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Characterization of the retromer complex of proteins in gastric parietal cells
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Characterization of the retromer complex of proteins in gastric parietal cells
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Content
CHARACTERIZATION OF THE RETROMER COMPLEX OF
PROTEINS IN GASTRIC PARIETAL CELLS
by
Asmiti Vivek Sura
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2013
Copyright 2013 Asmiti Vivek Sura
i
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my graduate advisor, Dr Curtis
Okamoto for his constant encouragement and patience. I have been extremely privileged
to work under his guidance and his insights about the subject have only led to increase
my curiosity in research.
I would also like to thank my committee members, Dr. Zoltan Tokes and Dr.
Vijay Kalra for their valuable time and helpful comments.
Last but not the least; I would like to thank my parents and my family for their
constant love and motivation. I am also grateful to my friends for their unassuming
friendship and willingness to help at all times. And finally, a special thanks to my roomie
who has always been a great source of support.
ii
TABLE OF CONTENTS
Acknowledgements i
List of Figures iv
List of Abbreviations vi
Abstract vii
Chapter 1 – Introduction
1.1 Anatomy of Human Stomach 1
1.2 Parietal cell morphology and ultrastructure 3
1.3 Gastric H,K-ATPase 5
1.4 Signal transducation leading to stimulation of acid secretion 6
1.5 Translocation of H,K-ATPase associated with acid secretion –
The membrane recycling hypothesis and vesicular membrane
trafficking machinery
8
1.6 The “Retromer” 12
1.7 Significance 14
Chapter 2 – Materials and Methods
2.1 Gastric Microsomal preparation 16
2.2 SDS PAGE and Immunoblot 16
2.3 Immunoprecipitation & Co-immunoprecipitation 17
2.4 Stripping of gastric microsomes & In-vitro polymerisation 18
iii
2.5 Preparation of isolated rabbit glands 19
2.6 Immunofluorescent staining of isolated gastric glands 19
Chapter 3 – Results
3.1 Immunodetection of retromer on Western blots of purified
gastric microsomes
21
3.2 Immunoprecipitation & Co-immunoprecipitation 27
3.3 Immunofluorescent staining of isolated gastric glands 30
3.4 In-vitro polymerization and reassembly studies 37
Chapter 4 – Discussion 40
Bibliography 44
iv
LIST OF FIGURES
Figure
No.
Figure Title
Pg.
No.
Fig 1.1 Anatomy of human stomach 1
Fig 1.2 Ultrastructure of oxyntic gland 2
Fig 1.3 Parietal cell morphology & ultrastructure 5
Fig 1.4 Gastric H,K-ATPase 6
Fig 1.5 Signal transduction leading to stimulation of acid secretion 7
Fig 1.6 Translocation of H,K-ATPase associated with acid secretion 8
Fig 1.7 Change in parietal cell morphology 10
Fig 1.8 Vesicular membrane trafficking 11
Fig 1.9 Structure of retromer 14
Fig 3.1
Coomassie blue stained gels of purified microsomal fractions
from rabbit gastric mucosa
23
Fig 3.2
Immunoblots of purified microsomal fractions from rabbit
gastric mucosa
24
Fig 3.3
Immunoblot of of purified microsomal fractions from hog
gastric mucosa
26
Fig 3.4
VPS35 secondary antibody alone background control
immunoblot
27
Fig 3.5 Co-IP of VPS35 and H,K-ATPase 29
v
Fig 3.6
Immunoblot showing efficiency of anti-VPS35 antibody to IP
VPS35
29
Fig 3.7 Immunostaining for clathrin (X22) 31
Fig 3.8 Immunostaining for vtiB 32
Fig 3.9 Immunostaining for WAVE1 32
Fig 3.10 Immunostaining for VPS35 34
Fig 3.11 Immunostaining for SNX1 35
Fig 3.12 Immunostaining for SNX2 36
Fig 3.13 Coomassie blue stained gel showing HKα & Clathrin 39
Fig 3.14 Immunoblot showing in-vitro reassembly of retromer 39
vi
LIST OF ABBREVIATIONS
BSA Bovine Serum Albumin
DTT Dithiothreitol
EDTA Ethylene Diamine Tetraacetic Acid
HCl Hydrochloric Acid
MES 2-(N-morpholino)ethanesulfonic acid
MSEP Mannitol Sucrose EDTA PIPES
NaCl Sodium Chloride
PBS Phosphate buffered saline
PBST Phosphate buffered saline with 0.1% Tween
PIPES Piperazine-N,N’-bis(2-ethanesulfonic acid)
SDS-PAGE
Sodium dodecyl sulphate – Polyacrylamide gel
electrophoresis
SNX Sortin Nexin
TDB Triton dilution buffer
TGN Trans Golgi network
TV Tubulovesicles
VPS Vacuolar protein sorting
vii
ABSTRACT
Gastric acid secretion involves the regulated recycling of the H, K-ATPase to and
from the apical membrane of parietal cells. All of the steps in the regulated recycling of
the H,K-ATPase should involve protein trafficking machinery. Based upon preliminary
mass spectrometric analysis of isolated, H,K-ATPase-rich gastric microsomes, subunits
of the retromer complex were identified. We sought to test the hypothesis that the
retromer complex may play an important novel role in regulating the formation of
tubulovesicular membranes in parietal cells and regulate the trafficking of the H, K-
ATPase through this population of membranes. To begin to test this hypothesis, we
characterized the retromer complex in gastric parietal cells. The mass spectrometry
analysis was validated by Western blot of gastric microsomal membranes,
immunoprecipitation, and immunofluorescent localization of retromer subunits.
Moreover, we sought to characterize binding partners of the retromer by co-
immunoprecipitation. Finally, we tried to develop a protocol for the in vitro assembly of
the retromer complex. Taken together, the novel data obtained in this study support the
presence of the retromer complex in a population of H,K-ATP-rich membranes in parietal
cells. However, further study will be required to characterize the functional role of the
retromer in parietal cell function.
1
INTRODUCTION
1.1 Anatomy of Human Stomach
The human stomach can be broadly divided into four regions: (1) cardia, where the
contents from the esophagus get emptied into the stomach; (2) fundus, comprising the
upper curvature of the organ; (3) corpus or body which forms the main central region; (4)
pylorus, the lower section of the organ that facilitates emptying the contents into the
small intestine. The wall of the stomach is organized into numerous concentric tissue
layers consisting of: (1) the mucosa which lines the lumen of the stomach; (2) the
muscularis mucosae which is a thin layer of smooth muscle; (3) the submucosa
comprising of connective tissue and blood vessels; (4) the tunica muscularis, composed
of several layers of smooth muscle; and, (5) the serosa which faces the peritoneal cavity.
Fig 1.1 – Anatomy of human stomach
Image Courtesy: 2009 Encyclopedia Britannica
2
A layer of columnar epithelial cells, called the surface mucous cells, which secrete
mucus and bicarbonate cover the luminal surface of gastric mucosa. These are said to
play a role in protecting the surface from direct acid exposure. The luminal surface is
studded with numerous invaginations, or pits, that serve as conduits for secretions from
the sub-adjacent gastric glands.
The gastric gland is organized in vertical tubular units consisting of an apical pit
region, followed by an isthmus, and then the gland region that forms the lower part of the
vertical unit. A small population of pluripotent stem cells located in the upper neck region
of the glands represents the progenitor cells which differentiate into all functionally
distinct epithelial cell types in gastric glands.
Fig 1.2 – Ultrastructure of oxyntic gland
Image Courtesy: Physiology of the Gastrointestinal Tract by Johnson, L. R. et al (2012)
3
The digestive juice is secreted from mucosa in which several types of secretory
epithelial cells make up the highly organized tubular gastric glands. In mammals, gastric
glands are composed of three major types of secretory epithelial cells: mucous neck cells,
chief cells, and parietal cells. Mucous neck cells are small cells and secrete a mucous
glycoprotein distinct from that of surface epithelial cells. They are located in the region
of transition at the base of the pits extending throughout the neck of the glands. Chief
cells, also known as zymogenic cells, secrete pepsinogen, the inactive precursor form of
pepsin, and predominate at the base of the glands. The large acid-secreting parietal cells,
also known as oxyntic cells, are found throughout the length of the gland, but tend to be
more abundant in the neck region of the gland. Parietal cells represent about 50–60% of
the mass of the secretory mucosa because of their large size and relative abundance.
Subadjacent to the glandular epithelial cells, there are a number of endocrine and
paracrine cells that play important regulatory roles in gastric secretory function. These
include enterochromaffin cells (serotonin), enterochromaffin-like (ECL) cells
(histamine), D cells (somatostatin), and G cells (gastrin-secreting cells) which are also
present at the base of the gland (Johnson, Ghishan et al. 2012) (Wyllie and Hyams 2010).
1.2 Parietal Cell Morphology and Ultrastructure
Oxyntic cells peripherally project onto the walls of the gastric gland, in the
mammalian gastric mucosa; thus they are also called parietal cells. The cell is generally
pyramidal in shape with tight junctions surrounding the apex of the pyramid. The parietal
4
cell is a highly specialized epithelial cell with several distinctive morphological features
that are fundamentally significant to its functional activities (Forte, Machen et al. 1977).
A series of small canals called canaliculi, forming frequent interconnections,
constitute the apical plasma membrane. The canaliculi invaginate from the surface and
project into the cell interior. In the resting parietal cell, these apical canalicular surfaces
are lined with short microvilli that are supported by actin microfilaments as well as
accessory actin-binding proteins. The cytoplasmic space is abundant with membranous
structures, commonly called tubulovesicles (TV), that take the morphological form of
vesicles, tubules, and cisternal sacs (Yao and Forte 2003). The most abundant protein in
this enormous pool of tubulovesicular membranes is the H,K-ATPase, the proton pump
responsible for gastric acid secretion. Tubulovesicles constitute ∼50% of the total
membrane mass (exclusive of mitochondria) and comprise the resting pool of H,K-
ATPase.
Consistent with the energy requirements and high oxidative capacity of these
cells, numerous large mitochondria occupying 30–40% of the cellular mass are also
characteristic of the parietal cell (Johnson, Ghishan et al. 2012) (Forte and Zhu 2010).
5
Fig 1.3 – Parietal Cell morphology and ultrastructure
1.3 Gastric H, K-ATPase
The parietal cell actively pumps out H
+
against a large concentration gradient
(greater than 10
6
-fold) in an obligatory exchange for an equal number of K
+
into the
cytoplasm coupled to the hydrolysis of ATP, at the apical membrane, via the enzyme H,
K-ATPase (Forte, Ganser et al. 1974) (Forte and Zhu 2010). The gastric H, K-ATPase is
an integral membrane protein and belongs to the diverse family of P-type ATPase
transporters that transport a wide variety of substrates such as cations, heavy metals, and
phospholipids.
The enzyme is a heterodimer and is the product of two genes, ATP4A and
ATP4B. The former encodes the 110 kDa α-subunit that has 10 membrane-spanning
segments and has an estimated 73% of its mass residing on the cytoplasmic side of the
membrane (Shin, Besancon et al. 1997). The latter encodes the 32 kDa β-subunit that has
only a single transmembrane segment with more than 80% of its peptide mass, which
Image Courtesy: John G Forte Annu. Rev. Physiol. 2010
6
also includes seven sites of N-linked glycosylation, residing on the extracellular side of
the membrane (Chow and Forte 1995). The two subunits are non-covalently linked.
The α-subunit carries out the catalytic and transport function of the enzyme and
may also encode sorting and targeting sequences responsible for apical membrane
localization (Spicer, Miller et al. 2000). The β-subunit stabilizes and protects the enzyme
from degradation and plays an important role in the precise folding of the enzyme during
synthesis and α/β assembly (Asano, Kawada et al. 2000) (Scarff, Judd et al. 1999). The
small cytoplasmic tail of the β-subunit also contains a putative tyrosine-based endocytotic
motif that may regulate membrane retrieval and recycling of the pump after withdrawal
of stimulation (Courtois-Coutry, Roush et al. 1997).
Fig 1.4 – Gastric H, K - ATPase
1.4 Signal Transduction Leading to Stimulation of Acid Secretion
Acid secretion by the parietal cell is triggered by neurocrine, paracrine, and
endocrine stimuli. Gastric acid secretion is a tightly regulated process triggered by ligand
receptor binding at the basolateral plasma membrane with ultimate output of H
+
, Cl
-
and
Image Courtesy: Michael J Caplan Current Opinion in Cell Biology 1998
7
H
2
O across the apical plasma membrane of the parietal cell (Yao and Forte 2003). The
cholinergic M3 receptor, gastrin CCK-B receptor and histamine H2 receptor localized on
the basolateral membrane bind acetylcholine, gastrin and histamine respectively to
provide the physiological stimuli for acid secretion (Forte and Zhu 2010).
Fig 1.5 – Signal transduction leading to stimulation of acid secretion
The inactive form of the proton pump resides in cytoplasmic tubulovesicles in the
resting parietal cell which is apparently caused by the low permeability of the membranes
to K
+
. Stimulation of acid secretion typically involves an initial elevation of cyclic
adenosine monophosphate (cAMP) and in some cases intracellular calcium, followed by
cascades that activate downstream protein kinases, which trigger the translocation and
insertion of H,K-ATPase from the cytoplasmic tubulovesicles to the apical plasma
membrane of the parietal cell, with concomitant activation of luminal membrane
conductances for K
+
and Cl
-
, leading to active proton pumping (Yao and Forte 2003).
Image Courtesy: Physiology of the Gastrointestinal Tract by Johnson, L. R. et al (2012)
8
Hydrolysis of ATP provides the energy required in pumping H
+
out of the parietal
cells in exchange for K
+
. Cl
−
is also extruded down its electrochemical gradient through
channels that are activated by cAMP in the apical membrane (Wolosin and Forte 1984,
Heitzmann and Warth 2007). Ultimately, acid is generated from the dissociation of two
molecules of water to form H
3
O
+
and OH
−
. The H
3
O
+
is secreted via the proton pump in
exchange for K
+
, while the corresponding OH
−
combines in the cell with carbon dioxide
to form HCO
3
−
. This reaction is catalyzed by the enzyme carbonic anhydrase, which is
enriched in the parietal cells. The formed HCO
3
−
is extruded across the basolateral
membrane in exchange for Cl
−
ions (Wyllie and Hyams 2010).
Fig 1.6 – Translocation of H, K – ATPase associated with acid secretion
1.5 Translocation of H,K-ATPase Associated with Acid Secretion: The Membrane
Recycling Hypothesis and Vesicular Membrane Trafficking Machinery
The ability to conserve energy and mass by recycling is a fundamental process of
all living organisms. This occurs at every level of organization, for example, recycling of
ions across membranes to establish electrochemical gradients, molecular recycling for
energy conservation. Cells bringing about bidirectional intracellular membrane
Image Courtesy: John G Forte Annu. Rev. Physiol. 2010
9
trafficking allows them to execute their specialized functions while conserving energy
(Forte and Zhu 2010).
In non-secreting cells, H,K-ATPase is retained in a quiescent state in cytoplasmic
TVs. In response to extracellular stimuli, parietal cells undergo a profound
morphological transition: the H,K ATPase rich TV’s migrate to the apical canalicular
membrane and fuse with it, giving rise to elongated microvilli and thereby greatly
increasing the apical membrane surface area, with a corresponding decrease of
cytoplasmic TV membrane area (Forte, Machen et al. 1977). The process of apical
membrane expansion from fusion of tubulovesicles continues until the steady state of
secretory activity is achieved. The maximally stimulated oxyntic cell has an apical
membrane surface area that is 5–10 times that of the resting cell (Johnson, Ghishan et al.
2012). Morphometric studies have shown that the total membrane surface area is
conserved; the large apical plasma membrane expansion is quantitatively matched by the
disappearance and decrease in the surface area of cytoplasmic TVs (Helander and
Hirschowitz 1972). It is generally believed that both homotypic fusion among TVs and
heterotypic fusion between TVs and apical membrane occurs during parietal cell
activation of acid secretion (Johnson, Ghishan et al. 2012). The translocation of TV
membranes are effected through protein machinery involving TV docking, priming, and
fusion, and ultimately expanding the apical canalicular surface with H,K-ATPase-rich
membrane and resulting in the secretion of voluminous, isotonic HCl (Wolosin and Forte
1981). This morphological transformation is a highly coordinated process achieved by
10
collaboration of regulated vesicle trafficking, membrane mixing, and the actin
cytoskeleton rearrangement (Okamoto and Forte 2001).
Consequently, large numbers of pump molecules are allowed access to deliver
HCl at the secretory membrane of the cells as long as the stimulus is applied. When the
stimulus is withdrawn, much of the massive amount of apical secretory membrane gets
reincorporated back into TVs within the cytoplasm, hence establishing an efficient switch
off mechanism for when the acid is not required (Forte, Black et al. 1981, Forte and Zhu
2010).
Fig 1.7 – Change in parietal cell morphology
Thus, the phenomenon by which the activation of acid secretion results in a
fusion-based recruitment of H,K-ATPase-rich tubulovesicles into the apical plasma
membrane followed by a complex process of endocytic retrieval of the H,K-ATPase from
the canalicular membrane and the reestablishment of the tubulovesicular compartment of
the resting cell upon cessation of secretion, has contributed to a general consensus for the
membrane recycling hypothesis (Agnew, Duman et al. 1999, Yao and Forte 2003)
Image Courtesy: John G Forte Annu. Rev. Physiol. 2010
11
(Lapierre, Avant et al. 2007). A number of proteins implicated to have a role in directing
the trafficking and recycling of H,K-ATPase-rich membranes have been identified in
parietal cells (Okamoto, Li et al. 2002). These include cytoskeletal proteins such as ezrin
and LASP-1 (LIM and SH3 domain protein 1), small guanine triphosphatases (GTPases)
such as rab11(Calhoun and Goldenring 1996) and ADP-ribosylation factor 6(Matsukawa,
Nakayama et al. 2003), actin-based microfilaments such as F-actin, soluble N-
ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as
VAMP(Karvar, Yao et al. 2002), syntaxins 1 and 3, and SNAP25 and proteins involved
in endocytotic recycling such as clathrin, its adaptors (Okamoto, Karam et al. 1998,
Okamoto, Duman et al. 2000) and dynamin.
Fig 1.8 – Vesicular membrane trafficking machinery
Image Courtesy: John G Forte Annu. Rev. Physiol. 2010
12
1.6 The “Retromer”
Several coat proteins such as clathrin, caveolin, coat protein (COP)I, COPII and
the endosomal sorting complex required for transport (ESCRT) complexes regulate the
process of intracellular membrane trafficking. A peripheral membrane protein assembly,
the retromer, is a relatively recent addition to this list. It plays a key role in retrograde
endosomal-to-trans-Golgi network membrane trafficking (Collins 2008).
The first evidence implicating retromer in the process of protein sorting came
from examining the model eukaryote Saccharomyces cerevisiae. In yeast the retromer is
comprised of five proteins encoded by vacuolar protein sorting (VPS) genes. The
heteropentameric retromer complex can be functionally dissected into two subcomplexes:
a cargo-selective complex formed from a conserved trimer of Vps35p, Vps29p and
Vps26p and a ‘structural complex’ formed from a dimer of the sorting nexin (SNX)
proteins Vps5p and Vps17p (Seaman, McCaffery et al. 1998). The mammalian retromer
complex is comprised of a cargo recognition trimer of Vps26, Vps29, and Vps35, and a
sorting nexin dimer composed of a still undefined combination of SNX1, SNX2, SNX5
and SNX6. SNX1 and SNX2 are largely interchangeable Vps5p orthologs, while SNX5
and SNX6 appear be the functional mammalian orthologs of Vps17p (Rojas, Kametaka et
al. 2007, Wassmer, Attar et al. 2007).
The SNX subunits are characterized by the presence of a phox homology (PX)
domain that allows binding to phosphatidylinositol phosphate membrane lipids (Seet and
Hong 2006). They also contain a Bin/Amphiphysin/ Rvs (BAR) domain immediately C-
terminal to their PX domain, which drives dimerization, assists in membrane recruitment
13
and is able to drive or stabilize membrane tubulation (Carlton, Bujny et al. 2004).The
Vps26-Vps29-Vps35 trimer is responsible for cargo selection and binding and is hence
known as the ‘cargo recognition complex’(Bonifacino and Hurley 2008).
Recruitment of the retromer subcomplexes to the endosomal membrane precedes
or occurs concomitantly with the selection of cargo. At the same time, the SNX dimer
assembles to promote formation of endosomal membrane tubules into which cargo
proteins are sorted. In order to facilitate further rounds of cargo sorting, the retromer
proteins dissociate from the membrane following tubule formation. Recruitment of the
retromer subcomplexes to the endosome is therefore a key element of the regulation of
retromer function (Seaman 2012).
The best characterized transmembrane proteins sorted by the retromer are the acid
hydrolase receptors (Vps10) in yeast (Seaman, McCaffery et al. 1998) and the cation
independent-mannose 6-phosphate receptors (CI-MPR) in mammals (Arighi, Hartnell et
al. 2004). The other prominent retromer cargoes include the Wnt transport protein,
Wntless/MIG-14 (Eaton 2008), and Sortilin-related receptor (SorLA), a protein that binds
amyloid precursor protein (APP) (Nielsen, Gustafsen et al. 2007). Retromer has also been
implicated in transport of the polymeric immunoglobulin receptor (pIgR) and its cargo
IgA from the basolateral to the apical surface of polarized epithelial cells (i.e.
‘transcytosis’) (Vergés, Luton et al. 2004). By facilitating the trafficking of many
membrane proteins, the retromer complex has been linked to processes such as lysosome
biogenesis and pathological conditions like Alzheimer disease (AD) (Seaman 2012).
14
Fig 1.9 – Structure of Retromer
1.7 Significance
So far the retromer had been largely recognized as a key component of the
endosomal protein sorting machinery. However, ever since then, the retromer has been
implicated in a wide range of diverse membrane trafficking pathways, no longer
confining its function to mediating retrograde transport of transmembrane proteins from
endosomes to the trans-Golgi network (TGN). Moreover, it has been proposed that
topologically the role of retromer may be similar to that of clathrin coats (Edeling, Smith
et al. 2006). However, in contrast to the vesicular membrane transport facilitated by
clathrin, retromer appears to regulate trafficking of receptors within morphologically
Image Courtesy: Juan S Bonifacino Current Opinion in Cell Biology 2008
15
distinct tubulovesicular membrane structure, thereby forming a putative ‘coat’ for
tubulovesicular structures (Hierro, Rojas et al. 2007).
Gastric microsome samples had been previously analyzed by mass spectrometry
and peptides corresponding to the retromer subunits had been identified (Rosa
Puertollano, unpublished). Thus, we sought to test the hypothesis that the retromer
complex may play an important novel role in regulating the formation of tubulovesicular
membranes in parietal cells and regulate the trafficking of the H, K-ATPase through this
population of membranes. To begin to test this hypothesis, we characterized the retromer
complex in gastric parietal cells. We sought to validate the mass spectrometry analysis
by Western blot of gastric microsomal membranes, immunoprecipitation, and
immunofluorescent localization of retromer subunits. Moreover, we sought to
characterize binding partners of the retromer by co-immunoprecipitation. Finally, we
tried to develop a protocol for the in vitro assembly of the retromer complex. The novel
data obtained in this study are support the presence of the retromer complex in a
population of H, K-ATP-rich membranes in parietal cells. However, further study will be
required to characterize the functional role of the retromer in parietal cell function.
16
MATERIALS AND METHODS
2.1 Gastric microsomal preparation
Gastric mucosal subcellular membrane fractions were prepared from rabbit and
hog gastric mucosae by differential centrifugation and discontinuous sucrose density
gradient centrifugation according to established protocols(Wolosin and Forte 1981).
Briefly, gastric mucosae were scraped, and the scrapings were minced with surgical
scissors, in a small volume of ice-cold MSEP buffer (125 mM mannitol, 40 mM sucrose,
1 mM EDTA-Tris and 5 mM PIPES-Tris, pH 6.7). The tissue was then diluted with 7
volumes of buffer and homogenized by 15 passes through a Teflon piston homogenizer
(Potter-Elvehejm). The total homogenate was first centrifuged at 15,000 x g for 10
minutes yielding a pellet (P
0
) and a supernatant that was again centrifuged at 100,000 x g
for 60 minutes to obtain the pellet (P100) and the supernatant (S100). P100 was
resuspended in MSEP and loaded on top of a 27%, 32% and 40% or 45% discontinuous
sucrose density gradient and centrifuged at 27,000 rpm overnight in a Beckman SW 41 Ti
rotor. The microsomes sedimenting at each interface were collected and stored at -80º C
in aliquots of 300µl. Protein concentration was measured using Coomassie Plus Protein
Assay Reagent (Pierce) and using BSA as standard.
2.2 SDS-polyacrylamide gel electrophoresis and immunoblot
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to the protocol of Laemmli (Laemmli 1970). Protein samples were
solubilized in a sample buffer containing 2% SDS (w/v), 2% β-mercaptoethanol, 10%
17
glycerol and 10 mM Tris-HCl, pH 6.8, and applied to 10% SDS-polyacrylamide gel.
Proteins were then transferred to a nitrocellulose membrane in a transfer buffer
containing 20% methanol, 192 mM glycine and 25 mM Tris (pH8.3) for 60 minutes at
400 mA. For Western blots, blocking of nitrocellulose was done in a 5% solution of
nonfat milk in PBS-Tween-20 (0.08%) (PBST) for 1 hour at room temperature and then
incubated with the appropriate primary antibody (1: 1000 dilution) in a heat-sealed plastic
pouch, overnight at 4
o
C under rotation. The membrane was washed with PBST, thrice for
15 minutes, and then incubated with the appropriate IRdye labeled secondary antibody
(Rockland Immunochemicals, PA) (1: 2000 dilution), in a heat sealed plastic pouch for 1
hour at room temperature. The membrane was washed again with PBST, thrice for 10
minutes, and the signal was detected using the LICOR Odyssey imaging system.
2.3 Immunoprecipitation and co-immunoprecipitation
Purified gastric microsomes from rabbit or hog were solubilized in 5% Triton
dilution buffer (TDB) consisting of 5% Triton X-100, 100 mM NaCl, 5 mM Na-EDTA,
0.02% NaN
3
, 100 mM triethanolamine-HCl (pH 8.6) and protease inhibitors [4-(2-
aminoethyl)benzenesulfonyl fluoride HCl, phenylmethylsulfonyl fluoride, leupeptin,
antipain and pepstatin]. For denaturing immunoprecipitation, the microsomes were
solubilized in 0.5% SDS and then diluted with 5% TDB to a final Triton X-100:SDS ratio
of 5:1. The samples were incubated with the appropriate antibody and 40 μl of protein G
sepharose overnight at 4
o
C with end-over-end rotation. The immunoprecipitate was
washed 3 times with mixed micelle wash buffer (1% Triton X-100, 0.2% SDS, 150 mM
18
NaCl, 5% w/v sucrose, 5 mM Na-EDTA, 0.2% NaN
3
, 20 mM triethanolamine-HCl (pH
8.6) and protease inhibitors) and once with final wash buffer (150 mM NaCl, 5 mM Na-
EDTA, 0.2% NaN
3
, 20 mM triethanolamine-HCl (pH 8.6) and protease inhibitors).
Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the
appropriate primary and secondary antibodies.
2.4 Stripping of gastric microsomes and in vitro polymerization
For in vitro polymerization of retromer, firstly, purified gastric microsomes were
stripped essentially according to the protocol of Keen, et al. (Keen, Willingham et al.
1979). The retromer and other peripheral membrane proteins were stripped from the
gastric microsomes by incubating the microsome samples in a stripping buffer containing
1 M Tris-HCl (pH 7.0), 4 mM EDTA and 0.4 mM DTT for 20 minutes on ice. The above
mixture was subjected to ultracentrifugation at 200,000 x g, 4ºC for 30 minutes a mini
ultracentrifuge (Sorvall RC M120EX). The stripped proteins, obtained in the supernatant
were dialyzed overnight, against three changes of dialysis buffer (100 mM MES, pH 6.0).
The following day, the dialysate was spun at 200,000 x g, 4ºC for 20 min. The
supernatant was collected and the pellet was resuspended in 100µl of dialysis buffer and
stored at -20ºC until further use. The samples were further analyzed by SDS-PAGE (by
Coomassie blue staining) and Western blot.
19
2.5 Preparation of isolated rabbit glands
The procedures for preparation and isolation of gastric glands followed Berglindh and
Öbrink (1976). Animals were anaesthetised by i.v. administration of nembutal
(Mebumal® 30–60 mg/kg). The gastric mucosa was perfused by high pressure via the
gastric artery with oxygenated phosphate buffered saline prewarmed to 37°C. The
stomach was quickly removed and the mucosa was bluntly dissected from the muscular
layer. After thorough mincing with scissors, the mucosal pieces were digested by
collagenase (Sigma-Aldrich) at 37°C until the suspension became homogenous as judged
by visual inspection. After filtration through nylon mesh, the glands were washed three
times with incubation medium before the volume was adjusted to about 10 mg dry wt/ml.
2.6 Immunofluorescent staining of isolated gastric glands
Isolated rabbit gastric glands were fixed in 3.7% formaldehyde in PBS at room
temperature and subsequently permeabilized in 0.1% Triton X-100 in PBS, or fixed and
permeabilized in cold (-20°C) methanol. The glands were immobilized on polylysine-
coated coverslips. After immobilization, glands were blocked for 30-60 minutes at room
temperature with 1 mg/ml bovine serum albumin (BSA) in PBS-0.5% Triton X-100. The
coverslips were washed thrice by dipping in a solution of PBS. Glands were then
incubated with appropriate primary antibody (1:100 dilution) for 60 minutes at room
temperature, followed by washing of the coverslips as above. Further the glands were
incubated with the appropriate secondary antibody (1:100 dilution) for 60 minutes at
room temperature. Glands were counterstained with Rhodamine phallaoidin (Invitrogen,
20
Molecular Probes, Oregon) (for staining of F-actin) simultaneously with the secondary
antibody. Next, the glands were washed and mounted in Pro-Long Gold antifade reagent
(Invitrogen, Molecular Probes, Oregon). Confocal images of glands were taken with a
Bio-Rad MRC-1024 equipped with a krypton/argon laser using a Zeiss Axioskop
epifluorescence microscope (1.4 oil immersion objective).
21
RESULTS
3.1 Immunodetection of retromer on Western blots of purified gastric microsomes.
Towards assessing the potential role of the retromer in the recycling of the H, K-
ATPase from the apical canalicular membrane back to the TV’s of the resting parietal
cells, and to validate the mass spectrometric detection of retromer subunits in gastric
microsomal membranes (Rosa Puertollano, unpublished), the presence of retromer in
density gradient fractions of H, K-ATPase rich gastric microsomes derived from
tubulovesicles of parietal cells was confirmed by immunoblotting. The gastric microsome
samples were isolated by differential centrifugation and discontinuous sucrose density
gradient centrifugation according to well established protocols as mentioned before.
Along with the traditional sucrose density gradients of 27% and 32%, the samples were
subjected to two more gradients, namely either 40% or 45% sucrose. The inclusion of the
additional gradient step was an attempt to isolate another distinct H,K-ATPase-rich
fraction, that might perhaps represent membranes relatively enriched in retromer
complexes. The microsomes sedimenting at each of the sucrose density gradient
interfaces were collected and analyzed further by SDS-PAGE and immunoblotting.
The Coomassie blue-stained gels of the purified microsomal fractions from rabbit
gastric mucosa are shown in Fig. 3.1 with the position of the α-subunit of the H,K-
ATPase indicated. The H,K-ATPase is clearly present in varying amounts in all of the
fractions, even the crude P100 microsomal fraction. Microsomes sedimenting at the
newly investigated 40% and 45% sucrose interfaces appeared to be more enriched in the
22
H,K-ATPase, relative to the other fractions. Hence the 40% and 45% layers of density
gradient purified microsomes also represent distinct membrane fractions enriched in
parietal cell tubulovesicles, in addition to the traditional 27% and 32% fractions.
As per previous findings, it is known that clathrin is present in these purified
gastric microsomes. A prominent protein band visible in the Coomassie blue-stained gels
is that of clathrin heavy chain, corresponding to approximately 170 kDa. In addition, we
observed here that the 27% and 32% fractions were relatively more enriched in clathrin
as compared to the either the 40% or 45% fractions.
We next looked at the distribution of the retromer in the purified gastric
microsomal fractions. A goat polyclonal Ab was used to detect the VPS35 subunit of the
retromer. A single relatively strong, sharp band migrating around 90 kDa, which is the
predicted size of VPS35 on SDS gels, was detected in all of the purified gastric
microsome fractions. Thus the Western blots (Fig. 3.2) confirmed the presence of this
subunit of retromer in these H,K-ATPase-rich membranes, therefore validating the mass
spectrometric data. However, the level of anti-VPS35 staining varied only slightly, if at
all.
Hence, by comparing the protein profile in the Coomassie blue-stained gels with
the Western blots, we could conclude that the 40% and 45% fractions were relatively
more enriched in the retromer over clathrin, as well as by being enriched in the H,K-
ATPase, consistent with the possibility of the retromer being involved in the recycling of
the H, K-ATPase. This membrane fraction may represent a good source to isolate and
characterize the retromer from tubulovesicles, without significant levels of clathrin.
23
Fig 3.1 – Coomassie blue-stained gels of the purified microsomal fractions from rabbit
gastric mucosa showing HKα and clathrin bands
24
Fig 3.2 – Immunoblots of the purified microsomal fractions from rabbit gastric mucosa
showing VPS35 bands
25
Western blot analysis of density gradient purified microsomes from hog gastric
mucosa was also performed. While a 90 kDa band could be detected with the polyclonal
Ab, it was not as distinct as that observed in the rabbit microsomal membrane fractions.
Thus, the overall immunoreactivity of the anti-VPS35 Ab was observed to be less in the
hog fractions as compared to the rabbit microsomes. This difference in immunoreactivity
could represent species-specific sequence differences in the epitope that is recognized by
the anti-VPS35 antibodies. On the other hand, the blot when reprobed for clathrin heavy
chain, to assess the overall immunoreactivity of other proteins known to be common to
rabbit and hog microsomes, a clear, distinct band corresponding to the molecular weight
of clathrin heavy chain was recognized by the anti-clathrin MAb TD.1 (Fig. 3.3),
suggesting that the Western blotting technique with hog microsomes was sound,
particularly when also compared to the secondary antibody alone background control
Western blot (Fig. 3.4).
Next, we sought to analyze the biochemical behavior of the VPS35 retromer
subunit on gastric microsomes. VPS35 is a peripheral membrane protein, so it should be
subject to being stripped from membranes by high salt washes. The fractions were
subjected to a stripping protocol by incubating the membranes with a 1.0 M Tris-HCl-
based stripping buffer used also to strip clathrin from membrane vesicles. VPS35 was
absent from microsomes when the membranes were subjected to the stripping protocol
(Fig 3.3), consistent with its predicted behavior as a peripheral membrane protein.
26
Fig 3.3 - Immunoblot of the purified microsomal fractions from hog gastric mucosa
showing VPS35 band and stripped microsome sample shows absence of VPS35. The blot
was reprobed for clathrin.
27
Fig 3.4 – VPS35 secondary antibody alone background control Western blot
Hence we confirmed the presence of the VPS35 subunit of the retromer in
purified gastric microsomes from both rabbit and hog and set the stage for evaluation of
the possible involvement of the retromer in the recycling of the H,K-ATPase.
3.2 Immunoprecipitation and co-immunoprecipitation
With the finding that the 40% and 45% fractions of purified microsomes from
rabbit gastric mucosa were co-enriched with H,K-ATPase and the retromer, and
relatively depleted in clathrin heavy chain, our next consideration was to verify whether
there was any interaction between the two, for which a co-immunoprecipitation
experiment was carried out. The 40% microsomal fractions were solubilized in Triton
28
dilution buffer and incubated overnight at 4
o
C with the goat polyclonal anti-VPS35
antibody and protein G sepharose beads. After washing the beads several times, the
immunoprecipitate was analyzed by immunoblotting, wherein the blot was probed with
the both the anti-VPS35 antibody and the mouse monoclonal anti-H,K-ATPase antibody.
The same was carried out for the negative control, except that the anti-VPS35 Ab was
eliminated.
As expected, the 40% rabbit gastric microsome fraction, the starting material for
the immunoprecipitation, showed a very high intensity band for the H,K-ATPase. On
repeating the experiment twice, an overall trend showed a relatively higher intensity band
for the H, K-ATPase in the test-IP as compared to the control IP, although there was
always some slight background for the anti-H,K-ATPase immunoreactivity (Fig 3.5).
Further, the efficiency of the anti-VPS35 Ab to IP VPS35 was confirmed by reprobing
the blot with the same, and a sharp 90 kDa band corresponding to the VPS35 subunit was
seen in the gastric microsome fraction as well as the test-IP (Fig 3.6). Thus the antibody
could specifically recognize the VPS35 subunit of the retromer in immunoprecipitation
experiments and additionally potentially co-immunoprecipitate interacting proteins.
Although a weak signal was observed for the H,K-ATPase, the results suggested
that the retromer may be interacting with the H,K-ATPase. However additional data may
be required to support this possibility.
29
Fig 3.5 – Co-IP of VPS35 and H,K-ATPase showing weak signal indicating possible
interaction of the two (left image: high sensitivity, right image: low sensitivity)
Fig 3.6 – Immunoblot showing efficiency of the anti-VPS35 Ab to IP VPS35.
30
Finally, immunoprecipitation experiments for VPS35 with purified gastric
microsomes from hog were also carried out (data not shown). However, the the ability to
immunoprecipitate VPS35 from hog microsomes, and co-IP the H,K-ATPase from these
membranes was not convincingly validated. However, the inability to immunoprecipitate
VPS35 from hog membranes was consistent with the poor immunoreactivity of the goat
polyclonal anti-VPS35 antibody in immunoblotting of hog gastric microsomal fractions.
3.3 Immunofluorescent staining of isolated gastric glands
To provide supportive evidence that the retromer may reside on the parietal cell
tubulovesicles, and to assess the relative levels of expression of retromer in parietal cells
versus other cells in gastric glands, isolated, fixed rabbit gastric glands were
immunostained with the following antibodies: goat polyclonal antibody for VPS35; and
mouse Mab for SNX1 and SNX2. Gastric glands can be isolated by collagenase digestion
of gastric mucosa. These isolated rabbit gastric glands are primarily composed of larger,
bulging HCl-secreting parietal cells interspersed with the smaller mucous neck cells and
chief cells. In isolated glands, the canalicular (apical) membranes of resting parietal cells
are delineated by intense staining of F-actin with rhodamine –phalloidin. The canaliculi
within parietal cells are clearly identifiable as a network of tubular structures projecting
from the gland lumen into the cell. The apical membranes of parietal cells as well as all
other cell types in the gastric gland form a central lumen which is also prominently
stained with rhodamine-phalloidin. Thus, rhodamine phalloidin can be used as a marker
31
to distinguish the apical membrane of parietal and other cells in the gastric gland, versus
the cytoplasm and the localization of proteins can thereby be deduced.
Fig 3.7 – Left panel: Immunostaining for clathrin (x22) as a reference showing
tubulovesicular and canalicular staining. Central panel : showing F-actin staining at the
apical canalicular membrane and gland lumen. Right panel: shows overlap of left and
central panel. Bar corresponds to 10µ.
32
Fig 3.8 - Left panel: Immunostaining for vtiB as a reference showing only
tubulovesicular staining. Central panel : showing F-actin staining at the apical canalicular
membrane and gland lumen. Right panel: shows overlap of left and central panel. Bar
corresponds to 10µ.
Fig 3.9 - Left panel: Immunostaining for WAVE1 as a reference showing only
canalicular staining. Central panel : showing F-actin staining at the apical canalicular
membrane and gland lumen. Right panel: shows overlap of left and central panel. Bar
corresponds to 10µ.
33
Most of the VPS35 immunoreactivity appeared to be concentrated in the
cytoplasm of the parietal cells. Presence of comma shaped, elongated or circular
structures, resembling tubulovesicular structures was observed. However, we could not
clearly distinguish between labeling of VPS35 on tubulovesicles or other intracellular
membranes and labeling of a cytoplasmic pool of VPS35. No significant immunostaining
for vps 35 was observed within chief cells and mucous neck cells suggesting that the
distribution of immunoreactive VPS35 appeared to predominate in the parietal cells (fig
3.10).
Isolated rabbit gastric glands were also immunostained for other putative subunits
of the retromer complex, sortin nexin-1(SNX1) and sorting nexin-2 (SNX2). The
canonical retromer complex is thought to be comprised of a sorting nexin dimer
composed of a still undefined combination of SNX1 and SNX2 or SNX5 and SNX6.
These SNX subunits can bind to highly curved membranes and tubules or may possess
membrane-bending functions and may be involved in recruiting the VPS26-VPS29-
VPS35 trimer of the retromer complex to the membrane. As with VPS35,
immunoreactivity of SNX-1 and SNX-2 was observed mainly, if not exclusively, in
parietal cells. However, the immunostaining pattern for SNX-1 and SNX-2 within
parietal cells was different from that for VPS35. Some of the distribution of
immunoreactivity of SNX-1 (fig 3.11) and SNX-2 (fig 3.12) overlapped with that of
VPS35 with respect to cytoplasmic (tubulovesicular) staining, but SNX-1 and SNX-2
were also localized near or at canalicular membranes. It is not clear why the distribution
34
of SNX-1 and SNX-2 was different from that of VPS35. However, there are reports that
the sorting nexins may form subcomplexes independent of the entire canonical retromer
holo-complex; the staining observed in parietal cells may reflect the existence of these
distinct subcomplexes.
Fig 3.10 – Immunostaining for VPS35 showing tubulovesicular (cytoplasmic) staining.
Bar corresponds to 10µ.
35
Fig 3.11 – Immunostaining for SNX1showing both tubulovesicular and canalicular
staining. Bar corresponds to 10µ.
36
Fig 3.12 - Immunostaining for SNX2showing both tubulovesicular and canalicular
staining. Bar corresponds to 10µ.
These immunofluorescence data are consistent with the results from the Western
blot analysis of gastric microsomes, in that the retromer, especially VPS35, is present on
membranes that are enriched in the gastric H,K-ATPase. Together, these data represent
the first demonstration of VPS35 on parietal cell membranes. The biochemical and
immunofluorescence data are consistent with the possible interaction of VPS35 (and the
retromer complex) with the H, K-ATPase.
37
3.4 In vitro polymerization and reassembly studies
After confirming the presence of the retromer by immunoblotting and
immunostaining, in the purified gastric microsomes and in isolated rabbit gastric glands,
respectively, followed by proposing its probable interaction with the H,K-ATPase by co-
immunoprecipitation studies, we could conclude that, so far, retromer had appeared to
fulfill a number of criteria fulfilled by other coat proteins. Hence, our next consideration
was to study the in vitro reconstitution and assembly of the retromer, since insight into
the structure and function of other coat protein complexes have been obtained from in
vitro assembly of such complexes.
In an attempt to reveal the nature of assembly of the retromer, we tried to
modulate the conditions such that VPS35 could be obtained in a complex which could
then be precipitated, much like for the case of clathrin (Okamoto, Duman et al. 2000).
The first step in this direction was to strip the gastric microsomes of their peripheral
membrane proteins. Knowing that VPS35 is a peripheral membrane protein, it should be
subject to being stripped from membranes by high salt washes. Thus the fractions were
subjected to a stripping protocol by incubating the membranes with a 1.0 M Tris-HCl
based stripping buffer, also previously used to strip clathrin from membrane vesicles
(Okamoto, Duman et al. 2000). Next, the stripped proteins, obtained in the supernatant
after ultracentrifugation, were dialyzed overnight against three changes of dialysis buffer
(100 mM Na-MES, pH 6.0), while the pellet constituted the stripped microsome fraction.
The dialysate was then centrifuged, to obtain a supernatant and a pellet which was
38
resuspended in the dialysis buffer. The samples were analyzed further by SDS-PAGE and
immunoblotting and the following results were obtained.
Visible in the Coomassie blue-stained gel were the following: The 40% fraction
of the purified microsome from rabbit gastric mucosa, which was the starting material,
was highly enriched in the H, K-ATPase and showed a band corresponding to the clathrin
heavy chain as expected. The pellet after dialysis was comprised of a number of proteins,
one of which corresponded to the molecular weight of the VPS35 subunit of the retromer.
What was encouraging to observe in the pellet was the absence of a significant band
denoting the clathrin heavy chain, indicating enrichment in the retromer over clathrin.
The supernatant was too dilute and did not show any significant protein bands (Fig 3.13).
Further, the samples were analyzed by immunoblotting and were probed with the
antibody against VPS35. The results were as follows: A distinct, sharp band for the
VPS35 subunit was observed in the 40% gastric microsome fraction as well as in the
pellet, whereas, as expected the band was not seen in the stripped microsome sample. In
addition, the stronger signal for VPS35 in the pellet after dialysis suggested an
enrichment of VPS35 in the set of proteins that comprised the pellet after dialysis (Fig
3.14).
Thus the results suggest that the above conditions possibly favored the
purification and in vitro reassembly of the retromer. However, it would be interesting to
observe whether a functional assembly of the retromer was achieved, as was shown for
clathrin (Okamoto, Duman et al. 2000). This may be achieved by further analyzing the
pellet by electron microscopy.
39
Fig 3.13 – Coomassie blue stained gel showing HKα and Clathrin bands.
Fig 3.14 – Immunoblot showing VPS35 band in the pellet and possible purification and
in-vitro re-assembly of retromer
40
DISCUSSION
Gastric parietal cells represent a unique biological model to study vesicular
trafficking. In the resting parietal cell, the gastric H-K-ATPase is sequestered in
intracellular tubulovesicular membranes. When cells are stimulated to secrete HCl, the
parietal cells undergo a profound morphological transition during which the
tubulovesicular membranes fuse with the apical canalicular membrane, thus delivering
the H-K-ATPase to the apical membrane. When the stimulus is withdrawn, the H-K-
ATPase is retrieved from the canalicular membrane, and the tubulovesicular
compartment is reestablished. Thus gastric acid secretion involves the regulated recycling
of the H, K-ATPase to and from the apical membrane of parietal cells. All of these steps
are likely to be regulated by families of protein and vesicle trafficking machinery.
Thus, we sought to test the hypothesis that the retromer complex may play an
important novel role in regulating the formation of tubulovesicular membranes in parietal
cells and regulate the trafficking of the H, K-ATPase through this population of
membranes.
To begin to test this hypothesis, we first characterized the presence of the
retromer complex in H,K-ATPase-rich gastric microsomal membranes, which are derived
from tubulovesicles, by immunoblotting and immunoprecipitation.. The immunoblotting
results clearly demonstrated the presence of the retromer subunit in the H, K-ATPase rich
gastric microsomes, forming the basis for evaluation of the possible involvement of the
retromer in the recycling of the H, K-ATPase. In addition, membrane fractions enriched
41
in the retromer over clathrin could also be obtained, providing a good source to isolate
and characterize the retromer from tubulovesicles without significant levels of clathrin.
To provide supportive evidence that the retromer may reside on the parietal cell
tubulovesicles, and to assess the relative levels of expression of retromer in parietal cells
versus other cells in gastric glands, isolated, fixed rabbit gastric glands were
immunostained with the anti-VPS35 antibody. Most of the VPS35 immunoreactivity
appeared to be concentrated in the cytoplasm of the parietal cells. Presence of comma-
shaped, elongated, punctate, or circular structures, possibly resembling tubulovesicular
structures was observed. However, we could not clearly distinguish between labeling of
VPS35 on tubulovesicles or other intracellular membranes and labeling of a cytoplasmic
pool of VPS35. No significant immunostaining for vps 35 was observed within chief cells
and mucous neck cells suggesting that the distribution of immunoreactive VPS35
appeared to predominate in the parietal cells.
Isolated rabbit gastric glands were also immunostained for other putative subunits
of the retromer complex, sorting nexin-1 (SNX1) and sorting nexin-2 (SNX2). As with
VPS35, immunoreactivity of SNX-1 and SNX-2 was observed mainly, if not exclusively,
in parietal cells. However, the immunostaining pattern for SNX-1 and SNX-2 within
parietal cells was different from that for VPS35. Some of the distribution of
immunoreactivity of SNX-1 and SNX-2 overlapped with that of VPS35 with respect to
cytoplasmic (tubulovesicular) staining, but SNX-1 and SNX-2 were also localized near or
at canalicular membranes. It is not clear why the distribution of SNX-1 and SNX-2 was
different from that of VPS35. However, there are reports that the sorting nexins may
42
form subcomplexes independent of the entire canonical retromer holo-complex; the
staining observed in parietal cells may reflect the existence of these distinct
subcomplexes. However, overall, the immunofluorescence data are consistent with the
results from the Western blot analysis of gastric microsomes, in that the retromer,
especially VPS35, is present on membranes that are enriched in the gastric H,K-ATPase.
Together, these data represent the first demonstration of VPS35 on parietal cell
membranes. The biochemical and immunofluorescence data are consistent with the
possible interaction of VPS35 (and the retromer complex) with the H,K-ATPase.
Our next consideration was to verify whether there was any interaction between
the retromer and the H, K-ATPase, for which the co-immunoprecipitation results
suggested that the VPS35 subunit of the retromer could be specifically recognized and
additionally potentially co-immunoprecipitate interacting proteins. Thus although a weak
signal was observed for the H, K-ATPase, the results suggested that the retromer may be
interacting with the H,K-ATPase. However additional data may be required to support
this possibility.
Finally, structural and functional characterization of a number of coat protein
complexes has depended in part upon the ability to isolate and re-assemble in vitro these
coat complexes. Thus, we tried to develop a protocol for the in vitro assembly of the
retromer complex. In an attempt to reveal the nature of assembly of the retromer, we tried
to modulate the conditions such that VPS35 could be obtained in a complex which could
then be precipitated, much like clathrin. The results suggest that the set conditions
investigated here possibly favored the purification and in vitro reassembly of the
43
retromer. The presence of VPS35 in the pellet fraction after dialysis of stripped
microsomal peripheral membrane proteins denoted that under the said conditions, the
retromer could be reconstituted. However, it would be interesting to observe whether a
functional assembly of the retromer was achieved, as was for clathrin (Okamoto, Duman
et al. 2000). This may be achieved by further analyzing the pellet by electron microscopy.
In summary, the novel data obtained in this study support the presence of the
retromer complex in a population of H,K-ATPase-rich membranes in parietal cells.
However, further study will be required to characterize the functional role of the retromer
in parietal cell function. We would like to characterize the other subunits of the retromer
similarly by immunoblotting, immuoprecipitation, and immunofluorescence. We would
also like to provide stronger evidence for the interaction between the retromer and H,K-
ATPase and may want to identify the sequence of interaction motifs in the H,K-ATPase
involved in retromer mediated transport. All of these data would implicate the retromer
complex in a novel role of regulating the trafficking of the gastric H,K-ATPase during
the secretory cycle of the parietal cell.
44
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Abstract (if available)
Abstract
Gastric acid secretion involves the regulated recycling of the H, K-ATPase to and from the apical membrane of parietal cells. All of the steps in the regulated recycling of the H,K-ATPase should involve protein trafficking machinery. Based upon preliminary mass spectrometric analysis of isolated, H,K-ATPase-rich gastric microsomes, subunits of the retromer complex were identified. We sought to test the hypothesis that the retromer complex may play an important novel role in regulating the formation of tubulovesicular membranes in parietal cells and regulate the trafficking of the H, K-ATPase through this population of membranes. To begin to test this hypothesis, we characterized the retromer complex in gastric parietal cells. The mass spectrometry analysis was validated by Western blot of gastric microsomal membranes, immunoprecipitation, and immunofluorescent localization of retromer subunits. Moreover, we sought to characterize binding partners of the retromer by co-immunoprecipitation. Finally, we tried to develop a protocol for the in vitro assembly of the retromer complex. Taken together, the novel data obtained in this study support the presence of the retromer complex in a population of H,K-ATP-rich membranes in parietal cells. However, further study will be required to characterize the functional role of the retromer in parietal cell function.
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Asset Metadata
Creator
Sura, Asmiti Vivek
(author)
Core Title
Characterization of the retromer complex of proteins in gastric parietal cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
07/31/2013
Defense Date
06/20/2013
Publisher
University of Southern California
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University of Southern California. Libraries
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Tag
apical membrane recycling,gastric microsomes,H K-ATPase,hydrogen-potassium-adenosinetriphosphatase,OAI-PMH Harvest,retromer,tubulovesicles
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English
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Okamoto, Curtis Toshio (
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), Kalra, Vijay (
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), Tokes, Zoltan A. (
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asmitisura@gmail.com,sura@usc.edu
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Tags
apical membrane recycling
gastric microsomes
H K-ATPase
hydrogen-potassium-adenosinetriphosphatase
retromer
tubulovesicles