii

SNARE SNAP receptor
TfR Transferrin receptor
TGF-α Transforming growth factor- α
TIP30 30-kDa HIV-1 Tat-interacting protein
VAMP Vesicle-associated membrane protein
V-ATPases Vacuolar (H+)-ATPases
Ypt Yeast protein transport
α alpha
α- anti
β beta
μ micro

xiv

Chapter 1 Introduction

1

1.1 Receptor-mediated endocytosis
Receptor-mediated endocytosis is utilized by eukaryotic cells to rapidly internalize nutrients
and other macromolecules and to reduce receptor number and signaling at the plasma membrane
(Figure 1.1). Upon binding ligands, internalized receptors are initially delivered to the sorting
station early endosomes, where they are either recycled back to the plasma membrane or
transported to late endosomes and lysosomes for degradation (Maxfield and McGraw, 2004; Seto
et al., 2002; Wiley, 2003).
1.1.1 Endosomes as a platform for receptor signaling
Although early endosomes have been long considered as a conduit for the degradation or
recycling of cell surface receptors, accumulating evidences shows that they are also an essential
site for signal transduction. Activated receptors in early endosomes can transmit signals that are
different from those that arise from the plasma membrane (Murphy et al., 2009b; von Zastrow
and Sorkin, 2007; Wang et al., 2002). For instance, phospholipase Cγ–1 (PLC-γ) is activated by
the epidermal growth factor receptor (EGFR) at the plasma membrane, whereas the activation of
the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the phosphoinositide 3-kinase
(PI3K) requires the normal endocytic trafficking of EGFR (Vieira et al., 1996). Other receptors,
such as the insulin receptor, the high-affinity nerve growth factor (NGF) receptor TrkA, plateletderived growth factor receptor (PDGFR), and the vascular endothelial growth factor receptor-2
(VEGFR2), also transmit signals to ERK1/2 and the PI3K/AKT pathway from endosomes
(Delcroix et al., 2003; Grimes et al., 1996; Kelly and Ruderman, 1993; Lampugnani et al., 2006;
Wang et al., 2004; Watson et al., 2001; Wu et al., 2001).

2

PM

EGF
Recycling endosome
pH 6.4-6.5
H+

EGFR
Clathrin and
adaptor proteins

H+

V-ATPase

H+
H+

Early endosome
pH 5.9-6.0

H+
Late endosome
pH 5.0-6.0

H+

H+

H+

H+

Lysosome
pH 5.0-5.5

Transporting
vesicles

H+

H+

TGN
H+

H+

Figure 1.1 The classic view of the endocytic pathway.
Upon ligand binding, receptors are rapidly internalized and enclosed in clathrin coated endocytic
vesicles, which fuse with early endosomes. Receptors dissociate from their ligands due to lower
pH in the early endosome and are further sorted into recycling endosomes for reuse or sent to
late endosomes and lysosomes for destruction. Drawing not to scale.
For interpretation of the references to color in this and all other figures, the reader is referred to
the electronic version of this dissertation.

3

The specific physiological significance of signaling from the endosome membrane remains a
challenging question. Given that endosome-based signals functionally distinct from those
emanating from the plasma membrane (Murphy et al., 2009b; Vieira et al., 1996; Watson et al.,
2001; Wu et al., 2001), endosomes may serve as a platform to assemble and activate certain
downstream signal transducers and to provide another layer of receptor signaling control.
1.1.2 Dissociation of receptor-ligand complexes
Both the recycling of unliganded receptors to the cell surface and the delivery of receptors to
lysosomes for degradation via multivesicular bodies (MVB) require the dissociation of receptorligand complexes. It is becoming increasingly clear that early endosomes host the dissociation of
many receptor-ligand complexes (Forgac, 2007; Maxfield and McGraw, 2004; Mellman, 1996a).
Dissociation of internalized insulin was shown to occur in the endosomes of rat hepatoma cells
(Backer et al., 1990; Murphy et al., 1984). Insulin receptors and other digestible molecules are
then transported to lysosome for degradation. Transporting receptors, such as the transferrin
receptor (TfR) and the low density lipoprotein receptor (LDLR), release their cargoes in early
endosomes, allowing for the recycling of receptors to the plasma membrane (Brown and
Goldstein, 1986; Yamashiro et al., 1984).
The endocytic trafficking of EGF-EGFR complexes was considered different from other
receptor-ligand complexes because earlier studies suggested that they traveled to lysosomes for
demolition as a unit in human foreskin fibroblasts (Carpenter and Cohen, 1976, 1979). However,
recent studies argue against this view by demonstrating that EGFR is inactivated before being
degraded and that EGF dissociates from EGFR prior to lysosomal transfer (Authier and Chauvet,
1999; Burke et al., 2001; Murphy et al., 2009b; Umebayashi et al., 2008). That signaling
4

receptors must be deactivated before being recycled or degraded is emerging as a common theme
during receptor-mediated endocytosis.
1.1.3 Vacuolar (H+)-ATPases (V-ATPases) and the termination of receptor endosomal
signaling
The V-ATPases are proton pumps that are central to pH homeostasis. They reside on the
membrane of many intracellular organelles, including endosomes, lysosomes and exocytotic
vesicles, and at the plasma membrane of some cells. Therefore, the V-ATPases play crucial roles
in various cellular processes, such as membrane trafficking, protein degradation, bone resorption
and sperm maturation, by regulating luminal acidification of diverse organelles and the acidity of
extracellular environment (Forgac, 2007; Marshansky and Futai, 2008).
Due to the action of V-ATPases, the lumen of endocytic vesicles becomes increasingly acidic
when going from early endosome (pH 5.9-6.0) to late endosome (pH = 5.0-6.0) and lysosomes
(pH 5.0-5.5) (Mellman et al., 1986). Acidic luminal pH created by V-ATPases contributes to the
dissociation of receptor-ligand complexes and the termination of receptor endosomal signaling in
early endosomes.
Despite the important roles played by V-ATPases on the endocytic pathway, little is known
about how these multi-subunit transmembrane complexes are transported to the early endosomes.
Proteins destined for residence at the membrane of endosomes are transported in membraneenclosed vesicles (Bonifacino and Glick, 2004; Palade, 1975). Therefore, it is reasonable to
assume that V-ATPases is transported by vesicles and that the loading of V-ATPases on the early
endosomes is mediated by vesicle membrane fusion.

5

1.2 Vesicle membrane fusion
Vesicles are constantly forming at the plasma membrane during endocytosis, and at the
endoplasmic reticulum (ER) and the Golgi during exocytosis (Griffiths and Simons, 1986;
Mellman, 1996a). They selectively fuse with recipient vesicles or the plasma membrane, thus
play a central role in the transport of molecules between a variety of specific membrane-enclosed
compartments.
Vesicle membrane fusion is the crucial step in all vesicular trafficking events and a
fundamental cellular activity for the transfer of proteins and lipids between different
compartments. Two types of events are considered essential for the fusion of a transport vesicle
with its target membrane. First, transport vesicles must recognize its specific target membranes.
Second, fusion must occur between transport vesicles and target membranes to accomplish the
transactions. It is believed that proteins are in charge of the mutual recognition between the
fusion partners and the initiation of membrane fusion. However, the merger of the two lipid
bilayers is the central event during membrane fusion. Two membranes must overcome two
dominant forces, a repulsive hydration force arising from water tightly bound to the hydrophilic
lipid headgroups and an attractive hydrophobic force between the hydrocarbon interiors of the
bilayers, to get closer enough to make fusion happen (Bonifacino and Glick, 2004; Burger, 2000;
McMahon et al., 2010).
1.2.1 Fatty acids and vesicle membrane fusion
The last two decades witnessed an explosion of knowledge on a number of factors involved
in vesicle membrane fusion, including proteins, fatty acids, acyl-CoA esters and membrane lipids.

6

Arachidonic acid, a long-chain polyunsaturated fatty acid (PUFA), has been demonstrated to be a
very effective fusogen. It can significantly promote Ca2+-triggered fusion of isolated chromaffin
granules (Creutz, 1981), endosome-endosome fusion (Mayorga et al., 1993) and GTP-dependent
fusion of microsomes (Paiement et al., 1994). Moreover, membrane-bound arachidonic acid can
drive annexin II-mediated membrane fusion of the lamellar body with the plasma membrane
during the exocytosis (Chattopadhyay et al., 2003).
How arachidonic acid promotes membrane fusion remains unclear. In neuronal cells,
arachidonic acids were found capable of removing the inhibitory Munc18 protein from syntaxin
in vitro, thus allowing for the formation of the soluble N-ethylmaleimide-sensitive factor
attachment protein receptor (SNARE) complexes, which are composed of vesicle-associated
membrane protein 2 (VAMP2), SNAP-25, and syntaxin1 (Rickman and Davletov, 2005).
However, a later in vitro study show that Munc18 remained attaching to syntaxin1 after
arachidonic acid stimulated formation of the SNARE complexes. The function of arachidonic
acids in SNARE complex formation need to be further investigated.
Fatty acyl-coenzyme A (CoA) esters are substrates for β-oxidation, for the synthesis and
remodeling of lipids, and for protein acylation reactions. One acyl-CoA, palmitoyl-CoA, was
shown indispensable for the budding of transport vesicles from Golgi cisternae and fusion of
transport vesicles with Golgi cisternae (Glick and Rothman, 1987; Pfanner et al., 1990b; Pfanner
et al., 1989). Vesicle transport was blocked by inhibitor of long-chain acyl-CoA synthetase and a
nonhydrolyzable analogue of palmitoyl-CoA, suggesting that fatty acid has to be activated by
CoA to stimulate transport and that the acyl group has to be transferred on other molecules.

7

Many of the proteins that mediate synaptic vesicle fusion and trafficking were indicated as
the recipients of the acyl groups (Huang and El-Husseini, 2005; Linder and Deschenes, 2007).
Palmitoyl groups covalently link to cysteine residues of synaptotagmin, α-SNAP (soluble Nethylmaleimide-sensitive-factor-attachment protein-α) and SNARE proteins Ykt6, VAMP and
SNAP-25 (synaptosome associated protein-25). Palmitoylation of these proteins was thought to
function in anchoring them to membranes or in their sorting to particular membrane
microdomains such as lipid rafts. Palmitoylation of Ykt6 has been suggested to provide a method
of regulating the rate of intracellular membrane flow and vesicle fusion in the secretory pathway
(Fukasawa et al., 2004). However, the significance of palmitoylation of these proteins in vesicle
membrane fusion is unclear.
Phosphatidic acid (PA) is another fusogenic lipid that plays important roles in vesicle
transport. It is proposed that PA, with a very small negatively charged headgroup, induces
negative membrane curvature at the inward membrane curve (Kooijman et al., 2003).
Phospholipase D (PLD) hydrolyzes membrane phosphatidylcholine to produce PA. The two
isoforms of PLDs, PLD1 and PLD2, are involved in vesicle trafficking during endocytosis and
exocytosis (Cazzolli et al., 2006; Donaldson, 2009). Depletion of PLD2 inhibited recycling of
transferrin receptors in HeLa cells (Padron et al., 2006). Endocytic trafficking and endosomal
signaling of EGFR are also regulated by PLD1 and its regulators, protein kinase Cα and RalA
(Shen et al., 2001). The role of PLD derived PA has been shown to be required for key
exocytotic processes in a variety of cells, including adipocytes (Huang et al., 2005a),
neuroendocrine cells (Humeau et al., 2001), mast cells (Peng and Beaven, 2005) and pancreatic
beta-cells (Hughes et al., 2004). These observations strongly suggest that lipid modifications are
essential for various vesicle membrane fusion events.
8

1.2.2 Role of SNARE and SM (Sec1/Munc18-like) proteins in membrane fusion
SNARE proteins are receptors for SNAP (soluble NSF attachment protein) and NSF (Nethylmaleimide-sensitive factor). They belong to a family of membrane-tethered coiled-coil
proteins that are required for vesicle membrane fusion. SNARE proteins have been shown to
mediate fusion of lipid bilayers in assays using reconstituted liposomes; therefore they are
considered the best candidates for the cellular fusogens. It was proposed that vesicle-associated
v-SNARE proteins syntaxin and SNAP-25 pair with cognate t-SNARE protein VAMP on the
target membrane to form four-helix bundle (SNAREpin) that brings lipid bilayers into close
proximity. The pairing starts at the N termini of the SNARE proteins and then proceeds in a
zipper-like manner towards the C-terminal trans-membrane regions (Figure 1.2). The resulting
mechanical force might overcome the energy barrier and bring the lipid bilayers close enough for
fusion to occur (Jahn and Scheller, 2006; Weber et al., 1998).

VM
v-SNARE

t-SNARE

TM

Figure 1.2 Proposed mechanism of SNARE protein function in membrane fusion. VM,
vesicle membrane; TM, target membrane. Drawing not to scale.
Fusion assays using in vitro reconstituted lipid bilayers have played a key role in supporting
the hypothesis that SNARE proteins are the minimal fusion machinery. It has been shown that
when synaptic vesicle membrane protein VAMP2, a v-SNARE protein, and two plasma
membrane t-SNARE proteins syntaxin1A and SNAP25 were reconstituted into phospholipids to
9

form donor and acceptor vesicles, respectively, they were sufficient to promote specific fusion
between the two types of vesicles (Weber et al., 1998). However, it is clear that in vivo many
proteins also play key roles in vesicle fusion apart from the SNARE proteins (McMahon et al.,
2010; Weber et al., 1998). In neuroendocrine cells, for instance, Munc-18 (mammalian
uncoordinated-18) protein, a member of the SM protein family, was shown to facilitate syntaxin
trafficking to the cell surface by interacting with syntaxin and preventing premature SNARE
complex formation between syntaxin and SNAP-25 (Medine et al., 2007).
Although plenty of information has been obtained using artificial membranes, studies also
indicate that artificial membrane fusion is far from being able to represent fusion between
biological membranes. Recently, endosome-endosome fusion was successfully mimicked using
reconstituted proteoliposomes with up to 17 recombinant proteins purified from bacteria (Ohya
et al., 2009). These proteins include Rab5, Rab5 effectors, SNARE proteins and SNARE
accessory factors, and other proteins that are currently known to be important for vesicle fusion.
They can promote fusion of proteoliposomes to physiologically meaningful rate; however, they
are not capable of promoting efficient fusion between biological intact endosomes, suggesting
that the SNARE proteins are not the fusogenic factors that are minimally required for membrane
fusion. In addition, high fusion rate was reached with reconstituted proteoliposomes utilizing
bacterially expressed proteins that essentially lack posttranslational modifications, whereas many
proteins that are involved in membrane fusion are posttranslationally modified in vivo, such as
the palmitoylation of synaptotagmin, α-SNAP and SNARE proteins Ykt6, VAMP and SNAP-25
(Fukasawa et al., 2004; Huang and El-Husseini, 2005; Linder and Deschenes, 2007), as well as
the isoprenylation of Rab5 (Kinsella and Maltese, 1991). This further indicates that fusion of
biological membrane has very different requirements than artificial lipid bilayers.
10

The data presented by Ohya et al also imply that some components critical to vesicle fusion
are missing in the in vitro assay system and that these components most likely function prior to
SNARE proteins (McMahon et al., 2010; Ohya et al., 2009). One important molecule that is
obviously not needed for artificial membrane fusion is arachidonic acid, which has been proven
to be essential for fusions between many kinds of vesicles, including endosome-endosome fusion
(Chattopadhyay et al., 2003; Creutz, 1981; Mayorga et al., 1993; Paiement et al., 1994).
1.2.3 Endophilins and membrane fusion
Endophilins are a group of proteins that contain an N-terminal amphipathic helix, a BAR
(Bin/Amphiphysin/Rvs) domain and a C-terminal SH3 domain. They play important roles in
endocytotic membrane trafficking. Endophilin is required for synaptic vesicle recycling in
Drosophila and in Caenorhabditis elegans (Guichet et al., 2002; Ringstad et al., 1999; Schuske
et al., 2003; Verstreken et al., 2002). In mammalian cells, endophilin is localized to synaptic
vesicles and is required for neurotransmitter release from endocytic vesicles (Gad et al., 2000;
Ringstad et al., 1999; Simpson et al., 1999).
Important mechanistic insights into endophilin functions have been gained from the studies
on the BAR domains, which are highly conserved in many proteins that involve in membrane
dynamics. The dimeric BAR domains of endophilins are banana shaped and can sense and bind
membrane curvature via its concave face to remodel liposomes structure (Powell, 2009). The
structural features of BAR domains and their ability to turn liposome into the “spaghetti-like”
tubular structures in vitro are well documented (Gallop et al., 2006; Masuda et al., 2006a; Wang
et al., 2008; Weissenhorn, 2005).
1.3 Rab GTPases in vesicle transport
11

Rab GTPases were originally identified as Ras-like from brain (Salminen and Novick, 1987).
They constitute the largest family of the Ras GTPase superfamily. The yeast S. cerevisiae
genome sequence encodes 11 Rab proteins, incuding Sec4p and the Ypt (Yeast protein transport)
proteins. Human cells contain approximately 70 Rab proteins that are localized to distinct
subcellular compartments and are key to membrane-trafficking events (Schwartz et al., 2007).
1.3.1 Rabs proteins are critical for vesicle transport
Transmembrane proteins that reside in the endoplasmic reticulum (ER), Golgi, endosomes,
lysosomes, and plasma membrane are synthesized on the rough ER and remain embedded in the
membrane as they move to their final destinations. These proteins are transported from one
membrane compartment to another by vesicles. It is well documented that Rab proteins regulate
discrete transport steps along the biosynthetic/secretory pathways, as well as the endocytic
pathways (Table 1).
Although many Rab proteins have been subjected to extensive studies, the precise function of
these proteins remained elusive. Rab proteins are anchored to the cytoplasmic face of all vesicles
involved in intracellular transport via prenyl groups covalently linked to two cysteines in the Cterminus. This special way of membrane anchorage makes Rab proteins the best candidates that
determine the identity of transporting vesicles and the specificity for their fusion with distinct
recipient membrane. Therefore, it was proposed that Rab proteins may act as identity tags for
distinct transporting vesicles and that they bring transporting vesicles to their specific recipient
membranes and tether those membranes by recruiting multitude of effectors (Grosshans et al.,
2006; Pfeffer, 2001; Takai et al., 2001; Zerial and McBride, 2001).

12

Table 1.1 A summary of known functions of Rab proteins
Name
Rab1, Rab2
Rab3
Rab3, Rab26,
Rab27, Rab37
Rab4, Rab35
Rab5
Rab5, Rab14,
Rab22
Rab6
Rab6, Rab33,
Rab7

Rab8, Rab10,
Rab14
Rab8, Rab11,
Rab17, Rab23
Rab9
Rab11, Rab15,
Rab17, Rab25
Rab13
Rab18
Rab22, Rab31
Rab23
Rab24, Rab33
Rab32
Rab32, Rab38

Implicated function
Transport of cargo between the ER and the Golgi apparatus (Griffiths
et al., 1994; Sannerud et al., 2006; Saraste et al., 1995; Tisdale et al.,
1992).
Synaptic vesicle exocytosis (Ng and Tang, 2008).
Various types of regulated exocytic events (Handley et al., 2007;
Masuda et al., 2000; Tsuboi and Fukuda, 2006; Yoshie et al., 2000).
Endosomal recycling back to the plasma membrane (Kouranti et al.,
2006; Sonnichsen et al., 2000).
Endosome fusion (Gorvel et al., 1991).
Maturation of early phagosomes (Duclos et al., 2000; Kyei et al.,
2006; Perskvist et al., 2002; Roberts et al., 2006).
Endosomes to the Golgi transport; Transport of secretory vesicles to
the plasma membrane (Grigoriev et al., 2007; White et al., 1999;
Young et al., 2005).
Intra-Golgi trafficking (Smith et al., 2007; Starr et al., 2010).
Endosomes and phagosomes maturation ; autophagy; melanosome
trafficking (Ceresa and Bahr, 2006; Feng et al., 1995; Gomez et al.,
2001; Gutierrez et al., 2004; Kinchen et al., 2008; Roberts et al., 2006;
Vitelli et al., 1997).
Constitutive biosynthetic trafficking from the trans-Golgi network
(TGN) to the plasma membrane (Huber et al., 1993; Junutula et al.,
2004; Schuck et al., 2007).
Ciliogenesis (Knodler et al., 2010; Yoshimura et al., 2007).
Trafficking from late endosomes to the TGN (Barbero et al., 2002).
Recycling from the TGN to the plasma membrane (Casanova et al.,
1999; Sonnichsen et al., 2000; Zacchi et al., 1998; Zuk and Elferink,
2000).
Endosomal recycling; junctional complex dynamics in epithelial cells
(Kohler et al., 2004; Marzesco et al., 2002).
Formation of lipid droplets (Martin et al., 2005).
Trafficking between the TGN and early endosomes (Kauppi et al.,
2002; Rodriguez-Gabin et al., 2001).
Ciliary trafficking (Boehlke et al., 2010).
Macroautophagy (Itoh et al., 2008; Munafo and Colombo, 2002).
Mitochondrial fission (Alto et al., 2002).
Biogenesis of melanosomes (Wasmeier et al., 2006).

13

1.3.2 Rab5 is required for endocytic trafficking
Rab5 is one of the best studied members of the Rab GTPases family. Functional studies
indicate that Rab5 is a key regulator of receptor-mediated endocytosis. Downregulation mediated
by siRNA against Rab5 inhibits the transition from early endosome to late endosome, leading to
delayed endocytic degradation of EGFR (Chen et al., 2009b). Rab5 is required for a form of
receptor tyrosine kinase induced actin remodeling and sub-compartmental organization and
sorting of early endosomes (de Renzis et al., 2002; Lanzetti et al., 2004). It is demonstrated that
Rab5 controls the endosomal release of amyloid precursor protein-like 1 (APPL1) and APPL2,
which then translocate to the nucleus where they activate a histone deacetylase (Miaczynska et
al., 2004a). More recently, Rab5 was identified as an essential regulator for phagosome
maturation (Kitano et al., 2008).
Ealier studies suggested that Rab5 is required for efficient fusion between biological
endosomes in vitro (Gorvel et al., 1991). A later study identified a Rab5 effector EEA1, whose
association with the endosomal membrane depends on Rab5 and phosphoinositide 3-kinase
(PI(3)K) activity (Simonsen et al., 1998). It was soon found that EEA1 can bypass Rab5 and
PI(3)K to promote endosomal tethering (Christoforidis et al., 1999). However, a recently study
showed that Rab5 and EEA can only increase endosomal fusion 3% and 10%, respectively
(Ohya et al., 2009), suggesting that both Rab5 and EEA are not capable of promoting significant
fusion between biological intact endosomes. This observation also leaves an important question
open again: what is the function of Rab5 on the endocytic pathways?
1.4 TIP30 is a tumor suppressor

14

TIP30, also called CC3 or HTATIP2, was initially identified as a metastasis suppressor by a
differential display analysis of mRNA from the highly metastatic human variant small cell lung
carcinoma (v-SCLC) versus less metastatic classic SCLC cell lines (Shtivelman, 1997). It was
independently isolated as an HIV-1 Tat-interacting protein that may enhance Tat-activated
transcription (Xiao et al., 1998). Tip30-deficient mice with C57BL6/J and 129SvJ mixed genetic
background spontaneously developed a spectrum of tumors, suggesting TIP30 as a novel tumor
suppressor (Ito et al., 2003). In the past several years, aberrant expression of TIP30 has been
associated with a variety of human cancers, including human liver (Ito et al., 2003; Lu et al.,
2008), lung (Tong et al., 2009), breast (Zhao et al., 2007), prostate (Singh et al., 2008; Zhang et
al., 2008), and gastric cancers (Li et al., 2009), as well as colorectal carcinoma (Chen et al.,
2010).
In mammary epithelial cells, TIP30 may function as a transcription repressor to inhibit ERαmediated c-myc transcription by interacting with ERα-interacting coactivator CIA (Jiang et al.,
2004a). Deletion of Tip30 in primary mammary epithelial cells leads to increased proliferation
and rapid immortalization relative to wild-type cells. Extensive ductal hyperplasia was observed
in Tip30-deficient mouse mammary glands with age (Pecha et al., 2007).
Approximately 30% of the tumors developed in Tip30-deficient mice are hepatocellular
carcinomas (HCC) (Ito et al., 2003). However, the mechanism of how TIP30 functions in liver
cells is largely unknown. It has been shown that TIP30 may upregulate other tumor

15

A

TIP30-EYFP

Mitotracker

overlay

B

TIP30
AKT-p473
AKT

Figure 1.3 TIP30 localizes in the cytosol and inhibits the AKT signaling pathway.
(A) The majority of TIP30 appear in the cytosol. Live human hepatocellular carcinoma Hep3B
cells expressing TIP30-EYFP (green) were imaged using LSM510 meta confocal microscope
after incubating with mitotracker (red).
(B) Ectopic expression of TIP30 inhibits the phosphorylation of AKT. Whole cell extracts were
prepared from human hepatocellular carcinoma HepG2 cells expressing TIP30HA or empty
vectors and were subjected to immunoblot analysis using the indicated antibodies.
suppressors such as p53 and E-cadherin and downregulate some oncoproteins such as N-cadherin
and c-MYC in HCC cells (Jiang et al., 2007; Zhao et al., 2006). Our preliminary results showed
that the majority of TIP30 was found in the cytoplasm (Figure1.3A) and that ectopic expression
of TIP30 inhibits the activation of the AKT signaling pathway in human HCC cells (Figure1.3B).
16

These observations link the TIP30 function directly with receptor signaling that originates from
the plasma membrane.
1.5 Objectives
The major objective of this study was to determine the molecular mechanism by which
TIP30 inhibits the AKT signaling pathway and to gain more insight into how TIP30 functions in
liver cells.
We performed co-immunoprecipitation to identify cellular proteins that interact with TIP30.
Three proteins, Rab5a, Endophilin B1 (Endo B1), acyl-CoA synthetase long-chain family
member 4 (ACSL4) have been found tightly associating with TIP30 to form a protein complex.
Since both Rab5a and Endo B1 are involved in receptor-mediated endocytosis, we examined
how these proteins regulate the endocytic trafficking of EGFR. Human liver cancer PLC/PRF/5
cells were subjected to EGFR internalization assay after knocking down the expression of each
of these genes with specific shRNAs. Compare to control cells, all cells with depleted TIP30,
Rab5a, Endo B1, or ACSL4 have much delayed EGFR degradation and sustained EGFR
signaling, suggesting that these proteins are required for EGFR endocytic trafficking. Further
studies revealed that these proteins facilitate the fusion of endocytic vesicles with Rab5a
vesicles, which is responsible for transporting vacuolar (H+)-ATPases (V-ATPases); thus are
required for the dissociation of EGF-EGFR complex in endosomes. Furthermore, in vitro
membrane fusion assay showed that in addition to the protein complex other molecules, such as
arachidonic acid, co-enzymeA and ATP are also required for the fusion of biological membranes.
Our membrane fusion assay using in vitro modified lipids suggest that the TIP30 complex may
promote membrane fusion through fatty acylating phosphatidic acid.
17

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30

Chapter 2 Rab5a vesicles transport vacuolar (H+)-ATPases to early endosomes via
membrane fusion

Chengliang Zhang, Aimin Li, Xinchun Zhang, and Hua Xiao
Submitted for publication

31

Abstract
Rab5a is a multifunctional GTPase crucial for endocytic trafficking. Here we show that a
novel protein complex consisting of TIP30, Endophilin B1 (Endo B1) and acyl-CoA synthetase
long-chain family member 4 (ACSL4) interacts with Rab5a and facilitates Rab5a localization to
early endosomes by promoting efficient fusion between Rab5a vesicles and endocytic vesicles.
Rab5a vesicles are EEA1-negative vesicles carrying vacuolar (H+)-ATPases (V-ATPases), an
endosome acidification enzyme that causes ligand-receptor dissociation. Inhibition of TIP30,
ACSL4 or Endo B1 impairs the fusion of Rab5a vesicles with early endosomes in response to
EGF, leading to delayed EGF-EGFR dissociation and prolonged EGFR signaling. Furthermore,
we show that TIP30, ACSL4 and Endo B1 act in concert to promote the fusion of Rab5a vesicles
with early endosomes via fatty acylating phosphatidic acid. Together, these results reveal a
critical role of TIP30, Endo B1 and ACSL4 in receptor-mediated endocytosis and suggest a
novel mechanism underlying membrane fusion.

32

Introduction
Receptor mediated endocytosis is a mechanism utilized by eukaryotic cells to rapidly take up
specific nutrients and reduce receptor signaling at the plasma membrane. Internalized ligandreceptor complexes are enclosed in early endosomes, also called sorting endosomes, where they
are either recycled or delivered to lysosomes for destruction (Maxfield and McGraw, 2004;
Mellman, 1996a). Signaling receptors continue to activate certain downstream pathways from
early endosomes (Miaczynska et al., 2004b; Murphy et al., 2009b; Polo and Di Fiore, 2006;
Sorkin and von Zastrow, 2009), until ligand-receptor complexes dissociate due to lower luminal
pH created by vacuolar V-ATPases, the major proton pump responsible for endosomal and
lysosomal acidification (Backer et al., 1990; Maxfield and McGraw, 2004; Nishi and Forgac,
2002; Yamashiro et al., 1984). Inactivation of V-ATPase blocks the transition from early to late
endosomes(Clague et al., 1994). Therefore, the proper endosomal targeting and activity of VATPases contribute to the tight regulation of both endocytic trafficking and receptor endosomal
signaling.
Membrane fusion is a cellular event at the center of intracellular organelle biogenesis and
membrane protein traffic. It generally requires cellular factors to bring donor and recipient
membranes into close proximity, to increase membrane curvature, and to disturb their lipid
bilayers (Martens and McMahon, 2008; McMahon et al., 2010). In addition to fusogenic lipids
such as acyl-CoA (Pfanner et al., 1990a; Pfanner et al., 1989), arachidonic acid (Chattopadhyay
et al., 2003; Mayorga et al., 1993), as well as phosphoinositides, phosphotidic acid and
diacylglycerol (Haucke and Di Paolo, 2007; Jun et al., 2004; Mima et al., 2008), many proteins
including Rab5, Rab5 effectors, SNARE proteins and SNARE accessory factors are necessary
for various membrane fusion events (Stenmark, 2009; Wickner and Schekman, 2008). Recently,
33

efficient endosome fusion was successfully mimicked using proteoliposomes reconstituted with
17 recombinant core fusion proteins in the absence of previously reported fusogenic lipids,
raising a critical question of whether fusogenic lipids or additional cellular factors are required
for efficient fusion between intact biological endosomes (McMahon et al., 2010; Ohya et al.,
2009).
One of the core fusion proteins is the small GTPase Rab5a, a membrane protein that
regulates early endosome fusion in vitro (Gorvel et al., 1991), motility of early endosomes on
microtubules (Nielsen et al., 1999), and the traffic between endosomes and lysosomes
(Rosenfeld et al., 2001). Deletion of Rab5a in cells inhibits the transport of EGFR from early
endosomes to lysosomes and consequently causes sustained EGFR signaling and delayed
degradation (Chen et al., 2009a). Despite its importance to endocytic transport, how Rab5a
reaches early endosomes and downregulates receptor signaling remains only partially
understood.
Here we show that Rab5a localizes to early endosomes through membrane fusion between
endocytic vesicles and Rab5a vesicles. A protein complex composed of TIP30, ACSL4, and
Endo B1 interacts with Rab5a and promotes efficient fusion of these two vesicles in vivo and in
vitro probably by acting in concert to transfer archidonyl group to PA. Furthermore, endosomal
targeting enzymes V-ATPases are carried to early endosomes by Rab5a vesicles and their proper
localization relys on Rab5a TIP30, ACSL4, and Endo B1. Thus, these studies identify a novel
function for Rab5a in endocytic trafficking and suggest a mechanism, in which addition of the
hydrophobic arachidonyl group to phosphatidic acid may bring membranes into close contact to
allow for membrane fusion.

34

Results
TIP30 interacts with Rab5a, Endo B1 and ACSL4
TIP30, also known as HTATIP2 or CC3 (Shtivelman, 1997; Xiao et al., 1998), is a tumor
suppressor that can act as a transcription cofactor to repress transcription in the nucleus (Jiang et
al., 2004b; Zhao et al., 2008a). However, TIP30 also localizes in the cytoplasm in which its
function is poorly understood (Ito et al., 2003; Lee et al., 2004; Tong et al., 2009; Zhao et al.,
2007). To identify cytosolic proteins that interact with TIP30, we stably expressed TIP30 protein
with an HA tag fused to its C-terminal end (TIP30-HA) in human hepatocellular carcinoma cells
(PLC/PRF/5). Co-immunoprecipitation (co-IP) assays were performed using whole cell extracts
generated from cells expressing TIP30-HA or control vector. Silver staining of the
immunoprecipitates resolved on SDS-PAGE revealed multiple major bands (Figure 2.1A). Mass
spectrometric analysis identified Rab5a, ACSL4, and EndoB1 (also known as Bif-1) in the
immunoprecipitates. Association of these proteins with TIP30-HA was confirmed by
immunoblot analysis using specific antibodies (Figure 2.1B). The interactions were further
confirmed by reciprocal IP. When Rab5a, Endo B1 or ACSL4 were HA-tagged, each could be
specifically coimmunoprecipitated with endogenous TIP30 and the other three endogenous
proteins (Figure 2.1C). Although Endo B1 failed to be immunoprecipitated with ACSL4-HA
possibly due to the interference of HA at its C-terminus, endogenous ACSL4 was detected in
Endo B1-HA immunoprecipitates. Notebly, Rab5a co-immunoprecipitated with the other
proteins in the presence of 0.2 mM EDTA, indicating that the interaction is independent of its
nucleotide binding status. Notebly, Rab5a co-immunoprecipitated with the other proteins in the
presence of 0.2 mM EDTA, indicating that the interaction is independent of its nucleotide
binding status.
35

Figure 2.1 TIP30 interacts with Rab5a, ACSL4, and Endo B1.
(A) Silver stain of TIP30 and its interacting proteins after co-immunoprecipitation (co-IP).
Whole cell extracts derived from cells expressing empty vector or TIP30-HA were
immunoprecipitated with α-HA agarose beads. The immunoprecipitates were eluted with HA
peptides, resolved on SDS-PAGE, and subjected to silver stain.
(B) Immunoblot confirmed the association of Rab5a, ACSL4, and Endo B1 with TIP30. The
immunoprecipitates were resolved on SDS-PAGE and analyzed by immunoblotting with the
indicated antibodies. The two polypeptides that reacted with anti-HA antibodies might result
from posttranslational modifications on TIP30. Input was 10% of the volume of lysates used in
the IP assays. The EGFR blot serves as a negative control.
(C) Reciprocal co-IP. Whole cell extracts made from cells expressing HA-Rab5a, ACSL4-HA,
Endo B1-HA, or empty vector were subjected to co-IP with α-HA agarose beads. The
immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. Input
was 10% of the volume of lysates used in the IP assays.
(D) BiFC analysis was performed on 293T cells by co-expressing TIP30-VC155 with VN173,
VN173-Rab5a, ACSL4-VN173, or Endo B1-VN173. Green indicates fluorescent signal from
Venus, a GFP variant. Bottom panels show overlays of DIC and representative confocal
microscope images. Scale bars, 10 μm.
(E) TIP30 and EGF-EGFR are colocalized in endosomes in response to EGF treatment. HepG2
cells co-expressing TIP30-CFP (green) and EGFR-DsRed (red) were treated with 10 ng/ml
Alexa647-EGF (blue) for 10 min and then analyzed by confocal microscopy. A typical image is
shown. Boxed areas are magnified. Scale bar, 10 μm.

36

Figure 2.1 Cont'd

A
kDa
250
150
100
75

ACSL4

50

Endo B1

37
TIP30-HA
Rab5a

25
20
15

TIP30HA

IP: α-HA
Vector

TIP30HA

Input

Vector

B

α-HA
α-Rab5a
α-ACSL4
α-Endo B1
α-EGFR

37

Figure 2.1 Cont'd

C
Input

IB: α-HA

IP: α-HA

kDa
80 -

α-Rab5a
α-ACSL4

45 -

α-Endo B1

25 -

α-TIP30

D
TIP30-VC155
+VN173

TIP30-VC155
+VN173-Rab5a

TIP30-VC155
+ACSL4-VN173

38

TIP30-VC155
+Endo B1-VN173

Figure 2.1 Cont'd

E
EGFR-DsRED
TIP30-CFP
EGF
overlay

39

Figure 2.2 TIP30, Rab5a, ACSL4, and Endo B1 promote the endocytic downregulation
of EGFR.
(A-E) Knockdown of TIP30, Rab5a, ACSL4, or Endo B1 results in delayed EGFR degradation
and sustained EGFR signaling. Control PLC/PRF/5 cells (A), TIP30 (B), Rab5a (C), Endo B1
(D), and ACSL4 (E) knockdown cells were collected at various time points after EGF
internalization and subjected to immunoblot analysis with the indicated antibodies. Results are
typical and representative of experiments on cells from two different shRNAs.
(F) Quantification of EGFR protein levels in (A-E) using Odyssey 2.1 software.
(G) Deletion of Tip30 in mouse primary hepatocytes leads to delayed EGFR degradation and
sustained EGFR signaling. Primary hepatocytes were isolated from wild type and Tip30-/- mice.
Endocytosis induced EGFR degradation was anylized as described in (A-E).
(H) Quantification of EGFR protein levels in (G) using Odyssey 2.1 software.

40

Figure 2.2 Cont'd

A

Control
hours

0 0.5 1 2

3

4

5

6

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

B

TIP30 knockdown
hours

0 0.5 1 2

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

41

3

4

5

6

Figure 2.2 Cont'd

C

Rab5a knockdown
hours

0 0.5 1 2

3

4 5 6

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

D

ACSL4 knockdown
hours

0 0.5 1 2

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

42

3

4 5 6

Figure 2.2 Cont'd

E

Endo B1 knockdown
hours

0 0.5 1 2

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

F

43

3

4 5 6

Figure 2.2 Cont'd

G

Tip30 -/-

Wild type
hours

0

0.5

1

2

EGFR-pY845
EGFR
AKT-pS473
AKT
β-actin

H

44

3

0

0.5

1

2

3

In addition, we observed that endogenous TIP30, ACSL4 and EndoB1 were co-sedimented at
a position between the 150 and 443 kDa marker proteins in a 15-35% glycerol gradient (Figure
S2.8A), and that bacterially expressed recombinant ACSL4 and Endo B1 were able to directly
bind purified baculovirus-expressed recombinant TIP30 (Figure S2.8B). These results indicate
that TIP30, ACSL4 and Endo B1 may form a protein complex to interact with Rab5a.
We next used bimolecular fluorescence complementation (BiFC) analysis (Hu et al., 2005) to
visualize the association of TIP30 with these proteins in living cells. We observed that
coexpressing TIP30-VC155 with VN173-Rab5a, ACSL4-VN173, or Endo B1-VN173 in cells
reconstituted fluorescence, whereas coexpression of TIP30-VC155 and control VN173 did not
(Figure 2.1D), indicating that TIP30 directly or indirectly interacts with these proteins in living
cells.
Rab5a colocalizes with EGFR in endosomes in response to EGF (Lakadamyali et al., 2006;
Leonard et al., 2008). To test if TIP30 is also targeted to endosomes, we coexpressed TIP30-CFP
and EGFR-DsRed fusion proteins and examined their localization in HepG2 cells lacking
detectable endogenous TIP30 and EGFR. Confocal microscopy analysis showed that TIP30-CFP
partially colocalized with EGF and EGFR-DsRed in endosomes 10 min after cells were treated
with Alexa-647 conjugated EGF (Figure 2.1E), suggesting that TIP30 was also targeted to
endosomes. Consistantly, immunostaining of endogenous TIP30 and EGFR revealed that TIP30
was partially localized to EGF-EGFR positive endosomes (Figure S2.8C). Taken together, our
protein-protein interaction and colocalization studies suggest that TIP30, ACSL4 and Endo B1
form a protein complex that interacts with Rab5a.

45

Inhibition of TIP30, ACSL4 or Endo B1 leads to delayed EGFR degradation and sustained
EGFR signaling
Knockdown of Rab5a expression in HeLa cells retards EGFR transport from early
endosomes to late endosomes and delays the degradation of EGFR (Chen et al., 2009a). The
interaction of TIP30, ACSL4, and Endo B1 with Rab5a raises the possibility that these proteins
may also function in endocytic pathways. We performed EGFR internalization analysis to
investigate whether the inhibition of TIP30, ACSL4, or Endo B1 affects the endocytic
downregulation of EGFR. Instead of continuous incubation with EGF, serum starved cells were
first incubated with EGF on ice to allow for ligand binding to receptor, and then washed to
remove unbound EGF before being moved to 37 ºC for internalization. Nascent protein synthesis
was blocked by cycloheximide in the culture medium. This approach eliminates the interference
from continuous ligand entrance and new receptor synthesis, thus enabling us to monitor both the
traffic and fate of EGF-EGFR complexes. We found that knockdown of TIP30, Rab5a, ACSL4,
or Endo B1 in PLC/PRF/5 cells (Figure S2.9A-S2.9D) resulted in a slower reduction of total
EGFR protein levels (Figure 2.2A-2.2F). To corroborate these findings, we further examined
whether phosphorylation of EGFR at Y845 (EGFR-pY845) and AKT at S473 (AKT-pS473), a
downstream target of EGFR signaling, were affected. Remarkably, the levels of EGFR-pY845
and AKT-pS473 sustained much longer in TIP30, ACSL4, Endo B1 or Rab5a knockdown cells
than in control cells (Figure 2.2A-2.2F).
To assess the effect of TIP30 deletion on EGFR endocytosis in normal cells and to exclude
off-target effects of shRNAs, we performed EGFR internalization analysis using primary
hepatocytes isolated from wild type and TIP30 knockout mouse littermates. Deletion of Tip30 in
primary hepatocytes delayed EGF-induced EGFR degradation (Figure 2.2G and 2.2H); and the
46

phosphorylation of EGFR at Y845 and Akt at S473 was higher and sustained longer in Tip30-/hepatocytes than in wild type hepatocytes. Together, these data provide strong evidence that
TIP30, ACSL4, Endo B1 are involved in the efficient endocytic downregulation of EGFR
protein level and signaling.
TIP30, ACSL4 and Endo B1 are involved in endocytic trafficking of EGF-EGFR
Despite the earlier studies indicating that dissociation and degradation of endocytic EGFEGFR complex occurred in lysososmes, cumulative evidence has clearly demonstrated that
EGFR is inactivated before being degraded and that EGF dissociates from EGFR prior to
lysosomal transfer (Authier and Chauvet, 1999; Burke et al., 2001; Murphy et al., 2009b;
Umebayashi et al., 2008). To test whether depletion of TIP30, Rab5a, ACSL4, and Endo B1
affects EGF dissociation from EGFR, we tracked Alexa-488 conjugated EGF (Alexa488-EGF)
and EGFR at different time points after internalization. As expected, fluorescent EGF partially
colocalizes with EGFR and Rab5a in control cells after 15 and 30 min internalization (Figure
S2.10A). Interestingly, EGF exited in membrane-bound vesicles from EGFR positive endosomes
in control cells after 60 min internalization (Figure 2.3A). In contrast, the majority of EGF
remained colocalized with EGFR in TIP30 or Rab5a knockdown cells (control cells: 7 ± 3%;
TIP30 knockdown cells: 36 ± 4%; Rab5a knockdown cells: 46 ± 5%; n = 60, p < 0.01 versus
control cells; Figure 2.3A and 2.3C). The EGF vesicles were devoid of early endosome markers
EEA1 and Rab5a, as well as late endosomal and lysosomal marker LAMP1 (Figure 2.3D),
indicating that they are neither early/late endosomes nor lysosomes.

47

Figure 2.3 TIP30 and Rab5a depletion delays the exit of EGF from early endosomes.
(A) Knockdown of TIP30 or Rab5a causes delayed exit of EGF from early endosomes. Cells
expressing control shRNA or shRNA against TIP30 or Rab5a were subjected to EGFR
internalization analysis. After internalization of Alexa488-EGF (blue) for 60 min, cells were fixed
and double-immunostained for EGFR (red) and EEA1 (green). Nucleus was stained by DAPI
(gray). Magenta results from overlap between red and blue. Results are representative of at least
three independent experiments on cells expressing two different shRNAs. Boxed areas are
magnified. Scale bars, 10 μm.
(B) Deletion of Tip30 in mouse primary hepatocytes leads to trapping of EGF in early
endosomes. Wild type and Tip30-/- primary hepatocytes were subjected to same EGFR
internalization analysis as decribed in (A). Boxed areas are magnified. Scale bars, 10 μm.
(C) Quantitative analysis of EGF-EGFR colocalization 60 min after EGF internalization. Sixty
cells represented by Figure 2.2A, 2.2B and Figure 2.4A in each group were analyzed using
MBF_imageJ. Pearson’s colocalization coefficients were calculated and converted to
percentages. Data represent means ± SEM. *P < 0.05, **P < 0.01, relative to control or wild
type cells; t test.
(D) EGF exits endosomes in vesicles. Cells were stained for EGFR and LAMP1 60 min after
EGF internalization. Scale bars, 10 μm.

48

Figure 2.3 Cont'd

A EGFR EEA1 EGF

B

EGFR EEA1 EGF

PLC/PRF/5 control, 60 min

WT Hepatocytes, 60 min

TIP30 KD, 60 min

Tip30 ‒/‒ Hepatocytes, 60 min

Rab5a KD, 60 min

C

EGF-EGFR colocalization

**

*
**
*

D EGFR LAMP1 EGF

49

**

We next performed EGFR internalization analysis using wild type and Tip30-/- primary
hepatocytes. Consistent with the preceding results, Tip30 deletion increased the colocalization of
EGF and EGFR nearly 2 fold (wild type primary hepatocytes: 15 ± 2%; Tip30-/- primary
hepatocytes: 29 ± 2%; n = 20, p < 0.01; Figure 2.3B and 2.3C).
Likewise, knockdown of ACSL4 or Endo B1 expression also increased the colocalization of
EGF and EGFR (ACSL4 knockdown cells: 40 ± 8%; Endo B1 knockdown cells: 23 ± 5%; n =
60, p < 0.05 versus control cells; Figure 2.3A and 2.3C). The majority of EGF colocalized with
EGFR even after 120 min internalization in knockdown and Tip30-/- cells (Figure S2.10AS2.10C). Taken together, these results suggest that TIP30, ACSL4, Endo B1, and Rab5a not only
physically interact but also function together in promoting EGF dissociation from EGFR during
endocytic trafficking.

50

Figure 2.4 TIP30, ACSL4, and Endo B1 are required for the localization of Rab5a to
early endosomes.
(A) Knockdown of TIP30, ACSL4, or Endo B1 inhibits the recruitment of Rab5a to early
endosomes. Control and TIP30, ACSL4, or Endo B1 knockdown cells were subjected to EGFR
internalization analysis.

Sixty min after internalization of Alexa488-EGF (blue), cells were

immunostained for EGFR (red) and Rab5a (green). Nucleus is stained by DAPI (gray). Results
are typical and representative of three experiments on cells from two different shRNAs. The
boxed areas are magnified. Scale bars, 10 μm.
(B) Tip30 deletion in primary hepatocytes inhibits the recruitment of Rab5a to early endosomes.
Primary hepatocytes were stained for EGFR (red) and Rab5a (green) after 60 min EGF (blue)
internalization. The boxed areas are magnified. Scale bars, 10 μm.
(C) Quantitative analysis of Rab5a-EGFR colocalization after 60 min EGF internalization was
performed as described in Figure 2.2C. Data represent means ± SEM. *P < 0.05, **P < 0.01,
relative to control or wild type cells; t test.

51

Figure 2.4 Cont'd

A

B EGFR Rab5a EGF

EGFR Rab5a EGF
PLC/PRF/5 control, 60 min

WT Hepatocytes, 60 min

TIP30 KD, 60 min

Tip30 ‒/‒ Hepatocytes, 60 min

ACSL4 KD, 60 min

C

Rab5a-EGFR colocalization

Endo B1 KD, 60 min

**

**

*
**

52

TIP30, ACSL4, and Endo B1 promote the recruitment of Rab5a to early endosomes
Endocytic vesicles gain Rab5a dynamically mostly by membrane fusion with Rab5-positive
endosomes in the course of cargo transport (Rink et al., 2005). This is further supported by the
observation that Rab5a is mainly localized on numerous membranous vesicles in the perinuclear
region of cells under steady state condition and is rapidly recruited to early endocytic vesicles at
the cell periphery in response to EGF (Lakadamyali et al., 2006; Leonard et al., 2008). The
similar effects of TIP30 and Rab5a knockdown on EGFR endocytosis prompted us to test
whether TIP30 is involved in the recruitment of Rab5a to early endosomes. In control cells,
Rab5a appeared in EGFR positive endosomes, from which EGF had exited after 60 min EGF
internalization (Figure 2.4A).

By contrast, the colocalization of Rab5a and EGFR was

significantly decreased in TIP30 knockdown cells (control cells: 72 ± 11%; TIP30 knockdown
cells, 18 ± 5%; n = 60, p < 0.01; Figure 2.4A and 2.4C) and Tip30-/- primary hepatocytes (wild
type hepatocytes: 34 ± 8%; Tip30-/- hepatocytes: 12 ± 5%; n = 20, p < 0.05; Figure 2.4B and
2.4C). Similar results were obtained with ACSL4 or Endo B1 knockdown cells (ACSL
knockdown cells: 3 ± 2%; Endo B1 knockdown cells: 27 ± 4%; n = 60, p < 0.01 versus control
cells; Figure 2.4A and 2.4C). Interestingly, there were a few Rab5a positive early endosomes in
Endo B1 knockdown cells, which was probably the result of incomplete knockdown. However,
these Rab5a positive endosomes appeared different from those in control cells. They did not
release EGF even after 120 min of internalization (Figure S2.10B), indicating that Endo B1 has
an additional function after Rab5a recruitment. This provides a possible explanation for the
observation that Endo B1 knockdown cells have less EGF-EGFR colocalization and more
Rab5a-EGFR overlap, but have longer lasting EGFR stability compared to TIP30 and ACSL4

53

knockdown cells (Figure 2.3C, 2.4C and 2.2A-2.2F). Together, these results indicate that TIP30,
ACSL4, and Endo B1 promote efficient Rab5a recruitment to early endosomes.
Rab5a vesicles transport V-ATPases to early endosomes
Curiously, we noted that Rab5a appeared in vesicles when it was not localized to EGFRpositive endosomes (Figure 2.4A and 2.4B). To further characterize those EGFR-negative Rab5a
vesicles, we co-stained EEA1 and Rab5a in wild type mouse primary hepatocytes 30 min after
EGF internalization. The EGFR-negative Rab5a vesicles were also negative for EEA1 and
transferrin receptor (Figure S2.11A and S2.11B), suggesting that they are neither plasma
membrane-derived endocytic vesicles nor recycling endosomes, but likely transporting vesicles
that originate from the trans-Golgi network.
Dissociation of ligand-receptor complexes inside endosomes is caused by the low luminal pH
created by V-ATPases. The previous observations that lack of Rab5a in early endosomes was
concomitant with delayed EGF-EGFR dissociation induced by loss of TIP30 or its interacting
proteins suggested the possibility that Rab5a vesicles deliver V-ATPases to early endosomes. To
address this issue, we examined the intracellular localization of V-ATPases for the regulatory
subunit H (ATP6V1H). A significant reduction of ATP6V1H localization to EGFR positive
endosomes was observed in TIP30, Rab5a, ACSL4, or Endo B1 knockdown cells (control cells:
46 ± 7%; TIP30 knockdown cells: 25 ± 4%; Rab5a knockdown cells: 19 ± 1%; ACSL
knockdown cells: 19 ± 2%; Endo B1 knockdown cells: 21 ± 1%; n = 60, p < 0.05 versus control
cells; Figure 2.5A and 2.5C) and in Tip30-/- primary hepatocytes (wild type hepatocytes: 61 ±
4%; Tip30-/- hepatocytes: 23 ± 6%; n = 20, p < 0.01; Figure 2.5B and 2.5C). ATP6V1H positive

54

staining was observed in Rab5a vesicles lacking EGF in TIP30 knockdown cells (Figure
S2.11C).

Figure 2.5 Rab5a vesicles transport V-ATPase to early endosomes.
(A) Depletion of TIP30, ACSL4, or Endo B1 inhibits the loading of V-ATPase on endocytic
vesicles. Cells were immunostained for EGFR (red) and a V-ATPase subunit ATP6V1H (green)
after 60 min EGF (blue) internalization. Results are typical and representative of three
experiments on cells from two different shRNAs. The boxed areas are magnified. Scale bars, 10
μm.
(B) Deletion of Tip30 in mouse primary hepatocytes results in V-ATPase mislocalization.
Immunostaining was performed as described in (B). Scale bars, 10 μm.
(C) Colocalization of V-ATPase and EGFR was analysed using MBF_ImageJ. Pearson’s
colocalization coefficients were calculated and converted to percentages. *P < 0.05, **P < 0.01,
relative to control or wild type cells; t test.
(D) EGF is released after EGF endocytic vesicles merge with Rab5a vesicles. Live cells
expressing ATP6V1H-DsRed (red) and EYFP-Rab5a (green) were imaged by confocal
microscopy at the indicated times after Alexa647-EGF internalization and images of a single focal
plane were acquired. A typical EGF endocytic vesicle movement was shown. Arrows points
toward the two vesicles undergoing merger and EGF release.

55

Endo B1 KD

ACSL4 KD

Rab5a KD

EGFR ATP6V1H EGF
WT
hepatocytes

Control

A

Tip30 -/hepatocytes

TIP30 KD

Figure 2.5 Cont'd

B
EGFR ATP6V1H

56

EGF

Figure 2.5 Cont'd

C

ATP6V1H‒EGFR colocalization

*

D

min

*

5

*

**

*

6.5

8

ATP6V1H-DsRed
EYFP-Rab5a
Alexa647-EGF
overlay

57

9.5

11

12.5

14

Moreover, in live PLC/PRF/5 cells co-expressing EYFP-Rab5a and ATP6V1H-DsRed, EGFpositive endosomes fused with Rab5a-ATP6V1H vesicles at 11 min after EGF internalization
and released EGF 3 min after the merge (Figure 2.5D). Taken together, these data indicate that
Rab5a may contribute to ligand-receptor dissociation by transporting V-ATPases to endocytic
vesicles and that TIP30, ACSL4 and Endo B1 are required for efficient transport.
TIP30 complex and arachidonic acid promote fusion between endocytic and Rab5a vesicles
in vitro
Since Rab5a on vesicles is transported to early endosmes by membrane fusion events
(Leonard et al., 2008; Rink et al., 2005), we sought to investigate whether the TIP30 complex
facilitates membrane fusion between Rab5a vesicles and early endosomes. First, we adopted a
previously validated in vitro cell free fusion assay (Bethani et al., 2009; Brandhorst et al., 2006)
with modifications. Instead of using two groups of early endosomes, we prepared Rab5a vesicles
from starved HepG2 cells expressing EYFP-Rab5a fusion proteins and endocytic vesicles from
EGF treated HepG2 cells expressing EGFR-DsRed fusion proteins (Fuchs and Ellinger, 2002;
Gorvel et al., 1995). The two types of vesicles that contain equal amount of proteins were
incubated in the fusion buffer at 37 oC followed by examination with confocal microscope.
Putative vesicle fusion was represented by the fluorescence overlap between EGFR-DsRed and
EYFP- Rab5a. Since ACSL4 is an acyl-CoA ligase with high substrate preference for
arachidonic acid (C20:4) (Cao et al., 1998), we also tested if arachidonic acid and coenzyme A
are needed. Vesicles resulting from reactions that were kept on ice evenly distributed on the
slides, appearing as small vesicles with low fluorescence intensity and virtually no overlap
between the two types of vesicles (Figure 2.6A, lane 1).

58

Figure 2.6 The TIP30 protein complex, arachidonic acid and coenzyme A promote the
fusion between endocytic and Rab5a vesicles.
(A) Aliquots of isolated EGFR-DsRed and EYFP-Rab5a vesicles (both contain 20 μg proteins)
were mixed and incubated in the reactions with the indicated components. Reaction mixtures
were spotted on glass slides and images were taken using confocal microscope. Panels 1, 4 and 6
were scanned with 3× amplification gain setting due to lower fluorescence intensity of individual
vesicles. Images are single plane and are representative for at least three independent
experiments. Scale bars, 5 μm.
(B) Signal overlap was quantified using MBF_ImageJ. Pearson’s colocalization coefficients
were calculated from three independent experiments and converted to percentages. Data
represent means ± SEM. **P < 0.01, ***P < 0.001; t test.
(C) Analysis of vesicle fusion with electron microscope. Vesicles incubated in the complete
reaction with or without arachidonic acid were stained with uranyl acetate and examined using
TEM. Scale bars, 500 nm.
(D) The graphs show the percentages of vesicles with different diameters. At least 6 images from
two independent experiments were counted. Data represent means ± SEM. n = 150; **P < 0.01, t
test.
(E) Bacterially expressed recombinant TIP30, ACSL4 and Endo B1 promote fusion between
endocytic and Rab5a vesicles. Vesicles (20 μg proteins) incubated with the indicated proteins (20
ng each) were examined using confocal microscope. Scale bars, 5 μm
(F) Quantification of images represented in (D) was performed similarly as in (B)
59

Figure 2.6 Cont'd

A

Triascin C

+
+
+
+
−
−

+
+
+
+
−
−

+
+
−
+
+
−

+
−
+
+
−
−

−
+
+
+
−
−

+
+
+
+
−
+

+
+
+
−
−
−

Temperature (oC)

0

37

37

37

37

37

37

1

2

3

4

5

6

7

Coenzyme A

Arachidonic acid
TIP30 complex
GTP
Control eluates

EYFP-Rab5a

EGFR-DsRED

overlay

C

B

**

***

**

**
***

‒ Arachidonic acid

+ Arachidonic acid

***

60

E

EYFPRab5a

EGFRDsRed

overlay
TIP30
TIP30
ACSL4
TIP30
ACSL4
Endo B1
TIP30M
ACSL4
Endo B1
Endo B1
ACSL4
Endo B1
ACSL4

D
Percentage of vesicles

Figure 2.6 Cont'd

‒ AA

+ AA

**

***
***
**

1
2
3
4
5
6
7

F

**

***

**

***

61

Similarly, no vesicle overlap was observed in the absence of arachidonic acid or in the presence
of triacsin C (10 uM), a potent inhibitor of long chain fatty acyl-CoA synthetase (Figure 2.6A
and 2.6B). In contrast, in the presence of arachidonic acid and coenzyme A immunopurified
TIP30 complex caused vesicle enlargement and significantly increased overlap between the two
vesicles (TIP30 complex: 50 ± 6%; control eluates: 20 ± 3%; omitting coenzyme A: 17 ± 2%;
omitting GTP: 16 ± 4%; n = 6 representative confocal images of 143 × 143 µm, p < 0.01 versus
TIP30 complex), thereby resulting in much intensified fluoresencence. Rab5a vesicles attached
to aggregated endocytic vesicles, but remained as small particles when the TIP30 complex,
coenzyme A or GTP was omitted. We next screened for other fatty acids that might promote the
putative fusion, including palmitic, palmitoleic, oleic, linoleic, linolenic, eicosapentaenoic, and
docosahexaenoic acids. None of these fatty acids could promote vesicle enlargement and vesicle
overlap (data not shown). Given that arachidonic acid is specifically needed for vesicle
membrane fusion (Creutz, 1981; Mayorga et al., 1993), these data imply that fluorescent overlap
between Rab5a and endocytic vesicles promoted by the TIP30 complex and arachidonic acid was
possibly due to membrane fusion.
To validate that the observed vesicle overlap was due to membrane fusion, we examined the
vesicles that resulted from fusion reactions by transmission electron microscopy (TEM). Without
the addition of arachidonic acid, all vesicles appeared spherical and ranged in diameter from 50
to 300 nm. In contrast, the complete fusion reaction led to the production of vesicles enlarged to
more than 1 m in diameter (Figure 2.6C); the larger vesicles were not found in reactions
without arachidonic acid. The captured ongoing fusion further confirmed that fusion actually
happened (Figure S2.12A). Quantitative analysis revealed a significant increase in the amount of
enlarged vesicles (with arachidonic acid: 300-500 nm, 24 ± 2%; > 500 nm, 16.5 ± 1.5%; without
62

arachidonic acid: 300-500 nm, 1 ± 0.2%; > 500 nm, 0; n = 150, p < 0.01; Figure 2.6D). Together,
these data indicate that the TIP30 complex promotes the fusion of Rab5a vesicles with endocytic
vesicles and that the synthesis of arachidonyl-CoA by ACSL4 is essential for vesicle membrane
fusion.
To exclude possible influences of other associated or contaminating proteins in
immunopurified TIP30 complexes, we replaced the complexes with bacterially expressed
recombinant TIP30, ACSL4, and Endo B1 in cell free assays. The three highly purified
recombinant proteins (Figure S2.12B) can efficiently promote overlap (75 ± 6%) between the
two vesicles, whereas lack of any of these proteins led to significantly less overlap (TIP30: 18 ±
1%; TIP30 and ACSL4: 23 ± 1%; Endo B1: 35 ± 3%; ACSL4 and EndoB1: 8 ± 2%; ACSL4: 20
± 5%; n = 6 representative images of 143 × 143 µm; p < 0.01 versus TIP30, ACSL4 and Endo
B1; Figure 2.6E and 2.6F). TIP30M, a TIP30 mutant with a mutated putative nucleotide binding
motif (GXXGXXG) (Xiao et al., 2000), only promoted 24 ± 2% overlap (n = 6 representative
images of 143 × 143 µm; p < 0.01 versus TIP30, ACSL4 and Endo B1; Figure 2.6D, panel 4).
These data suggest that TIP30, ACSL4 and Endo B1 together are capable of promoting the
heterotypic fusion between endocytic and Rab5a vesicles.
Vesicle tethering and stacking induced by acylation of phosphatidic acid
To gain further insight into how TIP30 and its interacting proteins mediate membrane fusion,
we first used 3H-arachidonic acid to label membrane lipids and found that at least one lipid
species in purified early endosomes was specifically labeled by the TIP30 complex (Figure
2.7A). The production of the labeled lipid was significantly less in the reaction using control
eluates or TIP30M immunoprecipitates, and was blocked by triacsin C. These data indicate that
63

lipids on early endosomes are the specific recipients of the arachidonyl group. Endophilins were
suggested as lysophosphatidic acid (LPA) acyl transferases (Modregger et al., 2003; Schmidt et
al., 1999). Consistent with a previous report (Gallop et al., 2005), we did not detect the acyl
transferase activity of Endo B1 when using LPA as the substrate (Figure S2.13A). To identify
the lipids that are modified, we performed protein-lipid overlay assays using membrane strips
prespotted with 15 cellular membrane lipids (Figure S2.13B). TIP30 and Endo B1 specifically
bind phosphatidic acid (PA) and cardiolipin, while Endo B1 also binds phosphatidylinositol 4phosphate (PtdIns(4)P; Figure 2.7B). ACSL4 and Rab5a did not bind any lipids spotted on the
membrane (data not shown). Cardiolipin is found predominantly in the inner mitochondrial
membrane, whereas PA has been implicated in fusion of various intracellular membranes
(Haucke and Di Paolo, 2007; Jenkins and Frohman, 2005). Therefore, we focused on PA and
tested whether the TIP30 complex could convert PA to PA-derivatives. Lipids extracted from the
acylation reactions with PA and arachidonic acid as substrates were subjected to MS/MS and
LC-MS/MS spectrometry analyses (Figure S2.13C-S2.13E). The flow injection precursor scan
in both modes revealed one predominant peak with an m/z (mass-to-charge ratio) value of 940.2,
which is close to the exact mass (940.85) of triacylglycerols (C61H112O6). Thus, we speculate that
triacylglycerol might be the PA derivative. To determine how PA derivatives would affect
membrane fusion, we carried out acylation reactions by incubating PA or phosphoinositide (PI)
with the TIP30 complex under the same condition as in vitro fusion assays. The resulting lipids
were purified, resuspended by sonication, and incubated with vesicles. Interestingly, the PA
derivatives promoted dramatic aggregation among endocytic and Rab5a vesicles on ice;
however, vesicles remained to be visibly individual particles (Figure 2.7C).

64

Figure 2.7 Acylation of phosphatidic acid promotes vesicle tethering and stacking.
(A) Fatty acylation of endosomal lipids by the TIP30 complex. [3H]-arachidonic acid can be
transferred to endosomal membrane lipids by TIP30 immunoprecipitates, but not by TIP30M
immunoprecipitates or control immunoprecipitates (left panel). The reaction was blocked by 10
µM triacsin C. Image was acquired by scanning lipids resolved on TLC plate with a Molecular
Dynamics Storm 860. * indicates the radiolabeled lipid.
(B) TIP30 and Endo B1 strongly bind phosphatidic acid (PA). Protein-lipid overlay assays were
carried out by incubating recombinant proteins with membranes containing 15 pre-spotted lipids.
Bound proteins were detected by immunoblot.
(C) Triacylglycerol and modified PA promote vesicle tethering. Lipids were extracted after
incubating 100 nmol PA and phosphatidylinositol (PI) with TIP30 complex or control eluates.
Lipids were resuspended in homogenization buffer by sonication and were mixed with EGFRDsRed and EYFP-Rab5a vesicles. Reaction mixtures were spotted on glass slides and images
were taken using confocal microscope. Scale bars, 5 μm.
(D-H) Effects of lipid acylation were determined using electron microscope. Lipids were
extracted after incubating PA with TIP30 immunoprecipitates (D), PA with recombinant TIP30,
ACSL4, and Endo B1 (E), PA with control immunoprecipitates (G), or PI with TIP30
immunoprecipitates (H). Vesicles were incubated on ice with the extracted lipids or
triacylglycerol (F) suspended in homogenization buffer and examined using electron microscope.
Scale bars, 500 nm.

65

Figure 2.7 Cont'd

A

Free fatty acid
*
*

Origin

B
TIP30

Endo B1

PtdIns(4)P
PA

Cardiolipin

Blue marker

66

Figure 2.7 Cont'd

C

Control eluates treated
PI

TIP30 complex treated

PA

PI

Triacylglycerol

PA

EYFPRab5a

EGFRDsRed

overlay

D

E PA+recombinant
TIP30, ACSL4, Endo B1

PA+TIP30 complex

G

PA+control eluates

H

PI+TIP30 complex

67

F
Triacylglycerol

In contrast, unmodified PA incubated with control eluates did not promote significant
membrane aggregation, nor did phosphoinositide (PI) that was prepared with the same procedure
as the PA derivative. A triacylglycerol (1,2-Dilinoleoyl-3-palmitoyl-rac-glycerol) with a
palmitoyl tail at the sn-3 position promoted significant aggregation (Figure 2.7C).
Finally, we used electron microscope to examine the vesicles after incubation with PA
derivatives on ice. The vesicle aggregation caused by PA derivatives generated by
immunopurified TIP30 complexes or recombinant proteins was apparently the result of both
tethering and stacking (Figure 2.7D, 2.7E and S2.6F). In contrast the vesicle aggregation caused
by the triacyglycerol seemed due only to tethering (Figure 2.7F), indicating that chain saturation
and length (C20:4 vs C16:0) at the sn-3 position is important. Consistent with the confocal
microscopy data, PA treated with control eluates (Figure 2.7G) or PI treated with
immunopurified TIP30 complex (Figure 2.7H) had no apparent effect. These data indicate that
the final product of the acyl transfer reaction, triacyglycerol, can promote aggregation among
vesicles, although intermediates in the reaction are not ruled out to have a similar function.
Nonetheless, we did not observe complete fluorescence overlap and enlarged endosomes (>
1m in diameter) as seen in Figure 2.6, suggesting that additional activities of the TIP30
complex or other proteins on the membranes, such as SNAREs, SNARE accessory factors,
Rab5a and its effectors, are needed to accomplish the fusion steps following close membrane
contact. Collectively, these data indicate that TIP30, ACSL4, and Endo B1 promote vesicle
membrane fusion by fatty acylating PA.
Discussion
Function of TIP30, ACSL4 and Endo B1 in endocytic trafficking
68

Elucidating the molecular basis of intracellular trafficking and sorting of internalized
receptors in early endosomes requires the identification of the critical components participating
in these processes. The present study led to the identification of three new players that act
together in controlling receptor-mediated endocytosis and revealed new insights into the
mechanism of vesicle membrane fusion. Specifically, we showed that TIP30, Endo B1, and
ACSL4 act as a functional unit to facilitate the Rab5a localization to early endosomes by
promoting the fusion of Rab5a vesicles with endocytic vesicles. TIP30 and Endo B1 have been
proposed to be tumor suppressors because their deficiency leads to spontaneous development of
tumors in mice (Ito et al., 2003; Takahashi et al., 2007). Thus, our results indicate that TIP30 and
Endo B1 suppress tumor growth, at least in part, through the regulation of the EGFR signaling
pathway.
Endo B1 is particularly interesting, since it belongs to a family of proteins that have Nterminal BAR domains, which may sense and stabilize membrane curvature (Masuda et al.,
2006b; McMahon and Gallop, 2005). Endo B1 and its homologue Endophilin 1 can directly bind
and envaginate lipid bilayers into narrow tubules, suggesting a role of Endo B1 in membrane
deformation (Farsad et al., 2001). It was proposed that Endophilins create membrane curvature
by transferring arachidonic acid to lysophosphatidic acid (Schmidt et al., 1999). However, our
data support the observation that Endophilins cannot transfer arachidonyl group to LPA (Gallop
et al., 2005). The strong binding of PA to Endo B1 (Figure 2.7B) raised the question of whether
Endo B1 takes PA as a potential substrate for its acyl transferase activity.
A role for Rab5a in transport of endosomal targeting proteins

69

Rab5a is one of the Rab GTPases that are key regulators in vesicle formation, motility,
tethering and docking, and membrane fusion (Grosshans et al., 2006; Pfeffer, 2001; Zerial and
McBride, 2001). Rab5 on early endosomes has been shown to be able to promote endosome
fusion in vitro (Gorvel et al., 1991). Our data showed that Rab5a vesicles can fuse with
endocytic vesicles in a GTP dependant manner, suggesting that Rab5a also actively participates
in the fusion of these two types of vesicles. Unlike homotypic endosome fusion, this type fusion
requires the TIP30 complex, arachidonic acid and coenzyme A, but does not require additional
NSF, SNARE proteins and Rab5 effectors. Given that high fusion efficiency of biological intact
endosomes was not achieved in vitro in a recent study (Ohya et al., 2009), it would be interesting
to further determine whether the TIP30 complex, arachidonic acid and coenzyme A could also
promote homotypic endosome fusion.
The Rab family proteins anchor themselves at the cytoplasmic face of vesicles by their Cterminal modification with hydrophobic geranylgeranyl groups (Takai et al., 2001). This special
way of membrane association makes them ideal candidates as identity tags that determine
specificity during vesicle transport (Jahn et al., 2003). Interestingly, our data revealed that
besides its function in early endosomes, Rab5a also appeared in vesicles carrying the integral
membrane protein V-ATPase, a proton pump responsible for ligand-receptor dissociation. The
localization of V-ATPase in early endosomes depends on Rab5a, suggesting that Rab5a is
involved in delivering V-ATPases to the early endosme. Thus, Rab5a may share similar
functions with many other Rab GTPases in determining specific destinations during vesicle
transport.
Lipids and membrane fusion

70

One of the unsolved issues in protein trafficking is to understand the mechanism underlying
membrane fusion. It is widely believed that membrane fusion events are facilitated by the core
protein machinery consisting of SNAREs, SNARE accessory factors, Rab5 and its effectors
(Ohya et al., 2009; Stenmark, 2009). The SNAREs are thought to bring membranes together,
whereas Rab5 recruits specific effectors to mediate vesicle tethering by controlling SNARE
complexes. However, it is not known how these proteins establish the minimal fusion events,
such as the local deformation of the membrane, the dehydration of lipid head groups, and
overcoming the energy barrier caused by opposing charges in the membranes (Sorensen, 2009).
Cumulative evidence also supports the idea that certain fatty acids including arachidonic acid
also play key roles in membrane fusion. For instance, arachidonic acid is required for endosome
fusion in vitro (Mayorga et al., 1993), and is the most effective fusogen in chromaffin granule
fusion (Creutz, 1981). In addition, membrane-bound arachidonic acid can drive annexin IImediated membrane fusion of the lamellar body with the plasma membrane during the
exocytosis (Chattopadhyay et al., 2003).
How does arachidonic acid promote membrane fusion? Arachidonic acid has been proposed
to stimulate SNARE complex assembly (Connell et al., 2007; Latham et al., 2007; Rickman and
Davletov, 2005). However, our data show that arachidonic acid is insufficient to support efficient
membrane fusion without the TIP30 complex (Figures 2.6). We show that the arachidonyl group
is transferred to endosomal PA to generate triacylglycerol that induces vesicle tethering and
stacking (Figure 2.7). Thus, we suggest that arachidonic acid promotes membrane fusion by
contributing to both PA acylation and SNARE complex assembly. Consistent with this, PA and
its synthetase phospholipase D (PLD) are known to participate in membrane fusion during
vesicle transport (Jenkins and Frohman, 2005; Jones et al., 1999). Moreover, as integral
71

components of many biological membranes, triacylglycerols have been demonstrated to possess
great potency to promote spontaneous curvature in an acyl chain length dependent manner (Lee
et al., 1996), which may alter membrane structure to support membrane fusion (Martens and
McMahon, 2008; McMahon et al., 2010). We speculate that the addition of the hydrophobic acyl
chain to endosomal PA may help to overcome the repulsive hydration force generated from
water bound to the lipid headgroups. In addition, triacylglycerol can exhibit an extended
conformation (Fahey and Small, 1986) with the 3-arachidonyl group in the opposite direction of
the other two acyl chains, thereby allowing for attachment and fusion between two membranes
(Kinnunen, 1992; Kinnunen and Holopainen, 2000). Our results suggest that as an initiation
event, acylation of endosomal PA enables the close contact between donor and recipient
membranes, thus allowing for fusion to be accomplished by SNAREs, SNARE accessory factors,
Rab5 and its effectors. Clearly, more work is needed to determine how acylation of PA and other
lipid derivatives are involved in the action of SNAREs, Rab5, and their effectors in membrane
fusion. It is expected that a more precise mechanism underlying membrane fusion will emerge
by integrating information from studies of both lipids and proteins.
Materials and methods
Cell culture, DNA constructs, shRNA, antibodies, and immunoprecipitation were described
in the Extended Experimental Procedures.
EGFR internalization and immunofluorescence
PLC/PRF/5 cells were infected by lentiviruses producing shRNA against indicated genes.
Cells were pooled after being selected for 4 days with 2 ug/ml puromycin. At least two
confirmed knockdown pools for each targeted gene were used for the experiments in figures 2.272

2.5. Control and knockdown cells were cultured on cover glass and serum starved for 24 hours in
DMEM. Wild type and Tip30-/- primary hepatocytes were starved for three hours. Cells were
incubated with 100 ng/ml Alexa488 EGF (Invitrogen) and 20 g/ml cycloheximide on ice for 1
hour and then washed 4 times with cold PBS and incubated in DMEM with 20 g/ml
cycloheximide at 37 ºC for different time periods. Cells were fixed in 4% paraformaldehyde in
PBS for 15 min, permeabilized with 0.1% Triton X-100 for 2 min and stained for the indicated
proteins. Images were obtained with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss)
using Plan-Apochromat 63×/1.40 Oil objective. Pinhole size was set to 1 airy unit for all
channels. All images are representative single optical sections. To determine EGFR stability
upon EGF treatment, cells were cultured in 6-cm dishes and treated as above except that
unlabeled EGF was used, and the cells were collected for immunoblot at various time points.
Purification of endocytic and Rab5a vesicles
HepG2 cells were transduced by the lentiviral vector pSin-EGFR-DsRed or pSin-EYFPRab5a and selected for 4 days with 2 g/ml puromycin. To prepare endosomes carrying EGFRDsRed, we treated cells with 10 ng/ml Alexa647-EGF at 37 ºC for 10 minutes. Early endosomes
were prepared on the flotation gradient essentially as described (Gorvel et al., 1995). Rab5
vesicles containing EYFP-Rab5a were prepared as described (Fuchs and Ellinger, 2002).
In vitro vesicle fusion assay
The assay was performed as described previously with modifications (Bethani et al., 2009;
Brandhorst et al., 2006) Briefly, EGFR-DsRED endocytic vesicles and EYFP-Rab5a vesicles
(aliquotes of both contain 20 g proteins) were gently mixed in a total volume of 20 l fusion

73

buffer (10 mM Hepes, pH 7.4, 1.5 mM MgOAc, 1 mM DTT, 50 mM KOAc, 100 nmol
arachidonic acid, 1 mM coenzyme A, 5 mM GTP, complemented with 4 l of an ATPregenerating system containing 1:1:1 mixture of 100 mM ATP, 600 mM creatine phosphate, and
4 mg/ml creatine phosphokinase). After incubating with indicated purified proteins at 37 °C for
40 min, a portion of the reactions were spotted on slides and examined using Zeiss LSM 510
Meta confocal microscope. All images are representative single optical sections. Colocalization
analysis was done using MBF_ImageJ. For reactions in Figure 2.6D, vesicles were incubated in
homogenization buffer (250 mM sucrose, 5 mM Hepes, pH 7.4) with indicated lipids. Electron
microscopy (TEM JEOL 100CX) was performed as described previously (Ohya et al., 2009).
Lipid acylation
Purified endocytic vesicles (aliquots containing 20 µg proteins) were incubated with
indicated proteins and [3H]-arachidonic acid (PerkinElmer) in the presence or absence of 10 M
triacsin C at 37 °C for 1 hour in total volume of 200 µl fusion buffer. For preparation of PA
derivatives, 100 nM 1,2-dioleoyl-sn-glycero-3-phosphate (Avanti Polar Lipids) were incubated
with immunopurified TIP30 complex or recombinant TIP30, ACSL4 and Endo B1 and
arachidonic acid (Sigma) in the fusion buffer. Lipids were extracted using Bligh-Dyer Method
(Bligh and Dyer, 1959).
Statistical analysis
All statistical tests were two-tailed t-test. Data represent means ± SEM. *P < 0.05, **P <
0.01, ***P < 0.001.
Acknowledgments
74

We are grateful to Stephen Prescott for generously sharing ACSL4 antibody and to Changdeng Hu for generously sharing BiFC plasmids. We thank Sandra Haslam, Christine Eischen and
Karen Friderici for critical reading of the manuscript and Alicia Pastor for electron microscopic
analysis. This work was supported by grants RO1 DK066110-01 and W81XWH-08-1-0377 (to
H.X.) from the NIDDK and Department of Defense.

75

Supplemental Figure 2.8 Related to Figure 2.1, TIP30, ACSL4 and Endo B1forms a
protein complex that may function on the EGFR endocytic pathway.
(A) Endogenous TIP30, ACSL4 and Endo B1 are co-sedimented in a glycerol gradient. Whole
cell extracts of PLC/PRF/5 cells and protein markers were loaded on the top of linear 10-35%
(V/V) glycerol gradients (10 ml) formed from the bottom of 12-ml Beckman tubes. The buffer
throughout the gradients was 20 mM Hepes-KOH (pH 7.0), 100 mM KCl, 1 mM dithiothreitol.
The gradients were centrifuged at 200,000 g in a SW41 Ti rotor (Beckman) at 4°C for 20 h. After
centrifugation, fractions were collected, concentrated and subjected to immunoblot analysis.
Albumin (66 kDa), alcohol dehydrogenase (150 kDa) apoferritin (443 kDa), thyroglobulin (670
kDa), and blue dextran (2,000 kDa) were run in parallel as molecular weight indicators.
(B) Direct interaction of TIP30, ACSL4, and Endo B1. GST-ACSL4 and His-Endo B1 (Lane 1),
Flag-TIP30 (Lane 2), or GST-ACSL4, His-Endo B1, and Flag-TIP30 (Lane 3) were subjected to
anti-Flag M2 immunoprecipitation. Aliquots of precipitates were resolved on SDS-PAGE and
analyzed by immunoblot with the indicated antibodies. Purified Flag-TIP30 was described
previously (Xiao et al., 2000). Bacterially-expressed GST-ACSL4 was purchased from Abnova.
His-Endo B1 was expressed in BL21 and purified as described in Figure S2.13A.
(C) TIP30 is localized to EGFR positive endosomes. PLC/PRF/5 cells were immunostained with
antibodies for EGFR (red) and TIP30 (green) after 30 min Alexa488 conjugated EGF (blue)
internalization. Boxed areas are magnified. Bars, 10 μm.

76

Supplemental Figure 2.8 Cont'd

A

(kDa) 66 150

443

15%
Fraction number
(kDa) 80

1

670
35%

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35
ACSL4

48
30

B

Endo B1
TIP30

1

2

3
α-ACSL4
α-Endo B1
α-TIP30

C
PLC/PRF/5 cells
EGFR
TIP30
EGF
overlay

77

Supplemental Figure 2.9 Related to Figure 2.2, Knockdown of TIP30, Rab5a, ACSL4,
and Endo B1 in PLC/PRF/5 cells.
(A-D) Immunoblot analyses show knockdown of TIP30, Rab5a, ACSL4 or Endo B1 in
PLC/PRF/5 cell. Whole cell lysates were made from cells expressing a scramble shRNA or
shRNA against TIP30 (A), Rab5a (B), ACSL4 (C), or Endo B1 (D). Two shRNAs against each
gene were used and equal amounts of protein were loaded per lane.

A

B
α-TIP30

α-Rab5a

α-β-Actin

α-β-Actin

C

D
α-ACSL4

α-Endo B1

α-β-Actin

α-β-Actin

78

Supplemental Figure 2.10 Related to Figure 2.3 and 2.4, Deletion of TIP30, ACSL4, or
Endo B1 Delays EGF-EGFR dissociation and inhibits the recruitment of Rab5a to
endocytic vesicles.
(A-B) TIP30, ACSL4, or Endo B1 knockdown delays EGF-EGFR dissociation and inhibits the
recruitment of Rab5a to endocytic vesicles. Localization of EGFR (red) and Rab5a (green) in
control, TIP30 KD, ACSL4 KD and Endo B1 KD cells were monitored 120 min after Alexa488EGF (blue) internalization. Nucleus is stained by DAPI (gray). Typical images of cells in each
group are shown. Boxed areas are magnified. Magenta represents red and blue overlap. Bars, 10
μm.
(C) TIP30 deletion in primary hepatocytes leads to delayed EGF-EGFR dissociation and inhibits
the recruitment of Rab5a to endocytic vesicles. Wild type and Tip30-/- primary hepatocytes were
subjected to EGFR internalization analysis and were immunostained for EGFR (red) and Rab5a
(green) 120 min after Alexa488-EGF (blue) internalization. Bars, 10 μm.

79

Supplemental Figure 2.10 Cont'd

80

Supplemental Figure 2.11 Related to Figure 2.5, Rab5a vesicles carry V-ATPases.
(A) EGFR negative Rab5a vesicles are not early endosomes. Wild type mouse primary
hepatocytes were immunostained for EEA1 (red) and Rab5a (green) after 30 min EGF (blue)
internalization. Scale bars, 10 μm.
(B) Transferrin receptor partially colocalizes with Rab5a. PLC/PRF/5 cells were immunostained
for transferrin receptor (red) and Rab5a (green) after 10 min EGF (blue) internalization. Scale
bars, 10 μm.
(C) V-ATPase colocalizes with Rab5a in transporting vesicles. TIP30 knockdown cells were
immunostained for V-ATPase (red) and Rab5a (green) after 30 min EGF (blue) internalization.
Scale bars, 10 μm.

81

Supplemental Figure 2.11 Cont'd

82

Supplemental Figure 2.12 Related to Figure 2.6, Examination of vesicle fusion using
TEM and the purification of recombinant proteins.
(A) Vesicles were stained and visualized with electron microscope after being incubated with
immunopurified TIP30 complex and arachidonic acid. Representative images are shown. Bars,
500 nm.
(B) Recombinant proteins were expressed in BL21 cells and purified using cobalt affinity resins.
Eluted proteins were dialyzed against BC300 and subjected to SDS-PAGE analysis followed by
coomassie blue staining. Images were acquired using Li-Cor scanner.

83

Supplemental Figure 2.12 Cont'd

A

B

250
150
100
75
50
37
25

84

Supplemental Figure 2.13 Related to Figure 2.6, Spectrometric analysis of PA
derivatives.
(A) Endo B1 cannot transfer arachidonic acid to LPA. LPA and [3H]-arachidonic acid were
incubated at 37 oC with or without bacterially expressed Endo B1 in fusion buffer. Image was
acquired by scanning lipids resolved on TLC plate with a Molecular Dynamics Storm 860.
(B) The schematic diagram shows the lipid species prespotted on membranes that are used in
lipid-protein overlay assays.
(C) The flow injection negative scan in the range of 400-1400 u. A predominant peak of 699.5
was detected. Atomic mass: arachidonic acid, 304.5 u; 18:1 PA, 699.5 u. The lipids in the
smaller peaks have molecular weights that do not match any of the expected PA derivatives.
(D) The flow injection negative precursor scan for 303.2 u over a mass range of 400-1400 u.
(E) The LC-C18 negative precursor scan for 303.2 u over a mass range of 400-1400 u. The
lipids after the acylation reaction was extracted and chromatographed on a C-18 column to
reduce possible adduction mechanisms of compounds detected in the flow injection negative
precursor scan of 303.2 u.
(F) Acylated PA causes tethering and stacking of endocytic and Rab5a vesicles. A representative
electron microscopy picture is shown. Bars, 1 μm.

85

Supplemental Figure 2.13 Cont'd

A

Free fatty acid

Origin

B
Phosphatidylinositol (PI)
PtdIns(4)P
PtdIns(4,5)P2
PtdIns(3,4,5)P3
Cholesterol
Sphingomyelin
3-sulfogalactosylceramide
Blue Blank

Triglyceride
Diacylglycerol (DAG)
Phosphatidic Acid (PA)
Phosphatidylserine (PS)
Phosphatidylethanolamine (PE)
Phosphatidylcholine (PC)
Phosphatidylglycerol (PG)
Cardiolipin

86

Supplemental Figure 2.13 Cont'd
C Flow injection MS scan

D

Flow injection MS precursor scan, 303.2u

87

Supplemental Figure 2.13 Cont'd
E

LC-C18/MS/MS precursor scan, 303.2u

F

88

Supplemental materials and methods
Cell culture, DNA constructs and shRNAs
The pSin-EF2 vector (Yu et al., 2007) was converted to destination vectors by cloning the
Gateway cassette rfA (Reading frame A, Invitrogen) with either N-terminal or C-terminal HA
tag, CFP, EYFP, or DsRed fluorescent proteins into blunted SpeI and EcoRI sites. For BiFC
assays, VC155 and VN173 (Hu et al., 2005) were cloned into pCDNA3.1 and pSin-EF2,
respectively, and both were also converted to destination vectors. Lentiviral plasmids producing
shRNAs against TIP30, Rab5a, and ACSL4 were from Sigma-Aldrich. Lentiviral plasmids for
shRNAs against Endo B1 were from Open Biosystems. See the supplemental experimental
procedures for antibodies, cell lines and other reagents.
PLC/PRF/5 and HepG2 cell lines were purchased from ATCC. cDNA clones of human
Rab5a, ACSL4 and EndoB1 were amplified using RT-PCR of mRNA isolated from PLC/PRF/5
cells. TIP30 was subcloned from pFlag7-TIP30 and pFlag7-TIP30M (Xiao et al., 2000).
Antibodies against HA (HA-7), β-Actin (AC-15) and Endo B1 were from Sigma. Antibodies
for AKT, AKT-pS473, EEA1 and Rab5a were from Cell Signaling. Anti-EGFR-pY845, antitransferrin receptor, Alexa Fluor 546 goat anti-mouse, Alexa Fluor 594 goat anti-rabbit
antibodies were from Invitrogen. Anti-EGFR antibodies were from Millipore. Anti-ATP6V1H
antibody was from Santa Cruz. Anti-LAMP1 was from The Developmental Studies Hybridoma
Bank at University of Iowa. Triacylglycerol (1,2-Dilinoleoyl-3-palmitoyl-rac-glycerol) and
phosphoinositide (PI) were purchased from Sigma.
Immunoprecipitation

89

PLC/PRF/5 cells were transfected with indicated constructs. Whole cell extracts were
prepared from pooled stable clones as described previously with modifications (Stringer et al.,
1990). Briefly, cells were homogenized by 20 strokes in two packed cell pellet volumes (p.c.v.)
of buffer A (10 mM Hepes, pH7.9, 10 mM KCl,0.5 mM DTT, protease inhibitor cocktail) using
Kontes homogenizer (B pestle). Another 20 strokes were applied after adding 1.5 p.c.v. of buffer
B (50 mM Hepes, pH7.9, 0.6 mM EDTA, 1.5 mM DTT, 1.26 M NaCl, 75% Glycerol) followed
by centrifugation at 100,000g for 1 hour. The supernatant was dialyzed against BC300 (20 mM
Hepes, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.3 M KCl) (Chiang and Roeder,
1995) and centrifuged at 15000 rpm for 20 min followed by rotating with anti-HA agarose beads
(Roche Diagnostics) overnight at 4 ºC. The beads were centrifuged and extensively washed using
BC300 buffer. Immunoprecipitates were eluted with HA peptides (Roche Diagnostics),
denatured, resolved on SDS-PAGE, and subjected to silver stain, immunoblot, or LC-MS/MS
spectral analyses (The MSU Proteomics Facility, Michigan State University).
Purification of recombinant proteins
Bacterially expressed TIP30, mutant TIP30, Endo B1 and ACSL4 proteins were His tagged
and purified as described (Xiao et al., 2000). TIP30, TIP30M, Endo B1, and ACSL4 were
subcloned into pRSETb vector. Purified proteins were dialyzed against BC300 and stored at -80
ºC.
Mouse hepatocyte isolation
Primary hepatocytes were isolated from 8 week-old wild type and Tip30-/- mice as described
(Seglen, 1972) with modifications. Briefly, the inferior vena cava was cannulated and the liver
was first perfused in situ with an oxygenated Krebs Ringer buffer (115 mM NaCl, 5.9 mM KCl,
90

25 mM NaHCO3, 10 mM glucose, 20 mM Hepes, pH 7.4) with 0.1 mM EGTA at 37 °C,
followed by perfusion with oxygenated Krebs Ringer buffer containing 0.25 mM CaCl2 and 20
µg/ml Liberase Blendzyme 3 (Roche Applied Science). Liver was removed and then gently
minced in ice cold Krebs Ringer buffer. Liver cell suspension was then filtered with Falcon cell
strainers (Becton Dickinson) and washed three times by centrifugation at 50 g for 2 min at 4 °C.
Cell viability was determined by trypan blue exclusion. Cells were cultured in DMEM (with
10% FBS and 1× Pen/Strep) at 37 °C with 5% CO2.
MS/MS spectrometry analysis
MS/MS spectrometry analysis of PA derivatives were performed in Avanti Polar Lipids Inc.
Briefly, PA derivatives were extracted according to Bligh-Dyer method and redissoved in
methanol/chloroform (85/18) with 10 mM NH4OAC and 1 ug/ml

NH4OH. First, standards of

17:0-20:4 PA and 17:0-20:4 PI were prepared in the above solution and infused in the API 4000
QTrap triple quadrupole with linear ion trap instrument for optimization of collisional energy to
provide mass fragments of arachidonic acid (20:4) at 303.2 u. Then samples were flow injected
into the MS/MS in negative scan mode to discover products from the described reaction. The m/z
value was used to search the most likely molecular species at http://www.lipidmaps.org.

91

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Chapter 3 Tip30 Deletion in MMTV-Neu Mice Leads to the Development of Estrogen
Receptor-Positive and Progesterone Receptor-Negative Mammary Tumors via Enhancing
EGFR Signaling

*Chengliang Zhang, *Mikito Mori, Shenglan Gao, Aimin Li, Isamu Hoshino, Mark D.
Aupperlee, Sandra Z. Haslam and Hua Xiao
Cancer Research, in press
* CL. Zhang and M. Mori contribute equally to this work

99

Abstract
Estrogen receptor-positive and progesterone receptor-negative (ER+/PR-) breast cancers
account for 15-25% of all human breast cancers and display more aggressive malignant
characteristics compared to ER+/PR+ cancers. However, the molecular mechanism underlying
development of ER+/PR- breast cancers still remains elusive. We show here that Tip30 deletion
dramatically accelerated the onset of mammary tumors in the MMTV-Neu mouse model of
breast cancer. The mammary tumors arising in Tip30-/-/MMTV-Neu mice were exclusively
ER+/PR- and exhibit estrogen and progesterone-dependent growth. Tip30 is predominantly
expressed in ER+ mammary epithelial cells (MECs) and its deletion leads to an increase in the
number of phospho-ER positive cells in mammary glands and accelerated activation of Akt in
MMTV-Neu mice. Moreover, we found that Tip30 regulates the PI3K/Akt pathway through
controlling endocytic downregulation of EGFR protein level and signaling. Together, these
findings suggest a novel mechanism in which loss of Tip30 cooperates with Neu activation to
enhance the activation of Akt signaling, leading to the development of ER+/PR- mammary
tumors.

100

Introduction
Despite considerable success in the treatment of ER+/PR+ breast cancers with therapies
directed at targeting estrogen and ER, a substantial fraction of patients with ER+/PR- tumors do
not benefit significantly from these therapies (Osborne et al., 2005; Ponzone et al., 2006). It is
estimated that 15-25% of all human breast cancers are ER+/PR- with more aggressive malignant
characteristics and poorer response to SERMs compared to ER+/PR+ breast cancers (Arpino et
al., 2005; Creighton et al., 2009; Ponzone et al., 2006). Moreover, 25% of ER+/PR- tumors are
found to have HER2/Neu overexpression; patients with this subtype of ER+/PR- breast cancer
have an extremely poor response to endocrine treatment. While several lines of evidence suggest
that ER+/PR- tumors can be derived from ER+/PR+ tumors by the loss of PR expression due to
anti-hormone therapy, other studies indicate that ER+/PR- tumors could arise by de novo
etiological factors (Kim et al., 2006). To date, the mechanisms underlying de novo and acquired
ER+/PR- breast cancer remain poorly defined. Thus, elucidation of the molecular basis of
ER+/PR- breast tumor development has the potential to reveal new therapeutic targets for the
treatment, and even prevention of the resistance to anti-estrogen therapy in breast cancer
patients.
There are several hypotheses to explain the generation of ER+/PR- breast cancers. These
include inhibition of PR transcription by aberrant ER cofactors or by nonfunctional ER, lower
circulating levels of estrogen leading to reduced ER activity, hypermethylation of PR promoter,
or by growth factor signaling pathways (Cui et al., 2005). Of particular interest are growth factor
signaling pathways, in which aberrations are common in many human cancers (Dowsett et al.,
2005; Konecny et al., 2003). Among the growth factor receptors, HER2/Neu is the most
frequently altered receptor in breast cancers. Most of HER2 positive breast cancers are ER-/PR-,
101

only a small fraction are ER+/PR+ or ER+/PR-, suggesting that HER2 may inhibit ER
expression as well as PR expression (Cui et al., 2005). This hypothesis is supported by the
observation that mouse models of breast cancer harboring a HER2/Neu transgene almost
exclusively develop ER-/PR- mammary tumors. Additionally, when transfected with HER2
expressing vectors, ER+/PR+ breast cancer cells exhibited a significant decrease in PR
expression (Konecny et al., 2003). Nevertheless, the mechanism by which activation of
HER2/Neu leads to development of ER-/PR-, but not ER+ breast cancer remains poorly
understood.
TIP30, also known as CC3, is a 30-kDa human cellular protein that was purified as a HIV-1
Tat interacting protein (Xiao et al., 1998) and its expression is altered in human liver, lung and
breast cancers (Ito et al., 2003; Lee et al., 2004; Tong et al., 2009; Zhao et al., 2007). Our
previous studies demonstrated that Tip30-deficient mice spontaneously develop tumors in
several tissues and mammary preneoplastic lesions, suggesting that TIP30 acts as a tumor
suppressor (Ito et al., 2003; Pecha et al., 2007). Its tumor suppressor activity is probably due to
multiple mechanisms. TIP30 functions as a transcription cofactor to repress expression of genes
that are involved in proliferation and apoptosis (Jiang et al., 2004a; Zhao et al., 2008b) and it can
induce apoptosis as an inhibitor of nuclear import (King and Shtivelman, 2004). In particular,
TIP30 can act as a repressor of ER-mediated c-Myc transcription in mammary glands and
breast cancer cells (Jiang et al., 2004a). Additionally, recent evidence has highlighted that TIP30
controls endocytic downregulation of the EGFR signaling pathway in primary hepatocytes and
hepatocellular carcinoma cells7.
The multiple functions of TIP30 have prompted the speculation that TIP30 loss may
contribute to mammary tumorigenesis induced by activation of oncogenes. Therefore, we aimed
102

to determine whether Tip30 deletion enhances mammary tumorigenesis in MMTV-Neu mice.
We report here that Tip30 deletion cooperates with HER2/Neu activation to promote
development of ER+/PR- mammary tumors, in part, through up-regulation of EGFR/Akt
signaling. The Neu+/Tip30-/- mouse model may help decipher the mechanisms leading to
ER+/PR- mammary tumors and identify therapeutic targets for this subgroup of tumors.
Results
Tip30 deletion significantly accelerates mammary tumorigenesis in MMTV-Neu mice.
To investigate whether Tip30 deletion cooperates with HER2/Neu to promote mammary
tumorigenesis, we generated Tip30 knockout mice with overexpression of Neu by crossing
MMTV-Neu transgene from MMTV-Neu mice (FVB/N-Tg; (Guy et al., 1992) into Tip30-/- FBV
mice. Neu+/Tip30-/- mice appeared similar to Neu+/Tip30+/+ mice in size and reached weaning
age at the expected Mendelian frequency. A cohort of Neu+/Tip30-/-, Neu+/Tip30+/- and
Neu+/Tip30+/+ female mice were monitored for 75 weeks. Mammary tumors were noted to
appear earlier in Neu+/Tip30-/- mice compared to Neu+/Tip30+/+ mice. Kaplan-Meier survival
curves (Fig. 3.1A) were generated based on the timing of mammary tumors becoming ≥ 0.5cm3
in volume (≥ 1 cm in diameter). We observed that 50% of Neu+/Tip30+/+ mice developed
mammary tumors with a relatively long median latency of 60 weeks. The relatively longer
median latency and lower frequency of tumors arising in Neu+/Tip30+/+ mice in comparison
with that in MMTV-Neu mice (Guy et al., 1992) are possibly due to only one MMTV-Neu wildtype transgene allele in Neu+/Tip30+/+ mice. By contrast, all Neu+/Tip30-/- mice developed
tumors at a median age of 39 weeks and 87% of Neu+/Tip30+/- mice developed tumors at a
median age of 47 weeks.
103

Figure 3.1 Tip30 deletion significantly accelerates the onset of mammary tumors in
MMTV-Neu mice.
Neu+/Tip30+/+ (n = 10), Neu+/Tip30+/- (n = 15) and Neu+/Tip30-/- (n = 10) female mice were
monitored weekly for a period of 75 weeks and sacrificed at the endpoint or when a tumor
became palpable ≥ 0.5 cm3. A, Kaplan-Meier curves show the timing of mammary tumors
became palpable ≥ 0.5 cm3 in mice. The data were plotted as percentage of tumor-free animals
against the time in weeks. P ≤ 0.0001 (log-rank test). B, Representative hematoxylin and eosin
(H&E) stained mammary tumors arising in Neu+/Tip30-/- mice. A poorly differentiated
adenocarcinoma with solid growth pattern (B1); A moderately differentiated adenocarcinoma
with glandular growth pattern (B2); A pulmonary metastasis (B3). Scale bar, 50 µm. C,
Representative immunohistochemical staining of mammary tumors for K8 (brown staining
indicates presence of K8) and SMA (lack of brown staining indicates lack of SMA). Scale
bar, 10 µm.

104

Figure 3.1 Cont'd

A

100

N+/Tip30 +/+
N+/Tip30 +/N+/Tip30 -/-

% tumor free mice

80

60

40

20

0
20

B

C

B1

40
60
Time (Week)
B2

N+/Tip30 +/+

80

B3

N+/Tip30 -/-

K8

SMA

105

Table 3.1 ER and PR staining in mammary tumors
Tumors

ER+/PR-/-

Neu+/Tip30
Neu+/Tip30+/Neu+/Tip30+/+

ER-/PR-

100% (8/8)
50% (3/6)
11% (1/9)

0% (0/8)
50% (3/6)
89% (8/9)

Figure 3.2 Mammary tumors arising in Neu+/Tip30 -/- mice are exclusively ER+/PR-.
A-C, Representative immunofluorescent staining of ER (red), PR-A (green) and PR-B (green)
in the positive control uterus (A) and mammary tumors arising in Neu+/Tip30-/- mice (B) or
Neu+/Tip30+/+mice (C). Tumor sections were stained with anti-ER, anti-PR-A (hPRa7) or antiPR-B (hPRa6) specific antibodies, followed by counterstaining with DAPI. Scale bar, 10 µm.

106

Figure 3.2 Cont'd

A Uterus
ERα

PRA

PRB

DAPI

DAPI

DAPI

B Neu+/Tip30-/- tumors
ERα

PRA

PRB

DAPI

DAPI

DAPI

C Neu+/Tip30+/+ tumors
ERα

PRA

PRB

DAPI

DAPI

DAPI

107

Histological analysis showed that tumors arising in Neu+/Tip30-/- mice were poorly or
moderately differentiated mammary tumors with solid growth or glandular growth patterns,
morphologically similar to the mammary tumors arising in MMTV-Neu mice (Fig. 3.1, B1 and
B2). Immunohistochemical staining revealed that mammary tumors arising in Neu+/Tip30-/- or
Neu+/Tip30+/+mice consisted of mostly cytokeratin (K8)-positive and -smooth muscle actin
(SMA)-negative tumor cells (Fig. 3.1C), indicating that Neu+/Tip30-/- tumors like MMTV-Neu
tumors (Huang et al., 2005b) are of the luminal cell type. The presence of metastasis in the lung
was observed in 4 out of 10 Neu+/Tip30-/- mice, whereas metastasis was detected in 1 out of 10
Neu+/Tip30+/+ mice. These results suggest that Tip30 loss accelerates the onset of mammary
luminal tumors in MMTV-Neu mice and possibly increases metastasis.
Deletion of Tip30 results in a shift from development of ER-/PR- tumors to ER+/PRtumors in the MMTV-Neu mouse model.
It is well known that MMTV-Neu transgenic mice develop mammary tumors composed
almost exclusively of ER-/PR- luminal epithelial cells. Surprisingly, we found that all
Neu+/Tip30-/- tumors (n = 8) and 50% of Neu+/Tip30+/- tumors (n = 6) examined showed an
ER+/PR- staining pattern (Fig. 3.2B), whereas 89% Neu+/Tip30+/+ tumors (n = 9) were ER-/PR(Fig. 3.2C), indicating that Neu+/Tip30-/- mice were more likely to develop ER+/PR- mammary
tumors compared to Neu+/Tip30+/+ mice (Table 3.1, 100% versus 11%; P = 0.004). These
results suggest that Tip30 loss combined with activation of Neu promotes development of
ER+/PR- mammary tumors.

108

Figure 3.3 Growth of ER+/PR- tumors arising in Neu+/Tip30 -/- mice depends upon
estrogen and progesterone.
A, Two ER+/PR- mammary tumors from Neu+/Tip30-/- mice were minced and inoculated
subcutaneously (s.c.) in the front flanks of ovary-intact (n = 16) or ovariectomized (n = 9) mice.
The graph represents the measurements of tumors by the end of three months after
transplantations or when the tumor volume reaches 1 cm3. B and C, Two ER+/PR- mammary
tumors from Neu+/Tip30-/- mice were minced and inoculated s.c. in the front flanks of
ovariectomized mice supplemented with Placebo (n = 10), estrogen (E2, n = 6), progesterone
(P4, n = 6) or E2 plus P4 (n = 7) pellets. P = 0.012. The graphs show the measurements of tumor
volumes by the end of three months after transplantations or when the tumor volume becoming 1
cm3 (B) and the timing of tumors becoming 1 cm3 in volume or by the end of three months of
tumors becoming  0.25 cm3 (C).  P = 0.009 (Placebo vs E2 + P4). Note that growth of tumors
in two mice of the placebo group were independent of ovarian hormones. D, Growth of ER+/PRtumors after being treated with saline/ethanol vehicle or RU486. Two ER+/PR- tumors arising in
Neu+/Tip30-/- female mice were minced and inoculated s.c. to nude mice. After transplanted
tumors reached approximately 0.5 cm in diameter, mice were divided into two groups. The
groups received either RU486 (6.5 mg/kg body weight) or saline/ethanol vehicle solution s.c.
daily for 7 days. Tumor size was measured by caliper (length and width) for another 7 days.
Tumor increase rate was calculated by comparing tumor volume (1/2 × length × width2) before
and after treatment. P = 0.025. E, Primary tumor cells derived from two ER+/PR- tumors (T1
and T2) were isolated and cultured in the presence or absence of MG132. Cell lysates were
subjected to immunoblotting with hPRa7 antibodies that detect both PR-A and PR-B by
immunoblot.
109

Figure 3.3 Cont'd

A
Tumor volume (mm3)

P = 0.012

OVX

Non-OVX

B
Tumor volume (mm3)

P = 0.039

Placebo

E2

110

E2 + P4

P4

Figure 3.3 Cont'd

C

Placebo
E2
E2 + P4
P4

% of tumor free mice

 P = 0.009



Time (weeks)

Time (weeks)
Tumor volume increase rate

D

P = 0.025

Saline

RU486

+ MG132

E

- MG132

100 kD
PR-A
75 kD
β-actin
T1

T2

111

T1

T2

Estrogen and progesterone promote growth of Neu+/Tip30-/- mammary tumors.
To determine whether ER+/PR- mammary tumors arising in Neu+/Tip30-/- were ovarian
hormone-dependent, we first transplanted small pieces of freshly dissected tumors into ovaryintact (Non-OVX) and ovariectomized (OVX) nude mice and then monitored the growth of
transplanted tumor tissues. Remarkably, removal of both ovaries from recipient mice drastically
reduced growth and progress of transplanted tumors, suggesting that ER+/PR- mammary tumors
that developed in Neu+/Tip30-/- mice are ovary-dependent (Fig. 3.3A). Next, we transplanted
small pieces of freshly dissected tumors into OVX mice supplemented with placebo, estrogen,
progesterone or estrogen plus progesterone pellets (Fig. 3.3B and 3.3C). Surprisingly, estrogen
plus progesterone strongly promoted tumor growth compared to placebo (P = 0.04), while
estrogen or progesterone alone only slightly increased tumor growth compared to placebo (P >
0.05). Moreover, the progesterone antagonist RU486 was able to significantly delay the growth
of Neu+/Tip30-/- tumors (Fig. 3.3D). These results suggest that both estrogen and progesterone
are required for promoting growth of ER+/PR- tumors arising in Neu+/Tip30-/- mice.
The effect of progesterone on the growth of ER+/PR- tumors from Neu+/Tip30-/- mice raises
the question of whether these tumor cells express any PR proteins. Because previous studies
have suggested that active PRs are rapidly degraded in breast cells (Lange et al., 2000), we
speculated that PR was expressed and then degraded rapidly in ER+/PR- tumors. To test this
hypothesis, we examined PR-A and PR-B expression in cultured tumor cells derived from
ER+/PR- tumors. Indeed, PR-A, but not PR-B, was clearly detected by Western Blot analysis
after treatment of cells with the proteasome inhibitor, MG132 (Fig. 3.3E), implying that PR-A is
expressed but rapidly turned over in these tumors. Together, these results suggest that estrogen

112

and progesterone play stimulating roles in the development of ER+/PR- mammary tumors in
Neu+/Tip30-/- mice.
Deletion of Tip30 leads to a progressively increased numbers of p-ER and p-Akt positive
cells in the mammary gland from MMTV-Neu mice.
The preceding data imply that Tip30 may play a key role in suppressing tumorigenesis in
ER-positive (ER+) epithelial cells. To test whether the Tip30 gene promoter is active in ER+
epithelial cells and ER+/PR- tumors, we performed immunofluorescent double staining for ER
and -galactosidase in the mammary glands and tumors derived from Neu+/Tip30+/- mice
harboring a knock-in -galactosidase (-Gal) gene at the Tip30 gene locus under the control of
Tip30 promoter. The -Gal protein was predominantly detected in ER+ mammary epithelial cells
and tumor cells (Fig. 3.4A), indicating that the Tip30 promoter is activated mainly in ER+ MECs
and tumor cells. Given that ER is activated through ligand binding and phosphorylation in
response to estrogen and growth factor induced signaling and that Tip30-/- mice do not exhibit a
significant increase in the number of ER positive cells in the mammary gland at the age of 4
months (Pecha et al., 2007), we therefore asked whether the proportion of p-ER positive cells is
altered in Neu+/Tip30-/- mammary glands. Immunohistochemistry analysis was used to examine
phospho-ER (p-ER) at Ser-171 (equivalent to Ser167 in human) in mammary glands and
tumors from Neu+/Tip30-/- and Neu+/Tip30+/+ mice (Fig. 3.4B). No significant difference in the
numbers of p-ER positive cells was detected in 2-month-old mammary glands from
Neu+/Tip30-/- and Neu+/Tip30+/+ mice (Fig. 3.4C). Strikingly, 12-month-old Neu+/Tip30-/mammary glands and tumors displayed an increase in the number of p-ER positive cells
compared to Neu+/Tip30+/+ mammary glands and tumors (P < 0.05).
113

Figure 3.4 Representative immunohistochemical staining of p-ERα in mammary glands
and mammary tumors from Neu+/Tip30 -/- and Neu+/Tip30 +/+ mice.
A, Representative immunofluorescent double staining of mammary gland and tumor sections
from a Neu+/Tip30+/- mouse for ER (red) and -Gal (green), following by counterstaining with
DAPI (blue). Scale bar, 10 µm. B. Representative immunohistochemical staining of p-ERα in 2month-old and 12-month-old mammary glands and mammary tumors from Neu+/Tip30-/- and
Neu+/Tip30+/+ mice. As a negative control, a uterus section was stained without using the
primary antibody (anti-pER). Scale bar, 10 µm. C, Data represent means  SEM of the
percentage of p-ER positive cells in the mammary glands and tumors derived from
Neu+/Tip30-/- and Neu+/Tip30+/+ mice. p-ER positive cells were counted in the sections of
mammary glands and tumors derived from three mice of each genotype (randomly selected fields
per section). 50 cells were counted per field and 10 fields were counted per mouse.

114

Figure 3.4 Cont'd
-Gal

A

ER

overlay

overlay

Normal
gland

Hyperplasia

Tumor

B

2 mo glands 12 mo glands

Tumor

Positive ctrl

N+/Tip30 +/+ N+/Tip30 -/-

Negative ctrl

% of p-ERα positive cells

C

Uterus

P < 0.0001
P = 0.006
N+/Tip30 -/- n = 3
N+/Tip30 +/+ n = 3

2 mo 12 mo
Tumor
glands glands

115

These results suggest that Tip30 deletion preferentially increases the number of p-ER positive
luminal cells in the mammary gland of MMTV-Neu mice.
Akt is one of the most important downstream factors in HER2/Neu and EGFR signaling
pathways that can phosphorylate ER and regulate MEC and apoptosis (Campbell et al., 2001;
Lannigan, 2003). Previous studies have demonstrated that activation of Akt-1 signaling
accelerates HER2/Neu-mediated mammary tumor formation (Hutchinson et al., 2004), whereas
disruption of Akt-1 delays HER2/Neu-mediated mammary tumorigenesis (Ju et al., 2007;
Maroulakou et al., 2007; Nardulli and Katzenellenbogen, 1988). To examine whether the
deletion of Tip30 affects the activation of Akt (p-Akt) in preneoplastic mammary glands from
MMTV-Neu mice, we performed the immunohistochemical analysis for p-Akt in preneoplastic
mammary glands from Neu+/Tip30-/- and Neu+/Tip30+/+ mice at the age of 2 and 12 months.
MECs at the different ages exhibit negative, weak, intermediate or strong staining for p-Akt (Fig.
3.5; A1, A2, A3 or A4, respectively).
Figure 3.5 Representative immunohistochemical staining for p-Akt in mammary tumors
and mammary glands from Neu+/Tip30 -/- and Neu+/Tip30 +/+ mice.
A, Representative immunohistochemical staining of p-Akt in mammary glands. p-Akt staining in
2-month-old mammary glands ranges from negative to weak (A1 and A2) and is more intense in
12-month-old mammary glands (A3 and A4, intermediate and strong, respectively). Scale bar,
10 µm. B-D, Data represent means ± SEM of the percentage of cells that were stained positive or
negative for p-Akt in 2-month-old (B) and 12-month-old (C) mammary glands and tumors (D)
derived from Neu+/Tip30-/- and Neu+/Tip30+/+ mice. Fifty cells were counted per field and 10
fields were counted per mouse. Data were analyzed by two-tailed t test.
116

Figure 3.5 Cont'd

A
A2

A3

A4

B

% of p-Akt staining cells in
mammary glands aged 2 mo

12 mo
glands

2 mo
glands

A1

N+/Tip30 -/- n = 5
N+/Tip30 +/+ n = 5

Negative

117

Weak

Figure 3.5 Cont'd

C
% of p-Akt staining cells in
mammary glands aged 12 mo

N+/Tip30-/- n = 5
N+/Tip30+/+ n = 5

P = 0.019

Weak or
Intermediate

D

Strong

N+/Tip30-/- n = 5
% of p-Akt staining cells
in tumors

N+/Tip30+/+ n = 5

Weak or
Intermediate
118

Strong

Figure 3.6 Deletion of Tip30 in MECs leads to delayed EGFR degradation.
A, mammary tumor cells isolated from Neu+/Tip30-/- and Neu+/Tip30+/+ mice were incubated
with 100 ng/ml of EGF for 1 hour on ice followed by washing with cold PBS and then incubated
in serum-free medium containing 20 µg/ml of cycloheximide for the indicated times. Whole cell
lysates were blotted with the indicated antibodies. B, Quantification of EGFR protein levels in
(A) using Odyssey 2.1 software. C, primary Tip30+/+ and Tip30-/- MECs were subjected to EGFR
internalization analysis. Representative confocal microscope images show the localization of
EGFR (red) in endosomes after two hours of Alexa488-EGF (green) internalization. Results are
typical and representative of three experiments on primary cells from two mice of each genotype.
Boxed areas are magnified. Representative cells are outlined in white. The colocalization of EGF
and EGFR (yellow) in Tip30-/- cells is indicative of delayed endocytic degradation of EGFR; the
nucleus was stained with DAPI (Grey). Scale bar, 10 μm. D, Quantitative analysis of EGF and
EGFR colocalization. Twenty cells in each group were analyzed using MBF_imageJ. Pearson’s
colocalization coefficients were calculated and converted to percentages. P = 0.035; t test.

119

Figure 3.6 Cont'd

A
Hours

Neu+/Tip30 -/0

1

3

7

Neu+/Tip30 +/+
0

1

EGFR
-Actin

B
Neu+/Tip30 -/Neu+/Tip30 +/+

120

3

7

Figure 3.6 Cont'd

Wild type MEC

EGFR

Tip30-/- MEC

C

EGFR

EGF
overlay

EGF
overlay

D
P = 0.0035

121

No significant difference in p-Akt expression levels and numbers of p-Akt-positive cells (P =
0.678 or 0.972, respectively) was detected between Neu+/Tip30-/- and Neu+/Tip30+/+ mammary
glands at 2 months of age (Fig. 3.5B). However, at 12 months, the number of MECs having
strongly positive p-Akt staining in Neu+/Tip30-/- mammary glands was significantly increased
compared to that in Neu+/Tip30+/+ mammary glands (Strong staining in Neu+/Tip30-/- mammary
gland: 41.4%; Strong staining in Neu+/Tip30+/+ mammary gland: 9.9%; P = 0.02; Fig. 3.5C).
However, there was no significant difference in the levels of p-Akt between mammary tumors
from Neu+/Tip30-/- and Neu+/Tip30+/+ mice (Fig. 3.5D). These data indicate that the relatively
earlier onset of enhanced activation p-Akt in the mammary glands due to Tip30 loss may
contribute to accelerated mammary tumorigenesis in Neu+/Tip30-/- mice.
Tip30 deletion leads to delayed EGFR degradation and sustained EGFR signaling.
Upon binding EGF, EGFR proteins are rapidly internalized and localized in early endosomes,
where they are either sent back to the plasma membrane or sorted into late endosomes and
lysosomes for destruction (Mellman, 1996b; Sorkin and Goh, 2009). Early endosomes serve as a
platform for signaling receptors to activate specific downstream signaling until ligand-receptor
dissociation occurs due to early endosomal acidification mediated by vacuolar (H+)-ATPases
(Forgac, 2007; Murphy et al., 2009a). Recently, we have demonstrated that TIP30 regulates
EGFR signaling by controlling endocytic downregulation of EGFR in primary hepatocytes and
liver cancer cells. Tip30 deletion impairs the fusion of Rab5 vesicles carrying vacuolar (H+)ATPases with early endosomes that contain internalized EGF and EGFR, leading to delayed
EGFR degradation and sustained EGFR signaling7. Therefore, we questioned whether the
increased phosphorylation of Akt and ER in Neu+/Tip30-/- mammary gland are also caused by
a similar mechanism. First, we measured the protein levels of EGFR in mammary tumors cells
122

isolated from Neu+/Tip30-/- and Neu+/Tip30+/+ mammary tumors in response to EGF treatment
at various times after EGF internalization. We used an experimental approach that eliminates the
interference from continuous ligand internalization and nascent protein synthesis to measure
endocytic degradation of EGFR. The comparison revealed that endocytic degradation of EGFR
was significantly delayed in Neu+/Tip30-/- mammary tumors cells compared to Neu+/Tip30+/+
mammary tumors cells, indicating that Tip30 deletion impairs endocytic degradation of EGFR
(Fig. 3.6A and B).
To determine whether Tip30 deletion can block EGFR trafficking from early endosomes to
lysosomes for degradation, we tracked Alexa-488 conjugated EGF (Alexa488-EGF) and EGFR in
normal primary MECs isolated from Tip30-/- and Tip30+/+ mice. The majority of internalized
EGF dissociated from EGFR in wild type MECs two hours after EGF internalization. In contrast,
they remained associated with EGFR in Tip30-/- MECs (EGF-EGFR colocalization in wild type
primary MECs: 11%; EGF-EGFR colocalization in Tip30-/- primary MECs: 55%; n = 20, P =
0.004; Fig. 3.6C and D), indicating that Tip30 deletion causes the trapping of EGF-EGFR
complex in endosomes and sustained endosomal EGFR signaling. To rule out the possibility that
Tip30 deletion increased Neu transgene expression at the level of transcription, we used
quantitative RT-PCR to examine the mRNA expression of Neu transgene in 5- to 9-week-old
Neu+/Tip30+/+ and Neu+/Tip30-/- mice and found no significant difference (data not shown).
Together, these results suggest that Tip30 loss may prolong EGFR signaling, which cooperates
with Neu activation to accelerate Akt activation and to promote the formation of ER+/PRtumors.
Discussion

123

This study was designed to investigate the relationship between HER2/Neu overexpression
and Tip30 deletion in mammary tumorigenesis by using genetically engineered mice containing
both Tip30 deletion and an MMTV-Neu transgene. Strikingly, the data demonstrate that Tip30
deletion cooperates with Neu overexpression to promote exclusive development of ER+/PRmammary tumors in mice. In addition, we show that Tip30 loss impairs endocytic trafficking of
EGF-EGFR, delays EGFR degradation in primary MECs and tumor cells and enhances Akt and
ER phosphorylation in the mammary gland.

These findings, combined with our recent

observation that TIP30 formed a protein complex with ACSL4 and EndoB1 to control EGFEGFR endocytic trafficking in hepatocytes7, strongly suggest a novel mechanism by which loss
of Tip30 contributes to the development of ER+/PR- tumors, at least in part, through enhancing
EGFR/Akt signaling in ER+ MECs.
It is not immediately obvious why Tip30 deletion combined with Neu overexpression cause
the exclusive development of ER+/PR- mammary tumors. The observation that the promoter of
Tip30 was predominantly active in ER+ MECs suggest that Tip30 deletion may mainly affect the
proliferation of ER+ cells by inducing enhanced ER activities, thereby selecting ER+ cells to
initiate tumorigenesis. Indeed, ER+/PR+ mammary tumors developed spontaneously in 22% of
aged Tip30 knockout female mice in the BALB/c genetic background8; and TIP30 was able to
inhibit ER-mediated transcription (Jiang et al., 2004a). The correlation between progressively
increased p-Akt and p-ER positive cells in Neu+/Tip30-/- mammary glands observed in this
study implies that Tip30 deletion may promote the development of ER+ mammary tumors by
enhancing Akt activation and increasing active ER positive cells. Consistent with this scenario,
a previous study showed that Akt overexpression can increase the intensity of ER staining, the
number of ER positive cells and the frequency of ER+ tumors in DMBA-treated mice (Blanco124

Aparicio et al., 2007), although it did not show whether these tumors were PR positive.
Moreover, expression and phosphorylation of ER in ER+ human breast cells is enhanced by the
activation of Akt (Blanco-Aparicio et al., 2007; Campbell et al., 2001). It should be noted that
enhanced Akt activation alone is insufficient for driving the tumorigenic process in mouse MECs
in vivo (Hutchinson et al., 2001); therefore, other mechanisms such as increased expression of cMyc and IGF-1 induced by Tip30 deletion may also contribute to the formation of ER+/PRmammary tumors in MMTV-Neu mouse models.
Although tumors arising in Neu+/Tip30-/- mice are stained negatively for PRs, these
ER+/PR- tumors were sensitive to progesterone stimulation and RU486 inhibition, and PR
proteins were detectable in cultured tumor cells from ER+/PR- tumors when proteasomes were
inhibited. One explanation for these observations is that PR-A is expressed in ER+/PR- tumors
but rapidly turned over due to enhanced activation of EGFR and HER2/Neu in vivo. This
explanation is supported by previous reports that PRs were targeted for degradation by the 26S
proteasome and regulated by MAPK-induced phosphorylation of PRs in cultured breast cancer
cells (Lange et al., 2000). Thus, it is possible that both activated EGFR and HER2 signaling
pathways induce PR hyperphosphorylation and rapid degradation, consequently resulting in a
negative staining for PRs in tumors.
Our data seem to support the hypothesis that ER+ breast cancers arise from ER+ or otherwise
estrogen-responsive progenitor cells (Allred et al., 2004). However, our data do not exclude the
possibility that Tip30 deletion may cause ER-/PR- cells to re-express ER or promote
transformed ER-/PR- luminal progenitor cells to differentiate to ER+/PR- epithelial cells. Studies
on the origin of tumor cells in MMTV-Neu mice have suggested that tumor cells from this model

125

originate from transformed luminal progenitor cells committing to ER-PR- cells (Vaillant et al.,
2008). Therefore, the cell origin of ER+/PR- tumors arising in Neu+/Tip30-/- mice remains to be
determined.
Currently there remains a profound need for more effective therapies for treating
HER2+/ER+/PR- breast cancers because of their poor response and development of resistance to
existing therapies. Nonetheless, the majority of pre-clinical studies of ER positive breast cancer
have relied on cultured cell lines or on xenograft tumor models, in which breast tumor
development and progression does not accurately represent clinical human breast cancer.
Alternatively, the use of genetically engineered mouse models of breast cancer has major
advantages for the investigation of the mechanisms fundamental to mammary tumorigenesis, as
well as the development of anticancer agents. To date, there have been many mouse models of
mammary cancer based on the overexpression or deletion of specific genes that are associated
with human breast cancer. Unfortunately, most mammary tumors arising in those animal models
are ER-/PR- and do not morphologically resemble the major subtype of human breast cancer
(ER+ ductal carcinoma); ER+ mammary tumors are observed in only a few geneticallyengineered mouse models (Liu et al., 2009; Medina et al., 2002; Rose-Hellekant et al., 2007;
Zhang et al., 2005). To our knowledge, there has been no animal model of HER2+/ER+/PRmammary tumors reported. Therefore, our mouse model of HER2+/ER+/PR- breast cancer
provides a valuable tool for deciphering the mechanisms of HER2+/ER+/PR- breast cancer
development and for testing single or combination therapies.
Materials and Methods
Mice, primary MECs and tumor cells
126

Tip30+/- mice in FBV genetic background were generated by backcrossing Tip30+/- C57BL/6
mice (Pecha et al., 2007) with FBV mice seven times. Tip30+/- mice in FBV background were
bred with MMTV-Neu mice (FVB/N-Tg, Jackson Laboratory) to generate Neu+/Tip30-/-,
Neu+/Tip3+/- and Neu+/Tip30+/+ mice. Primary MECs and tumor cells were isolated and
cultured as described previously (Medina, 2000). For tumor transplantation assays, all recipient
mice were 8-week-old Nu/Nu female nude mice (Charles River). For ovariectomized mice both
ovaries of mice were removed under anesthesia. Placebo (25 mg, 90 days release), 17-estradiol
(0.1mg E2, 90-days release), Progesterone (35 mg P4, 21 days release) or E2 + P4 pellets (0.1
mg E2 + 32.5 mg P4, 90 days release) were purchased from Innovative Research of America and
implanted subcutaneously in the front flanks of each mouse, respectively. RU486 was purchased
from Calbiochem. Mice were sacrificed in the end of three months or when the tumor volume
reached 1 cm3. All mice were housed and cared for in the Animal Facility at Michigan State
University according to institutional guidelines.
Immunofluorescence and immunohistochemistry
Immunofluorescent staining of mouse mammary tissues was performed as follow. After
deparaffinization and rehydration, tissue sections were autoclaved and then incubated with
primary antibody specific for ER (MC-20, 1:50; Santa Cruz Biotechnology), p-ER (Santa
Cruz Biotechnology), PR-A (hPRa7, 1:50; Labvision), PR-B (hPRa6, 1:50; Labvision), or -Gal
(Promega) at 4 oC overnight. After PBS rinse, tissue sections were sequentially incubated for 30
min at room temperature with diluted goat anti-rabbit or mouse Alex-488 or -594-conjugated
secondary antibody (1:200; Molecular Probes). Nuclei were counterstained with DAP1.
Immunohistochemical staining of mouse mammary tissues was described previously (Jiang et

127

al., 2007). Immunohistochemical analysis of p-Akt at Ser-473 (1:50; Cell Signaling
Technologies) and p-ER (Santa Cruz Biotechnology) were performed as described previously
(Jiang et al., 2007).
EGFR internalization assay
Tip30+/+ and Tip30-/- primary mammary epithelial cells were isolated from 2-month-old
Tip30+/+ and Tip30-/- female mice and cultured as previously described (Pecha et al., 2007) and
then serum-starved for three hours. Cells were incubated on ice with 100 ng/ml Alexa488-EGF
(Invitrogen) and 20 g/ml cycloheximide on ice for 1 hour and then washed 4 times with cold
PBS before being moved to 37 ºC and incubated in DMEM with 20 g/ml cycloheximide for two
hours. Cells were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1%
Triton X-100 for 2 min and stained for EGFR. Images were obtained with a Zeiss LSM 510 Meta
confocal microscope (Carl Zeiss) using Plan-Apochromat 63×/1.40 Oil objective. Pinhole size
was set to 1 airy unit for all channels. All images are representative single optical sections.
Statistical Analysis
Comparisons among groups were analyzed by two-sided t-test or Fisher’s exact test. A
difference of P < 0.05 was considered to be statistically significant. All analyses were done with
SPSS software, Version 11.5. Data are expressed as mean ± SEM.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
128

We are grateful to Jill Pecha for backcrossing Tip30 deletion allele into FBV mice and Ryan
Brooks, Adam C. Edmunds, George Chen for the excellent technical assistance. We thank Ying
Qin for histopathological expertise and Eran Andrechek for critical reading of the manuscript.
This work was supported by grants RO1 DK066110-01 and W81XWH-08-1-0377 (to H.X.)
from the NIDDK and Department of Defense and grant U01 ES/CA 012800 (to S.Z.H.) from the
National Institute of Environment Health Science (NIEHS) and the National Cancer Institute
(NCI), National Institutes of Health, Department of Health and Human Services. Its contents are
solely the responsibility of the authors and do not necessarily represent the official views of the
NIDDK, NIEHS or NCI, NIH.

129

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Chapter 4 Conclusions, Discussions and Future Directions

135

4.1 EGF dissociates from EGFR and exits the early endosomes in membrane-bound
vesicles
It has become a general theme that receptors release their ligands in the early endosomes
before being recycled back to the plasma membrane or being destroyed in lysosomes. In 1979,
based on a study that individually tracked EGF and EGFR, Carpenter et al suggested that EGFEGFR complexes travel together to lysosomes for degradation (Carpenter and Cohen, 1976,
1979). Our data generated in this study by simultaneously tracking multiple molecules showed
that EGF, like many other ligands, is shed by EGFR earlier in the endocytic pathway. More
strikingly, we demonstrated that EGF is enclosed in membrane-bound vesicles after leaving
endosomes. The EGF vesicles are devoid of the currently known early endosome makers Rab5a
and EEA1 and the late endosome/lysosome marker LAMP1, suggesting that they are previously
undescribed vesicles (Figure 4.1). Significantly, the discovery and characterization of this new
group of vesicles will revise the method that has been adopted to study EGFR trafficking by
tracking the behavior of EGF vesicles.
The physiological significance of secluding EFG in vesicles remains to be determined. It
would be interesting to know if under certain circumstances the enclosed EGF has an opportunity
of being transported back outside of the cell and being reused. Another important question that
needs to be answered is whether other ligands, such as insulin, IGF, TGF and LDL, are also
enclosed in vesicles after being released from the early endosome.

136

EGFR

PM

Endocytic vesicle

EGF
Rab5a
H+

TIP30
complex

H+
H+

PA
V-ATPase

H+
Transporting
vesicle
H+

H+
Late endosome H+
H+
TGN

Figure 4.1 Function of the TIP30 complex
The TIP30 protein complex transfers arachionic acid to PA to initiate membrane fusion between
Rab5a vesicles and endocytic vesicles. Drawing not to scale.

137

4.2 Rab5a vesicles deliver endosomal targeting proteins via membrane fusion
Rab5a is a well established early endosome marker. However, how Rab5a is recruited on
early endosomes remains unclear. It was proposed that Rab5a either comes from the plasma
membrane where it internalizes together with the incoming vesicles or is recruited from the
cytoplasm (Bucci et al., 1992; Pfeffer and Aivazian, 2004). Nonetheless, our Triton X-114
partition experiments (Bordier, 1981) with mouse liver samples showed that Rab5a proteins
exclusively appear in membrane fractions (Figure 4.2). Other members in the Rab protein family
were also revealed in insoluble membrane fractions (Guo et al., 1999; Khosravi-Far et al., 1991).
Moreover, Rab5a was localized in numerous smaller vesicles, which are redistributed from the
perinuclear region to the cell periphery region where they are recruited to early endosomes in
response to EGF (Bucci et al., 1992; Lakadamyali et al., 2006; Leonard et al., 2008; Nielsen et
al., 1999). Our data clearly showed that Rab5a indeed is recruited on early endosomes in
vesicles. Two populations of Rab5a-positive vesicles were characterized. One population is
EEA1-positive endocytic vesicles; another one is EEA1 negative transporting vesicles. More
significantly, we provided a direct mechanistic explanation of how Rab5a regulates endocytic
trafficking by showing that Rab5a vesicles are responsible for the delivery of V-ATPase, a
proton pump that is crucial for ligand-receptor dissociation. Failure to release ligands resulted in
the trapping of ligand-receptor complexes in endosomes.

138

D

A

D

A

D

A

D

A
anti-Rab5a

anti-β-actin

Figure 4.2 Phase separation of Rab5a in Triton X-114
Phase separation of integral membrane proteins in Triton X-114 was performed as described
(Bordier, 1981). Protein samples were prepared from two male mice and two female mice.
Detergent phase (D) and aqueous phases (A) were subjected to SDS-PAGE and immunoblot
using indicated antibodies.

During endocytosis, a number of proteins are recruited to endosomes and lysosomes to
facilitate the movement and maturation of these vesicles and to digest the contents of lysosomes.
It would be interesting to know what other proteins may be carried by the Rab5a vesicles. In
addition to endosome maturation, Rab5a was also required for the maturation of autophagosomes
(Kitano et al., 2008). This observation raised the possibility that Rab5a might also deliver VATPase to phagosomes to create the low pH environment that is needed for content digestion by
acidic proteolytic enzymes.
139

4.3 TIP30, ACSL4 and Endo B1 form a protein complex that interacts with Rab5a and
mediates the fusion of Rab5a vesicles with endocytic vesicles
In this study, we identified a novel TIP30 protein complex containing TIP30, ACSL4, Endo
B1 and Rab5a. Rab5a has been reported as regulator of the endocytic pathway (Bucci et al.,
1992; Chen et al., 2009b; Lanzetti et al., 2004; Miaczynska et al., 2004a; Nielsen et al., 1999;
Sonnichsen et al., 2000). Identification of these two proteins in the protein complex led us to
study the regulation of receptor-mediated endocytosis by these proteins. We first found that these
proteins are required for the endocytic downregulation of EGFR protein levels and signaling
levels. It was later discovered that without these proteins Rab5a vesicles cannot deliver VATPase to endocytic vesicles, leading to the trapping of EGF-EGFR complexes in early
endosomes. In vitro membrane fusion assays indicated that the TIP30 complex acts coordinately
to promote membrane fusion between Rab5a vesicles and endocytic vesicles likely by fatty
acylating phosphatidic acid (PA).
Although proteins in the TIP30 complex have been shown to be important for vesicular
membrane fusion, the precise roles of these proteins in this process are not known. How do these
proteins help the Rab5a vesicles find the specific target membrane to fuse? EEA1 was purified as
a Rab5a effector, which can promote endosome-endosome fusion in vitro. It was shown that the
recruitment of EEA1 to the early endosome membrane depends on its interaction with Rab5a and
its binding to phosphatidylinositol-3-phosphate (Simonsen et al., 1998; Stenmark et al., 1996).
However, a recent study showed that depletion of Rab5a in cells does not affect the distribution
of EEA1 on early endosomes (Chen et al., 2009b). Our data support this observation by
demonstrating that EEA1 appears on early endosomes that are devoid of Rab5a. Therefore, it is
possible that EEA1 is recruited to early endosomes through binding to phosphatidylinositol-3140

phosphate with its FYVE finger. Then specificity is achieved by the recognition and binding of
Rab5a to EEA1. In the meantime, Rab5a recruits the other proteins in the TIP30 complex to the
fusion sites for the initiation of lipid modification.
Another important question is how these proteins facilitate membrane fusion. The ability of
the TIP30 complex to initiate membrane fusion seems to rely on the transfer of an arachidonyl
group to endosomal PA. Of the four proteins that have been tested for membrane lipid binding,
TIP30 and Endo B1 strongly bind to PA, raising the possibility that they may be involves in the
transferring arachidonyl group to PA. This hypothesis is supported by the finding that PA
incubated with the TIP30 complex and arachidonic acid is capable of inducing dramatic
membrane tethering and stacking.
4.4 Arachidonic acid and the synthesis of arachidonyl-coA by ACSL4 is essential for
membrane fusion

The essential role of arachidonic acid in vesicle membrane fusion was first described by Carl
Creutz 30 years ago (Creutz, 1981). He also found that arachidonic acid loses its fusogenic
function if its head group is methylated, suggesting that the carboxyl group needs to be available
for other reactions during membrane fusion. In the following years, the essential role of
arachidonic acid in a variety of membrane fusion events has been reported (Chattopadhyay et al.,
2003; Freeman et al., 1990; Latham et al., 2007; Nishio et al., 1996; Paiement et al., 1994;
Williams et al., 1989). However, the underlying mechanism for its pivotal function remained a
mystery. Recently, in vitro protein binding studies indicated that arachidonic acid directly binds
to syntaxin 1 and stimulates its association with SNAP25 to form SNARE complexes (Connell et
al., 2007). A non-hydrolysable analogue of arachidonic acid was shown to have a similar effect
141

(Latham et al., 2007), raising the question of whether arachidonic acid needs to be activated for
its fusogenic function. Consistent with the Creutz report, our data showed that arachidonic acid is
essential for membrane fusion and that it must be activated by ACSL4 to produce arachidonylcoA.
In vitro membrane fusion experiments utilizing artificial membranes have demonstrated that
a set of proteins are capable of promoting fusion at a physiologically meaningful speed (Ohya et
al., 2009). Significantly, our data showed that at least three more proteins are required for
efficient fusion of biological intact membranes. The next important issue that needs to be
addressed is whether lipid modification by the TIP30 protein complex is universal to all vesicular
membrane fusion events.
4.5 Tip30 deletion in MMTV-Neu transgenic mice leads to the development of ER+/PRtumors
Approximately 5% of breast cancer patients are found with HER2+/ER+/PR- mammary
tumors, which have more aggressive malignant characteristics and an extremely poor response to
endocrine treatment. Mechanistic study of the development of tumors with this phenotype has
been hampered by the lack of mouse models. Significantly, we discovered that Tip30 deletion
resulted in not only earlier onset of tumors in MMTV-Neu transgenic mice, but also the
development of ER+/PR- mammary tumors. Similar to liver cells, deletion of Tip30 in mouse
mammary cells also delayed EGFR destruction and sustained EGFR signaling in response to
EGF treatment, suggesting that the regulation of endocytic trafficking by Tip30 can expand to
other cell types.

142

Enhanced EGFR/Akt signaling has been correlated with hyperphosphorylation of ER and
accelerated protein turnover of PR(Campbell et al., 2001; Kim et al., 2006). Consistently, our
data showed that the levels of pAktS473 and pERS167 were significantly elevated in Tip30-//MMTV-Neu. PR was undetectable by western blot analysis in mammary cells that were isolated
from these mice unless protease a inhibitor was added. Together, these data suggest a mechanism
of the development of HER2/ER+/PR- mammary tumors.
The clinical significance of this study points to the possibility of adding four genes, TIP30,
Rab5a, Endo B1, and ACSL4, as prognosis markers for HER2/ER+/PR- mammary tumors. The
activation sites for Akt have been restricted to early endosomes containing signaling receptors
(Murphy et al., 2009b; Vieira et al., 1996; Wang et al., 2002). Therefore, drugs that target
molecules downstream of the signaling receptor have to be taken into consideration for the
treatment of HER2/ER+/PR- breast cancer patients.

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