A novel sec7 domain containing protein BIG3 and its role in regulated secretory pathway

THE FUNCTIONAL ROLE OF BIG3
IN THE REGULATED SECRETORY PATHWAY






LI HONGYU

INSTITUTE OF MOLECULAR AND CELL BIOLOGY
DEPARTMENT OF BIOCHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2010



THE FUNCTIONAL ROLE OF BIG3
IN THE REGULATED SECRETORY PATHWAY



LI HONGYU
B.Sc.
FUDAN UNIVERSITY

A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

INSTITUTE OF MOLECULAR AND CELL BIOLOGY
DEPARTMENT OF BIOCHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2010

i
Acknowledgements
I would like to express my gratitude to my supervisor, Professor Hong Wan Jin for his
supervision, guidance and support to my work; and to my supervisory committee
members: Dr. Cai Ming Jie and Dr. Walter Hunziker, for their invaluable comments and
advices on my work. I am grateful to Dr. Jill Mae Lan Tham for her constant guidance
and advices. I would like to thank Dr. Vadim Atlachkine and Miss Huang Cai Xia for
their great contributions on generating the BIG3 knockout mice. I would also like to
thank Dr. Wang Cheng Chun for the help on mouse work, to Ng Chee Peng for the help
on electron microscopy work, to Dr. Wei Shun Hui for the help on capacitance
measurement assay, and to Dr. Guo Ke for the help on histological work. My
appreciation also goes to Dr. Tran Thi Ton Hoai and Dr. Ong Yan Shan for their
criticisms and advices on my work. I would like to thank my present and past colleagues
for their help and advices, Dr. Chan Siew Wee, Dr. Loo Li Shen, Dr. Liu Ning Sheng, Dr.
Zhang Xiao Qian, Dr. Paramjeet Singh, Dr. Wang Tuan Lao, Dr. Cai Lei, Dr. Lu Lei, Dr.
Lim Koh Pang, Dr. Tai Gui Hua, Miss Tan Yik Loo, Mr. Lim Chun Jye and Miss Chong
Yaan fun.
My appreciation also goes to the DNA sequencing unit and histology facility of IMCB,
the animal facility of Biological Resource Centre, and the Flow Cytometry Facility of
Biopolis Shared Facilities, for their excellent services.
I specially thank my family for their constant care and support, patience and
understanding.

Hongyu

2010

ii
Table of Contents
Summary
List of Tables
List of Figures
Abbreviations
Chapter 1 Introduction
1.1 General Secretory Pathway
1.1.1 ER, the entry station of secretory pathway
1.1.2 Golgi, the sorting station of secretory pathway
1.1.2.1 Golgi structure and function
1.1.2.2 Golgi biogenesis and intra-Golgi transport
1.1.2.3 TGN, the sorting station
1.1.2.4 Post-TGN carrier formation
1.1.3 Exocytosis
1.2 Regulated Secretory Pathway
1.2.1 Identity of secretory granules
1.2.2 Biogenesis Models of secretory granules from the TGN
1.2.3 ISG Maturation
1.2.4 Secretory Cargo Sorting Mechanisms
1.2.5 Prohormone processing enzymes
1.2.6 Balance of production and demand
1.3 Sec7 proteins
1.3.1 Arf proteins: properties and functions
1.3.2 Activating Arf proteins by Sec7-family GEFs

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1.3.2.1 The Sec7 domain of Arf-GEF

1.3.2.2 BFA inhibition
1.3.2.3 Arf-GEF family
1.3.2.4 BIG3, a putative member of Sec7 protein family
4. Aim of the Study
Figures of Chapter 1
Chapter 2 Materials and methods
2.1 Cloning
2.2 Expression and purification of recombinant fusion proteins
2.3 SDS-PAGE
2.4 Coomassie blue staining
2.5 Immunization of rabbits and affinity purification of antibodies
2.6 Antibodies used in the study
2.7 Cell lines used in the study
2.8 Preparation of tissue and cell lysates
2.9 Western blot
2.10 Indirect immunoflurescence microscopy
2.11 Histological analysis and immuno-staining
2.12 Pancreatic islet isolation and primary culture
2.13 K
+
/Ca
++
, glucose and cycloheximide treatment
2.14 Construct expression in mammalian cells
2.15 Electron microscopy
2.16 GST-GAT pulldown assay
2.17 Short hairpin RNA-mediated gene silencing

iv
2.18 Lenti-viral infection

2.19 Cell sorting
2.20 Quantitative Real-Time PCR
2.21 Pulse-Chase assay using
35
S labeling
2.22 Membrane capacitance measurement
2.23 Animal Welfare and housing
2.24 Generation of the BIG3
-/-
mice
2.25 DNA extraction and genotyping
2.26 Body fat composition measurement
2.27 Glucose, triglyceride, cholesterol, insulin and testosterone measurement

2.28 Glucose and insulin tolerance tests
2.29 Metabolic Cage Studies
2.30 Statistical Analysis
Chapter 3 Identification, expression and localization of BIG3
3.1 Identification of BIG3
3.2 Generation of rabbit polyclonal antibody against BIG3
3.3 Selective expression of BIG3 protein
3.3.1 Expression of BIG3 in mouse tissues
3.3.2 Expression of BIG3 in cultured cell lines
3.3.3 BIG3 is presented in the endocrine pancreatic islets
3.4 BIG3 is localized to secretory granules in MIN6 cells
3.4.1 BIG3 is partially localized to the insulin and CGA containing vesicles
3.4.2 BIG3 partially co-localized with gamma-adaptin
3.4.3 Minimal co-localization of BIG3 with Golgi or endosomal resident markers

v

3.4.4 Co-localization of BIG3 and CGA limited to the perinuclear region, and
sensitive to cycloheximide inhibition

3.5 Presence of BIG3 on dense core granules revealed by EM
3.6 Null catalytic function of BIG3 Sec7 on ARF activation
3.7 Discussion
Figures of Chapter 3
Chapter 4 Functional role of BIG3 in the regulated secretory pathway
4.1 Generation of stable BIG3 knockdown MIN6 cells
4.2 Increased secretory protein storage in BIG3-deficient cells
4.3 Elevated secretion in BIG3-deficient cells
4.3.1 Elevated betagranin secretion upon K+/Ca++ stimulation
4.3.2 Elevated total secretion upon K+/Ca++ stimulation
4.3.3 Increased exocytosis events in BIG3-deficient cells
4.4 Elevated betagranin production in BIG3-deficient cells
4.5 Generating BIG3 Knockout mice
4.6 Increased dense core granule number in BIG3-/- beta cells
4.7 Increased insulin secretion in BIG3-/- islets
4.8 Discussion
Figures of Chapter 4
Chapter 5 Metabolic abnormalities in BIG3 knockout mice
5.1 BIG3 knockout mice developed normally
5.2 Histological examination exhibited fatty liver disease in BIG3 knockout mice
5.3 Altered glucose homeostasis and insulin sensitivity in BIG3 knockout mice
5.4 Altered metabolic parameters in BIG3-/- mice on 129/Sv background

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5.5 Reduced testosterone level in BIG3-/- male mice
5.6 Discussions
Figures of Chapter 5

Chapter 6 General discussion and future perspectives
References


















vii
Summary
The beta cell dysfunction in insulin secretion is one of the key factors in the
pathophysiology of diabetes (Bell and Polonsky, 2001). However the mechanisms that
underline the development of the beta cell dysfunction in humans still remain elusive.
Arf-GEFs are a family of Sec7 domain proteins that regulate intracellular vesicle
trafficking. BIG3 is identified as a putative member of the Sec7 protein family and
distantly related to BIG/Sec7p subfamily. BIG3 mRNA expression profile shows it is
selectively expressed with high levels in the brain and islet. This study therefore
investigates the function of endogenous BIG3 in islet cells. BIG3 protein was found

highly expressed in beta cells, and predominantly localized to the secretory granules.
Investigations on BIG3 knockdown and knockout beta cells demonstrated that the
deficiency of BIG3 caused increased amount of secretory granules and secretory proteins
in the cells, and elevated secretion upon stimulation. The study on BIG3 knockout mice
revealed that in absence of BIG3, the mice exhibited relatively elevated blood insulin
level, disturbed glucose homeostasis, impaired glucose tolerance, reduced insulin
sensitivity, fatty liver disease, disturbed energy expenditure and activity, and reduced
testosterone level. Up-regulated insulin storage and secretion in beta cells may account
for the elevated blood insulin level. The metabolic abnormalities could be in part due to
the excessively secreted insulin. These data demonstrate for the first time that BIG3 has a
functional role in the regulated secretory pathway to modulate the insulin secretion, and
therefore to affect the whole body metabolic homeostasis.




viii
List of Tables
Table 5.1 Mortality of adult BIG3+/+ and BIG3-/- mice on mixed background.

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List of Figures
Figure1.1 Simplified illustration of the general secretory pathway.
Figure1.2 Simplified illustration of the constitutive and regulated pathways.

Figure3.1 Sequence analysis of BIG3 protein.
Figure3.2 Characterization of rabbit polyclonal antibody against BIG3.
Figure3.3 Selective expression of BIG3 in mouse tissues.
Figure3.4 Selectively expression of BIG3 protein in cultured cell lines.
Figure3.5 Expression of BIG3 protein in pancreatic islets.
Figure3.6 Expression of BIG3 in islet alpha and beta cells.
Figure3.7 Partial co-localization of BIG3 with insulin and CGA in MIN6 cells.
Figure3.8 Partial co-localization of BIG3 with gamma-adaptin in MIN6 cells.
Figure3.9 Minimal co-localization of BIG3 with Vti1a, Vti1b or GS28 on separated
vesicles in MIN6 cells.

Figure3.10 Minimal co-localization of BIG3 with Stx6 or GM130 on separated vesicles
in MIN6 cells.

Figure3.11 Dynamic localization of BIG3 and CGA upon K+/Ca++ or cycloheximide
treatment.

Figure3.12 Localization of BIG3 and gamma-adaptin upon K+/Ca++ or cycloheximide
treatment.

Figure3.13 Expression of BIG3 full length construct.
Figure3.14 Preferential localization of BIG3 to dense core granules by Immuno-electron
microscopy.

Figure3.15 Null catalytic function of BIG3 on Arf activation.
Figure4.1 Generation of BIG3 stable knockdown MIN6 cell line.
Figure4.2 Increased betagranin storage in BIG3-deficient MIN6 cells.
Figure4.3 Increased insulin storage in BIG3-deficient MIN6 cells.

x
Figure4.4 Increased betagranin secretion in BIG3-deficient cells.
Figure4.5 Increased total secretion in BIG3-deficient MIN6 cells.
Figure4.6 Increased exocytosis events in BIG3-deficient MIN6 cells.
Figure4.7 Up-regulated processing of CGA to betagranin in BIG3-deficent MIN6 cells.
Figure4.8 Generation of BIG3 targeted embryonic stem cells (E-14).
Figure4.9 Generation of BIG3 knockout mice.
Figure4.10 Increased granule number in beta cells of BIG3-/- mice.
Figure4.11 Increased insulin secretion by BIG3-/- islets.
Figure5.1 Body weight of BIG3+/+, BIG3+/- and BIG3-/- mice on mixed background.

Figure5.2 Body weight of high-fat-diet fed BIG3+/+ and BIG3-/- male mice on mixed
background.

Figure5.3 Body weight of BIG3+/+ and BIG3-/- on 129/Sv background.
Figure5.4 Survival rate of BIG3+/+ and BIG3-/- adult male mice on mixed background.
Figure5.5 Normal morphological appearance of pancreatic islets in BIG3-/- mice on both
backgrounds.

Figure5.6 Fatty liver in BIG3-/- female mice on both backgrounds.
Figure5.7 Progressed fatty liver in a sick BIG3-/- female mice on mixed background.

Figure5.8 Fatty liver in BIG3-/- male mice on both backgrounds.
Figure5.9 Body fat composition of female mice on 129/Sv background at ~4 months old.

Figure5.10 Blood triglyceride and cholesterol level of BIG3+/+ and BIG3-/- mice on
mixed background at 9 months old.

Figure5.11 Blood triglyceride and cholesterol level of BIG3+/+ and BIG3-/- mice on
129/Sv background at 6 months old.

Figure5.12 Blood glucose and insulin levels in BIG3+/+ and BIG3-/- female mice on
mixed background at 18-20 weeks old.

Figure5.13 Blood glucose and insulin levels in BIG3+/+ and BIG3-/- male mice on
mixed background at 16 weeks old.


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Figure5.14 Delayed glucose uptake in BIG3-/- male and female mice on 129/Sv
background at 12 weeks old.

Figure5.15 Delayed glucose uptake in BIG3-/- male and female mice on 129/Sv
background at 24 weeks old.

Figure5.16 Delayed glucose uptake in BIG3-/- female mice on mixed background at 32
weeks old.

Figure5.17 Insulin tolerance challenge in BIG3-/- male mice on 129/Sv background at 14
weeks old.

Figure5.18 Insulin tolerance challenge in BIG3-/- female mice on 129/Sv background at
14 weeks old.

Figure5.19 Insulin tolerance challenge in BIG3-/- male mice on 129/Sv background at 26
weeks old.

Figure5.20 Insulin tolerance challenge in BIG3-/- female mice on 129/Sv background at
26 weeks old.

Figure5.21 Reduced insulin sensitivity in BIG3-/- female mice on mixed background at
34 weeks old.

Figure5.22 Energy Expenditure (heat) and Respiratory parameters of BIG3+/+ and BIG3-
/- male mice on 129/Sv background.

Figure5.23 Food, water intake and activity of BIG3+/+ and BIG3-/- male mice on 129/Sv
background.

Figure5.24 Energy Expenditure (heat) and Respiratory parameters of BIG3+/+ and BIG3-
/- female mice on 129/Sv background.

Figure5.25 Food, water intake and activity of BIG3+/+ and BIG3-/- female mice on
129/Sv background.

Figure5.26 Decreased plasma testosterone level in male BIG3-/- mice on mixed
background at 6 months old.






xii
Abbreviations
aa amino acid
ACTH adrenocorticotropic hormone
AMPK AMP-activated Protein Kinase
AOX coenzyme A oxidase
AP-1 adaptor protein-1
ARF ADP ribosylation factor
Arg arginine
ARNO ARF nucleotide binding site opener
BAR Bin/amphiphysin/Rvs (domain)
BFA Brefeldin A
BIG1/2 BFA inhibited GEF1/2
BLAST Basic Local Alignment Search Tool
bp base pair
BRAG Brefeldin A resistant Arf GEF
BSA bovine serum albumin
Ca
++
calcium ion
CCV clathrin-coated vesicle

cDNA complementary DNA
CGA chromogranin A
CLAMS Comprehensive Lab Animal Monitoring System
COPI/II coat protein complex I/II
Cy3 Cyanine dye 3
DCB Dimerisation and Cyclophilin Binding domain

xiii
DME Dulbecco’s modified Eagles (medium)
DMP dimethyl pimelidate
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
DUF1981 domain of unknown function 1981
EDTA ethylenediaminetetraacetic acid
EFA6 exchange factor for Arf 6
EGFP enhanced green fluorescent protein
EM electron microscopy
ER endoplasmic reticulum
ERGIC ER-Golgi-intermediate compartment
ES embryonic stem
FACS Fluorescence Activated Cell Sorter
FBS fetal bovine serum
FITC fluorescein isothiocyanate
FSH Follicle stimulating hormone
GA Golgi apparatus
GAP GTPase activating protein
GBF1 Golgi-specific BFA resistance factor 1
GDP guanosine diphosphate
GEF guanine-nucleotide exchange factor

GFP green fluorescent protein
GGA1/2/3 Golgi-associated, γ-adaptin ear homology, ARF binding protein 1/2/3

xiv
GH growth hormone
GST Glutathione-S-transferase
GTP guanosine triphosphate
GTPase GTP hydrolase
GTT glucose tolerance test
GWAS genomic-wide association scans
H&E Hematoxylin & Eosin
HBSS Hanks' Balanced Salt solution
HDS Homology Downstream of Sec7
HEPES N-2-hydroethylpiperizine-N’-2-ethanesulfonic acid
His histidine
hr hour
HRP horseradish peroxidase
HUS Homology Upstream of Sec7 domain
i.p. intraperitoneal
IF immunofluorescence
IFG impaired fasting glucose
IP immunoprecipitation
IPTG isopropyl β-D-thiogalactopyranoside
IQ isoleucine-glutamine
ISG immature SG
ITT insulin tolerance test
K
+
potassium ion
kb kilo basepair

xv
kDa kilo Dalton
KRH Krebs-Ringer HEPES buffer
LB Luria Bertani
LH Luteinizing hormone
Lys lysine
M molar
mg milligram
min minute
ml milliliter
mM millimolar
MODY maturity-onset diabetes of the young
MSG mature SG
NAFLD nonalcoholic fatty liver diseases
neo neomycin
ng nanogram
nM nanomolar
O.C.T. Optimal Cutting Temperature (compound)
OD optical density
PAGE poly-acrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
PFA paraformaldehyde
Pfu Pyrococcus furiosus
PH Pleckstrin homology

xvi
PI3,4,5P3 phosphoinositide-3,4,5-trisphosphate
PI4,5P2 phosphatidylinositol 4,5-bisphosphate

PI4K phosphatidylinositol 4 kinase
PKA protein kinase A
PKD Protein kinase D
PLD phospholipase D
PM plasma membrane
PMSF phenyl-methyl-sulfonyl fluoride
POMC Proopiomelanocortin
PP1γ protein phosphotase 1 γ
PRL Prolactin
RIPA radioimmunoprecipitation
RNA ribonucleic acid
RNAi RNA interference
RPMI Roswell Park Memorial Institute (medium)
RSP regulated secretory pathway
SDS sodium dodecyl sulfate
SG Secretory granule
SHBG sex hormone-binding globulin
shRNA short hairpin RNA
siRNA small interfering RNA
SNARE SNAP receptor
TEA-Cl tetraethylammonium chloride
TG triglyceride

xvii
TGN trans-Golgi network
Tris Tris(hydroxymethyl)-aminomethane
TTI tubular transport intermediates
v/v volume to volume
VTC vesicular transport complex
μg microgram

μl microliter
μM micromolar

Chapter 1 Introduction
Balance of energy consumption and storage to adapt to environmental changes is crucial
to the maintainance of metabolic homeostasis in multicellular organisms. The ‘thrifty
genotype’ hypothesis suggests that humans are genetically adapted to the circle of feed
and starvation ( Neel, 1962; Joffe and Zimmet, 1998). However the rapid changes in
human behavior and lifestyle have resulted in worldwide epidemic outbreak of type 2
diabetes (Zimmet et al., 2001). It has been well established that this disease is a
multifactorial syndrome that shows heterogeneity in many respects (Ayres, 1977; Lisanti
et al., 1989; Pillay et al., 1995; Bastian, 2002). The combination of insulin resistance and
abnormal insulin secretion takes a centre role in this disease, although there are debates
on which one is more predominant ( Reaven, 1993; Ferrannini, 1998; Relimpio, 2003).
The balance between insulin action and insulin secretion is coordinated by insulin
sensitive tissues and endocrinal beta cells. Multiple genes and genetic loci responsible for
the genetic susceptibility to type 2 diabetes have been identified to be involved in insulin
signaling, insulin secretion, insulin resistance, glucose metabolism, obesity, and the
hormone processing ( Kahn et al., 1996; Bastian, 2002; Florez, 2008a). However the
mechanisms that underline the development of the insulin resistance and beta cell
dysfunction in humans still remain elusive.

The beta cell dysfunction in insulin secretion is one of the key factors in the
pathophysiology of diabetes (Bell and Polonsky, 2001). It is not only the determinant
factor of the final failure to compensate the insulin demand, but also a major contributor
to the development of the disease (Weir and Bonner-Weir, 2004). Multiple lines of
evidence have demonstrated that the defects in insulin synthesis, processing, and/or
1
secretion can often give rise to the disease. For example, studies on the monogenic
diabetes, maturity-onset diabetes of the young (MODY) have discovered a class of genes

that are expressed in beta cells and control insulin secretion (Velho and Froguel, 1997;
Stride and Hattersley, 2002;). They demonstrate that strong effects of a single gene can
lead to diabetes. However it is much more difficult to identify genes in polygenic
diabetes since their contributions to the diabetic phenotype may be modest, variable
among different populations, and dependent on interactions with other genes and the
environment. Recently genomic-wide association scans (GWAS) have revealed a number
of new genetic loci associated with type 2 diabetes in addition to a few well known genes
(Dupuis et al.; Zeggini et al., 2008). Interestingly, most of them appear to be involved in
insulin secretion rather than insulin resistance (Florez, 2008b). It implies that the genetic
control of insulin secretion may be stronger than that of insulin resistance. It also
suggests the necessity of further efforts to improve our understanding on the detailed
mechanisms of insulin secretion in beta cells.

Insulin release to the body tissues is modulated in amounts and time-dynamics to
maintain plasma glucose within a narrow concentration range (Ferrannini and Mari,
2004). The amount of insulin output must cope with glucose uprise after meals on the
one hand, and with target tissue sensitivity on the other. The rate of insulin secretion
must adapt to stimuli both on a minute basis and in the longer term as well. To achieve
this complex task, the pancreatic beta cells feature an array of specialised functions,
which are finely integrated to respond to highly variable environmental inputs. Endocrine
cells, including beta cells, usually produce and process protein hormones by a common
secretory pathway similar to other secreted proteins in all cells, but they also contain
2
specialized secretory vesicles capable of additional processing, large amount of storage,
and induced release of contents. This regulated secretory pathway allows the fine control
of cellular hormone production and secretion to meet the body’s demand.

Although specialized, the regulated secretory pathway possesses many common
mechanisms of the general secretory pathway, such as the secretory protein production
and modification, intracellular transport and exocytosis (Burgess and Kelly, 1987;

Bonifacino and Glick, 2004; Morvan and Tooze, 2008). The general secretory pathway
starts from the endoplasmic reticulum (ER); goes through Golgi apparatus (GA); then
seperates into distinct transport routes at the trans-Golgi network (TGN); and finally
targets to the plasma membrane (PM), endosomes and other compartments (Enns, 2001).
A simplified diagram demonstrates this pathway in Figure 1.1.

1.1 General Secretory Pathway
Along the secretory pathway, a cell is compartmentalized with a series of subcellular
organelles. The different compartments are defined by membrane-enclosed structures.
Maintenance of the characteristic of a compartment as well as the communications
between compartments is largely achieved by vesicular transport (Lee et al., 2004;
Munro, 2004; Bonifacino and Glick, 2004).

1.1.1 ER, the entry station of secretory pathway
The ER is the starting point of the secretory pathway, firstly described by Palade in 1975.
It is a large intracellular network consisting of differentiated subdomains. Translocan is
the gateway of entry to the ER. New proteins are translocated across or integrated into
3
the ER membrane via the translocan complex (Walter and Lingappa, 1986). The
translocation event is coupled with translation (Crowley et al., 1993). A series of
covalent modifications and conformational foldings take place during and after
translocation.

After proteins are properly processed, they are transported to the Golgi Aparatus via ER-
Golgi-intermediate compartment (ERGIC), either by small transport vesicles or by
prolonged tubular structures (Bonifacino and Glick, 2004; Martinez-Menarguez et al.,
1999). There are two major working models that are implicated for protein export from
the ER (Lee et al., 2004). In active transport model, secreted proteins are selectively
enriched at ER exit sites with specific receptors. ER export receptors would then interact
with coat proteins or their adaptors to trigger the vesicle formation. In the bulk flow

model, no specific receptor or signal sequence is required. By default, the aggregation of
proteins at ER exit sites itself triggers spontaneous vesicle formation. Thus both ER
resident and secreted proteins are transported to Golgi, where ER residents with signal
sequences such as KDEL are sorted back to ER (Lewis and Pelham, 1992). At the
ERGIC, COPI-coated (Coat Protein complex I) vesicular transport complexes (VTCs)
separate from ERGIC and move towards the Golgi (Watson and Stephens, 2005). Parallel
to above pathway, tubular transport intermediates (TTIs) carry cargo that are too large to
enter COPII (Coat Protein complex II) vesicles to the Golgi (Fromme and Schekman,
2005).

1.1.2 Golgi, the sorting station of secretory pathway
1.1.2.1 Golgi structure and function
4
The Golgi was firstly discovered by Camillo Golgi in 1898. Newly synthesized proteins
are further modified and sorted in the Golgi. The Golgi is a highly organized and polar-
distributed complex that consists of membrane-enclosed flat stacked layers of cisternae
with distinct resident enzymes (Mellman and Simons, 1992). In yeast, Golgi simply
exists as individual cisternae (Rossanese et al., 1999). In higher organisms like
mammals, Golgi is a stack of ordered layers, divided into cis, medial, trans faces and
TGN. The distribution pattern of the Golgi depends on cell types. In most plant and fungi
cells, the Golgi stacks scatter throughout the cell (Wooding and Pelham, 1998); while in
a mammalian cell, Golgi stack mainly clusters at the perinuclear region (Sciaky et al.,
1997).

1.1.2.2 Golgi biogenesis and intra-Golgi transport
The formation and maintenance of the Golgi structure is dynamically balanced, where
COPI coated vesicles play important roles in cargo protein sorting and resident protein
retention (Lee et al., 2004). COPI formation is ADP-ribosylation factor (Arf) dependent,
which is a class of small GTPases, and catalyzed by a specific group of guanine-
nucleotide exchange factors (GEFs) and GTPase activatng proteins (GAPs)(Fiedler et al.,

1996; Orci et al., 1993; Zhao et al., 1997).

The polarized Golgi is in the order of the entry, maturation and exit sites for secretory
proteins. There are two major models to explain how the secretory proteins pass through
the Golgi. In vesicular transport model (Rothman, 1994), secretory cargos are carried by
vesicles moving between layers or by bulk flow, while the Golgi cisternae as well as the
resident enzymes mainly remain unchanged. Cisternal maturation model is more widely
5
accepted nowadays(Anelli and Sitia, 2008; Bonfanti et al., 1998; Mironov et al., 1997;
Pelham, 1998). In this model, Golgi cisternae themselves gradually move forward,
carrying cargo proteins and resident enzymes; and resident components are sorted
backwards by tubular-visicular transport. The Golgi is continuously balanced by
consumption at trans face into post-Golgi intermediates and replenishment at cis face by
the pre-Golgi intermediates formed by fusion of ER-derived vesicles.

1.1.2.3 TGN, the sorting station
The outer phase of trans Golgi cisternae is connected to a scattered tubular-saccular
network as the TGN. The most distal layers of TGN are exclusively enriched with
clathrin coats(Ladinsky et al., 1994; Mogelsvang et al., 2004). The TGN is the main
sorting and packing station of newly synthesized protein. Cargos leaving the Golgi from
TGN are sorted into large tubular segments that are elongated and detached from the
TGN(Hirschberg et al., 1998; Polishchuk et al., 2000), and later packed into different
coated vesicles for constitutive secretion(Kornfeld and Mellman, 1989; Pearse and
Robinson, 1990), regulated secretion (Traub and Kornfeld, 1997), endosome pathway,
and others (Rodriguez-Boulan et al., 2005). The morphology and size of the TGN vary in
different cell types according to the amount and type of cargo. An example is that there is
less tubular TGN structure in the cells with regulated secretion compared to that in cells
without secretory granules (Clermont et al., 1995). The size of the TGN also dramatically
changes depending on cargo flux: it reduces when cargo is released and recovers when
cargo is reloaded (Trucco et al., 2004).

6