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Biochemical characterization of hydrogen,potassium-ATPase-rich membranes from the gastric parietal cell

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Biochemical characterization of hydrogen,potassium-ATPase-rich membranes from the gastric parietal cell

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BIOCHEMICAL CHARACTERIZATION OF H,K-ATPase-RICH MEMBRANES
FROM THE GASTRIC PARIETAL CELL
by
Michiko Taniguchi
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
May 2002
Copyright 2002 Michiko Taniguchi
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
U niversity Park
LOS ANGELES, CALIFORNIA 900894695
This th esis, w ritten b y
tfich:ko 'Tanr^uch]
U nder th e d irectio n o f Thesis
C om m ittee, an d app roved b y a il its m em bers,
has been p resen ted to an d accep ted b y The
G raduate School, in p a rtia l fu lfillm en t o f
requirem ents fo r th e degree o f
Dean o f Graduate Studies
&a te May 1 0 , 7 0 0 7______________
THESIS COMMITTEE
7p*k £ g L < t -1
on
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Acknowledgements
I would like to thank my adviser, Dr. Curtis T. Okamoto, who provided me with the
opportunity to discover the excitement o f pursuing the research. Dr. Okamoto’s
careful advice and guidance have greatly nourished my critical thinking during the
research process.
I am thankful to a laboratory technician, Young Y. Jeng, who spent much time with
me for my familiarizing with molecular biology techniques, and gave me precious
advice and encouragement.
I would like to thank my Committee members: Dr. James Ou and Dr. Gunther
Dennert for their time and insightful comments.
Thank you my fellow laboratory members for providing the heart-warming
laboratory atmosphere.
Finally, I would specially like to thank my parents, Man, and Sakura for their
enormous support and love.
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Table of Contents
CONTENTS PAGE
Acknowledgements ii
List of figures iv
List of abbreviations vi
Abstract viii
Introduction I
Chapter 1
1.1. CCV trafficking during gastric acid secretion 3
1.2. CCV formation 5
1.3. A role o f dynamin in vesicle fission 9
1.4. Vesicle transport to a destination 11
Chapter 2
2.1. Purpose of Thesis 17
2.2. Experimental models 17
2.3. Materials and methods 18
2.4. Results and Discussion 25
Conclusion 50
Bibliography 54
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List of Figures
FIGURES PAGE
1. Morphological transition between two parietal cellular states 2
2. CCV formation in the H,K-ATPase insertion and retrieval 4
3. Clathrin triskelion assembly into a lattice 6
4. AP-1 complex in clathrin lattice 8
5. Structure o f non-hydrolyzable GTP analogue GTP y - S 10
6. Domain structure o f dynamin 11
7. Vesicle transport by microtubule motor ATPase 16
8. Structure o f iodixanol 19
9. Vesicle fractionation in OptiPrep gradients (fractions 1-5) in two steps 21
10. Protein distributions through five budding assay fractions (fractions 1-5)
at three different iodixanol concentration 26
11. Cytosolic protein recruitment under three conditions: P100 and S100
mixture (PS), P100 alone (P), S100 alone (S) 29
12. Schematic cell-free budding assay protocol 30
13. CCV protein distributions through five budding assay fractions
(fractions 1-5) in 20% Iodixanol gradients 32
14. CHC distribution in five budding assay fractions (fractions 1-5)
in 20% iodixanol gradients 33
15. Uncoating o f clathrin-coat proteins 35
16. Protein complex formation in S100 38
iv
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17. Western blot analysis of proteins in budding assay SI 00 complex
formed in the absence of membrane 38
18. Dynamin's role in vesicle fission by GTP hydrolysis 39
19. Dynamin as a potential membranous factors for SI00 interaction 40
20. Dynamin immunoreactivity in five budding assay fractions
under various conditions 41
21. Kinesin (A), p50 (B) and dynein (C) distributions through 5 fractions 43
22. pl50G lu e d distribution through five fractions in various conditions 44
23. Immnoblot analysis o f Munc-18 in the five fractions
(20% Iodixanol gradients) in the presence (A), or absence (B) of S 100 47
24. Immunoprecipitation o f Munc-18 with clathrin y - adaptin 49
v
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List of Abbreviations
AP: Adaptor protein
Arp-1: Actin-reiated protein centractin
CCV: Clathrin-coated vesicles
CHC: Clathrin heavy chain
ECL: Enhanced chemiluminescence
ER: Endoplasmic reticulum
GED: GTPase effector domain
HC1: Hydrochloric acid
HKP(a): P(a)-subunit for the H,K-ATPase
Hsc: Heat shock cognate protein
MAbs: Monoclonal antibodies
PH: Pleckstrin homology
PI(4,5)P2: Phosphatidylinositol (4,5)-bisphosphate
PRD: Proline-rich domain
SCAMP: Secretory carrier membrane proteins
SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors
SNAP: Synaptosome associated protein
TGN: Trans-Golgi network
VAMP: Vesicle-associated membrane proteins
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Abstract
Gastric acid secretion is hypothesized to be regulated by the H,K-ATPase trafficking
in clathrin-coated vesicles (CCVs) derived from tubulovesicular (exocytosis) or
apical (endocytosis) membranes depending on the stimulatory status o f a parietal
cell. A significant progress has been made on the characterization o f CCV-associated
molecules; however, regulatory mechanisms by these factors in the gastric acid
secretory cycle remains unclear. To investigate distinct CCV protein recruitment
during the H,K-ATPase transport, we attempted to establish CCV-budding step
reconstitution in a cell-free budding assay, by which the formation o f different
vesicular types was analyzed in iodixanol gradients under various conditions.
Temperature-insensitive protein distribution indicated the presence o f pre-formed
and intermediate vesicular mixtures derived from tubulovesicles. Fractions 1 and 4 in
20% (w/v) iodixanol gradients contained vesicles rich in H,K-ATPase, GTPase-
dynamin, microtubule-dependent motors, and Seel homologue Munc-18. The
enrichment o f these factors suggests that interactions o f proteins from both
membrane-bound, and soluble, cytosolic pools regulate the H,K-ATPase-rich vesicle
trafficking.
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Introduction
Hydrochloric (HC1) acid secretion by a gastric parietal cell is regulated by the H,K-
ATPase (proton pump) trafficking (I I, 38, 42). The exocytic and endocytic cellular
states in the H,K-ATPase trafficking are determined in response to secretagogues
such as histamine and acetylcholine (21, 44). In nonsecreting resting parietal cells,
the H,K-ATPase is stored in tubulovesicles beneath the apical membrane (42, 44). In
secreting active cells, tubulovesicles fuse with the apical membrane, resulting in the
H,K-ATPase transport to the apical membrane with the subsequent H+ pumping into
the gastric lumen (42, 44). The H,K-ATPase is recycled back to the tubulovesicular
compartment to be “reestablished” upon cessation of stimulation (42). Accordingly,
the transition between the two states causes dramatic morphological changes in a
parietal cell (42, 44). A stimulated parietal cell contains a decreased number of
tubulovesicles due to tubulovesicular fusion with the apical membrane resulting in
the apical surface expansion. In a resting state, the apical membrane surface is
limited: intracellular canaliculi which are contiguous with the apical membrane are
surrounded by abundant tubulovesicles (40, 44) (Figure 1). The first chapter reviews
the potential mechanisms of clathrin-coated vesicle (CCV)-mediated H,K-ATPase
trafficking in the gastric acid secretory cycle by focusing on each of the membrane-
associated elements. In the next chapter, regulatory aspects of CCV formation are
discussed with a use of a cell-free system budding assay, in which proteins from both
membrane-bound, and cytoplasmic sources were suggested to be interactive for
regulatory roles.
I
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canaliculi
Resting
H Stimulated
0 canalic
w
apical
membrane
cytoplasm
tubulovesicles
Figure 1. Morphological transition between two parietal cellular states. Upon
stimulation, tubulovesicles are transported to fuse with the apical membrane,
resulting in apical and canalicular membrane surface expansion; the stimulatory
withdrawal accompanies tubulovesicular "re-establishment”.
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Chapter 1
1.1. CCV trafficking during gastric acid secretion
H,K-ATPase trafficking is likely to involve specific accessory (cytosolic) protein
recruitment in the H,K-ATPase recruitment-retrieval pathway (38, 40, 42). In
general, regulation of the trafficking process by CCVs consists of five steps (31, 50)
(Figure 2): I) initiation of clathrin-coated pit formation by the recruitment o f coaling
and accessory proteins, 2) coat assembly by clathrin-lattice docked on a membrane,
3) vesicle budding from a membrane by dynamin’s GTP hydrolysis, 4) vesicle
transport to the target membrane by microtubule-dependent motor proteins, and 5)
recycling of clathrin coat proteins mediated by uncoating proteins. This clathrin-
mediated process is regulated by interactions of a wide variety of molecules cycling
between cytosolic and membrane-bound pools, such as coat components (clathrin,
adaptors), dynamin GTPase, microtubular motor proteins, and proteins regulating
vesicle fusion such as Secl-related proteins.
The proteins discussed below comprise only a small part of the growing list of
specific proteins involved in vesicular trafficking. Our research focuses on the
characterization of distinct membrane trafficking-associated components and their
interactions with intracellular membranes during gastric acid secretory cycle.
3
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H , K-ATPase
Dynamin
Adaptin complex
Clathrin
Uncoating protein
SBfiiflftfeS . H + ggl Initiation S 8 H C oat
; a sse m b ly
v e sic le
b u d d in a
T ransport
Cytosol
l
T ran sp o rt
v esicle
uncoating
V esicle
budding
Clathrin-coated
ubulovesicle -
Figure 2. CCV formation in the H,K-ATPase insertion and retrieval.
4
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1.2. CCV formation
Structure and function o f the gastric H,K-ATPase
The H,K-ATPase is a P-type ATPase residing in tubulovesicles and canaliculi o f the
gastric parietal cell (1, 5, 38, 42, 46). The H,K-ATPase is a heterodimeric complex
of 2 subunits: catalytic a-subunit (U4kDa) with 10 transmembrane spans, and
highly glycosylated (5 -subunit (35 kDa) with a single transmembrane domain (38,
42, 49).
The importance of an a-subunit is implicated in its sites for ATP hydrolysis and
cation transport (42). The function of the [3-subunit is not clear. However, mutation
of a tyrosine residue in the cytoplasmic domain of the [3-subunit. when expressed in
transgenic mice, appeared to result in the development of hyper secretion followed
by gastric ulcers (5, 10, 52). This pathological outcome implicated that the H,K-
ATPase in tubulovesicles was recruited to the apical membrane upon the stimulation;
however, enzyme recycling could not be completed without appropriate tyrosine-
based signals from the [3-subunit (38), thus implicating a role for the [3-subunit in the
trafficking of the H,K-ATPase.
Assembly into an a(3 heterodimer, thereby the subunit interaction, is required for the
minimum enzymatic activity of the H,K-ATPase activity during gastric acid
secretion (38,44). The subunit interaction sites have been investigated, and it
5
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appears that a part of the regulation of gastric H,K-ATPase pump activities is
achieved by the structural association of the two subunits (38). It remains unclear
how the ion pump interacts with other proteins during its membrane trafficking
cycle.
Clathrin and adaptors in the initiation o f vesicular coat assembly
In the clathrin-dependent pathway, clathrin plays a role as a carrier of cargo
molecules such as proteins and lipids between the endosome or the TGN, and the
plasma membrane (6, 31, 50). Clathrin is a trimer of heavy chains (180 kDa) each of
which is associated with a light chain (25 kDa) to form a three-legged structure
(triskelion) (3, 26, 31). In the initiation of CCV formation, clathrin triskelions are
recruited from the cytosol and assemble into a flat lattice, which becomes curved and
invaginated to form the outside structure of a coated vesicle (9, 25, 3 1) (Figure 3).
terrr'
Figure 3. Clathrin triskelion assembly into a lattice [A image of clathrin lattice
from Brodsky et al. (3)].
dorr
heavy chain
light chain
Clathrin triskelion
Clathrin lattice
6
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Clathrin is capable of self-assembling into an empty lattice cage in vitro (26).
Clathrin, which does not interact with plasma lipid bilayers by itself, then binds to
the clathrin box motif (consensus sequence LLpL) in other specific proteins for its
recruitment on a membrane (31). The most abundant protein of this type is a
heterotetrameric adaptor protein (AP) complex (31). AP complexes are classified
into 4 types: TGN-associated AP-l (y, (31, p i, a l), plasma membrane-associated AP-
2 (a, (32, p2, c t 2). and newly discovered AP-3 (83, £3, p 3 ,83) and AP-4
(e4, (34, p4, 84) (26, 31, 41, 42). Adaptors in association with clathrin subsequently
function to mediate clathrin coat assembly into a uniform size, and to direct clathrin
to a specific membrane-docking site (26). There are several factors that determine
adaptor localization on a membrane including the G protein Arf-l association with
AP-l, and the interaction of the N-terminal segment of AP-2 a subunit with
membrane lipids (31). Purified clathrin was still capable of assembling into a cage
structure in vitro (7), thus clathrin-clathrin chain interactions appear to be sufficient
for coat assembly. However, it is believed that clathrin assembly in vivo may be
regulated by adaptins or other vesicular components.
H,K-ATPase as the major cargo in vesicular trafficking in the parietal cell
Tubulovesicles were the parietal sites where both clathrin and AP-l y-adaptin
subunit were identified (42). The AP-l localization on tubulovesicles supports the
tubuiovesicle-derivation from the Golgi apparatus (42). A regulatory role of AP-l in
7
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the H,K-ATPase trafficking was suggested by the co-purification of the AP-L
y-adaptin with the H,K-ATPase (42). On the other hand, AP-2 a-adaptin subunit
and dynamin (see below) in addition to clathrin co-localized with canalicular
membranes in the resting parietal cell (39). This observation indicates that endocytic
activities are occurred in the resting parietal state in which clathrin, AP-2, and
dynamin may play regulatory roles. The endocytic cargo in the resting cell is
currently unknown.
Figure 4. A P-l complex in clathrin lattice. Both yand [3 subunits of an adaptin
interact with two sites of the clathrin heavy chain: a proximal leg and a terminal
domain (50) [A picture of clathrin lattice from Brodsky et al. (3)].
S
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1.3. A role of dynamin in vesicle fission
Followed by the assembly o f coat proteins and membrane factors, CCVs are pinched
off from a membrane for cargo delivery. This process is believed to require GTP
hydrolysis by dynamin (37, 51, 53). Dynamin is a high molecular weight GTPase
(100 kDa) with high intrinsic GTPase activity (28, 37, 53), low GTP affinity (25),
and no activation switches by guanine nucleotides (53). Dynamin is classified into 3
subfamilies: dynamin I (neurons), dynamin II (ubiquitous), and dynamin III
(testis/brain/lung) (28, 53). It is an essential component of vesicle fission from a
plasma membrane in that it facilitates vesicle budding (vesicle closure and release)
by associating with a neck of the clathrin-coated pit (i.e., late stages of coated vesicle
fission) (53). However, the molecular mechanism of dynamin-mediated vesicle
formation remains unclear, especially in its regulatory role by GTP hydrolysis to
achieve CCV fission. The requirement for GTP hydrolysis, not merely GTP binding,
was suggested by GTP-dependent vesiculation, whereas no significant vesicle
formation with GTP-y-S (non-hydrolyzable analogue of GTP) (Figure 5) and other
nucleotides (e.g., ADP and GDP) (53). In addition, the GED (Figure 6) mutant
(T65A) failed to catalyze vesicle fission because of its defect in GTP hydrolysis (37).
Furthermore, self-assembly of dynamin increases GTPase activity (10 to 100 fold)
(9).
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o
HN
N
S O O n 2 '
q H2N N ‘ "^N
11
P . P. P .
HO | O | O | O |
OH OH OH OH
OH
Figure 5. Structure of non-hydrolyzable GTP analogue GTP
y-S (http://wvvw.biolog.de/g019.html).
In neurons, pre-synaptic dynamin that assembled around a neck of the clathrin-
coated pit was arrested in the presence of GTPy-S (25). The observations in neurons
suggest that vesicle fission by dynamirfs GTP hydrolysis was blocked by GTPy-S;
These observations hypothesize a dynamin as a “pinchase” (31, 62) that pinches off a
vesicle from the membrane through conformational changes that result in vesicular
neck constriction caused by GTP hydrolysis (force-generating mechanism).
Alternatively, dynamin may be a recruiter o f specific fission-catalyzing proteins to
membrane-budding sites by associating them with any of the five distinct dynamin
domains: N-terminal GTPase domain (GTP hydrolysis): N- and C-middle domain
(unknown functions); pleckstrin homology (PH) domain (interaction with membrane
lipids); GTPase Effector Domain (GED) (activation of a GTPase domain); proline-
rich domain (PRD) (interaction with cytoskeletal and SH3-containing proteins such
as Grb2, amphiphysin, endophilin, and PLCy) (9. 22, 23, 25, 3 1) (Figure 6).
10
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Figure 6. Domain structure of dynamin
Each domain preferentially interacts with specific molecules to enhance GTPase
activity: the binding of PH domain to phosphatidylinositol (4, 5)-bisphosphate
localizes dynamin to a membrane. GED interaction with the GTPase domain
increases dynamin assembly with a result from dynamin-dynamin interaction, which
subsequently oligomerizes dynamin (25. 37). A large body of research into dynamin
functions has revealed that altered coated pit formation results from mutated
domains.
1.4. Vesicle transport to a destination
The newly budded vesicles are ultimately transported to a target membrane. To
achieve this, the coated vesicle delivery process requires: 1) determination of
destination where vesicles are to be fused, and 2) generation of a driving force for
vesicles to reach its destination.
Vesicle targeting and fusion
Vesicle fusion involves the complex assembly of soluble N-ethylmaleimide-sensitive
factor attachment protein receptor (SNARE) proteins on transport vesicles (v-
1 1
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SNAREs) and ones on target membranes (t-SNAREs) (44, 48). Expression of
SNARE proteins was observed in the parietal cell including syntaxins 1, 2, 4 and 3,
synaptosome-associated protein (SNAP)-25, vesicle-associated membrane protein 2
(VAMP2) (44). Furthermore, H.K-ATPase-rich tubulovesicles were co-
immunoisolated with 2 SNARE proteins (syntaxin 3 and VAMP) (44), secretory
carrier membrane proteins (SCAMPs) (4); and, rabl 1 and rab25 (small GTP-binding
proteins) (4).
It has been suggested that SNARE complex formation is regulated by the S eel- and
rab-family proteins through high affinity binding with SNARE proteins (44, 48).
Munc-I8 is a homologue of the Seel proteins that interact with syntaxins during
vesicle fusion (47). Regulatory functions of Seel-syntaxin complexes are implicated
in epithelial cells (Munc-18-2 and syntaxin 3 complex) (44. 47): Munc-18-2 mutants
with defective binding to syntaxin switched as its interaction partner to SNAP-23
(another t-SNARE), and the Munc-18-2 overexpression inhibited the association of
appropriate SNARE pair complexes (syntaxin 3 and SNAP-23) (48). These data
together suggest that the regulatory factors of gastric vesicle fusion process may
include: 1) the association of SNAREs and Munc-18 with H.K-ATPase-rich
tubulovesicle, and 2) the involvement of small GTP-binding proteins. Accordingly, it
appears that intense protein-protein interactions occur for SNARE complex assembly
to achieve vesicle fusion. Seel-syntaxin association was also observed to regulate in
12
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GLUT-4 translocation in adipocytes (Munc-l8c/syntaxin 4), and in ER-to-Golgi
trafficking (Seel homologue r-Slyl/syntaxin 5) (44, 57).
Vesicle transport to a destination
Energetic force is required to drive the newly-formed vesicle to a target membrane
where t-SNARE and v-SNARE protein assembly occurs. The plasma membrane of
an epithelial cell contains two distinct plasma membrane domains, the apical and the
basolateral membrane surfaces, which imparts a structural and functional polarity to
these cell types (9, 34). Cytoskeletal structures known as microtubules serve as
tracks along which vesicles travel in a directed fashion, and these microtubules are
also organized in a polarized manner in epithelial cells with their "plus'* ends
oriented towards the basolateral side (34). Thus, microtubules can serve to direct
polarized vesicular traffic in epithelial cells. For example, in a stimulated parietal
cell, tubulovesicles move towards the apical membrane for H,K-ATPase insertion,
whereas during the transition from a stimulated state to a resting state, membranes
containing the H.K-ATPase move away from the apical membrane toward the
basolateral membrane (Figure 7).
The bi-directional vesicular transport is mediated by microtubule-dependent motor
proteins that transport vesicles towards either end of the microtubule by sliding along
the microtubular track (8, 34). The motor proteins generate the forces for vesicle
transport along a microtubule by associating with vesicles and through ATP
13
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hydrolysis for their microtubule-based mechanochemical motor activity (8, 34).
Based on the direction of movement of the motor relative to the polarity of the
microtubule, motor proteins may be classified into two groups: plus-end-directed
proteins (e.g., kinesin, kinesin-related proteins) or minus-end-directed proteins (e.g.,
dyneins) (34). However, particular motor proteins may be responsible for motion in
one direction or both directions.
Kinesin is a plus-end directed motor that utilizes the release of the product ADP for
its interaction with a microtubule (15, 19). Kinesin consists of a dimer of heavy
chains (110-130 kDa) and a pair of light chains (58-65 kDa) (34). Kinesins have
been shown to regulate vesicle transport and may be in involved in microtubule
crosslinking (19).
Dynein (120-150 kDa) is a minus end-directed motor that is classified into two types:
cytosolic and axonemal dyneins (18. 60). Cytoplasmic dynein consists of two heavy
chains (500 kDa); three intermediate chains (74 kDa); four light intermediate chains
(50-60 kDa): one or two light chains (8-11 kDa) (29. 34). Dynein functions in
mitosis in spindle organization, spindle orientation, and chromosomal movement
(18. 60). It is also involved in transport along the endocytic pathway such as protein
movement from early to late endosomes, regulation of endosome distribution within
the cell: and, axonal transport in neurons (18). Activation of dynein requires its
interaction with a multisubunit regulatory complex called dynactin (61).
14
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Dynactin is composed of a minifilament of an actin-related protein Arp-1
(centractin): the largest pl50G ,u cd (2 molecules); the second most abundant p50 (4 or
5 molecules); and other components (30, 34). Investigation of the role o f individual
subunits of dynactin in dynein-dynactin association has provided insight into the
regulation of dynein-driven motility by dynactin. For example, pl50G lu cd binds to
both dynein intermediate chains (IC), microtubules (35. 59), and a cargo molecule
(e.g., vesicle). Thus, the p i50 subunit may play dual roles in that it mediates dynein
recruitment to a microtubule, and facilitates dynein-driven vesiclular motor activity.
15
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B asolateral
H,K-ATPase packaged
tubulovesicle Apical
1
C W l ' l ! 1" U N i n M R H U W W M U
microtubule
kinesin dynein-
dynactin
complex
Js
f
% £ * * %
W %
✓
Figure 7. Vesicle transport by microtubule motor ATPases.
16
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Chapter 2
2.1. Purpose of Thesis
H,K-ATPase trafficking in the gastric acid secretory cycle may be mediated by
CCVs. CCV formation is regulated by a variety of membrane-bound and cytosolic
proteins that functions at distinct vesicle-formation steps. However, the regulatory
significance of these types of proteins including coat, accessory, and microtubule
motor proteins is unclear in the trafficking of the H.K-ATPase in the parietal cell, in
which extensive interactions among membrane-bound and cytosolic elements are
believed to occur. Thus, our research objective was to characterize in vitro the
mechanisms by which the CCV-mediated trafficking of parietal cell membrane
vesicles is regulated.
2.2. Experimental models
We attempted to reconstitute parietal cell CCV formation with a cell-free budding
assay to investigate the molecular mechanism of the regulation of CCV-mediated
vesicular trafficking. We attempted to evaluate our system by measuring in vitro
production of vesicles from a crude membrane preparation and to identify and
characterize proteins associated with newly formed vesicles.
17
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2.3. Materials and methods
Antibodies
Anti-clathrin heavy chain monoclonal antibody (MAbs) TD .l was obtained from
TD. I hybridoma (American Type Culture Collection) cell culture supernatants. Anti-
y-adaptin MAb 100/3 was purchased from Sigma Chemical (St. Louis, MO). Anti-
adaptin (3 , anti-adaptin a , anti-adaptin y, anti-p50, anti-pl50C I U L ‘ d, and anti-dynamin
MAbs were purchased from Transduction Laboratories (Lexington, KY). Anti-H, K-
ATPase P subunit MAb 2/2E6 was obtained from culture supernatant from 2/2E6
hybridoma. Anti-dynein monoclonal antibody and anti-kinesin heavy chain
monoclonal antibody DK 410.4.1 were purchased from Chemicon International, Inc.
(Temecula, CA).
Secondary IgG (goat anti-mouse and goat anti-rabbit) conjugated to horseradish
peroxidase (HRP) and SDS-PAGE pre-stained molecular mass standards, were
purchased from Bio-Rad (Hercules, CA). Enhanced chemiluminescence (ECL)
reagents were purchased from Pierce Chemical (Rockford, IL).
Preparation o f gastric microsomes
Rabbit gastric mucosa was minced and added to 5 volumes of ice-cold MSEP (125
mM mannitol, 40 mM sucrose, I mM EDTA, 5 mM PIPES, pH 6.7). which was
subsequently homogenized by a Potter-Elvehjem homogenizer (15 strokes).
Followed by centrifugation o f the homogenate (1000 x g, 10 min), the resulting
18
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supernatant was re-centrifuged (100,000 x g, 60 min) to generate microsomal
membrane pellets (P100) and cytosolic supernatant (S100). PlOO was resuspended
with MSEP; protein concentration in PlOO (mg/ml) was measured
spectrophotometrically using the BCA protein assay (Pierce Chemical Co.). Both
PlOO and S 100 were stored at -80°C.
Membrane vesicle separation using iodixanol gradients
lodixanol, 5, 5’ -[(2-hydroxy-1, 3-propanediyl)-bis(acetylamino)| bis-[N.N'-bis(2, 3-
dihydroxypreopyl-2, 4, 6-triiodo-l. 3-benzenecarboxamide)| (Figure 8) is a nonionic,
iodinated density gradient compound which isolates biological materials (9, 25).
r 7
HOCH*CHCH2NHn.c ^ ° NHCHjCHCH,OH
HOCHyCHCH2NHv.
Figure 8. Structure of iodixanol
(http://www.ps.toyaku.ac.jp/-dobashi/database/index.html).
Advantages of a use of iodixanol (Figure 9) include: lower osmolarity and viscosity;
no measurable cytotoxicity or enzymatic inhibition: rapid self-generating gradient
formation (i.e., in less than a few hour centrifugation) (2. 12). OptiPrep is capable of
shifting iodixanol densities up to 1.320 g/ml with an osmotic balancer such as
19
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sucrose for organelles (12, 43), resulting in iso-osmotic conditions for biological
material (1.329g/ml densities). Accordingly, physiological hydration properties of
vesicular components are maintained in iodixanol (12, 45, 58); hyperosmotility (cf.
high osmotic sucrose gradients) may cause cell rapture by changing physiological
hydrated state (16, 45).
The iodixanol gradients were established in two steps with OptiPrep (60% w/v
iodixanol stock in H^O): the formation of pre-formed iodixanol gradients; the
subsequent formation of self-generating iodixanol gradients by layering budding
assay sample on the pre-formed iodixanol gradient solution (Figure 8). This
iodixanol gradient preparation achieves the faster vesicle separation in comparison
with conventional discontinuous sucrose gradients that usually require overnight
centrifugation.
20
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20% iodixanol
budding
assay
sample
iodixanol
pre-formed
gradients
generated
from 20
% Iodixanol
Iodixanol
startin g
co n cen tratio n
300,000 x g, 2hrs
150,000 x g,
40 min
Pre-form ed Iodixanol
g rad ien ts
fraction 1
fraction 2
fraction 3
fraction 4
fraction 5
low
density
white interface band
Self-generating
Iodixanol grad ien ts
Figure 9. Vesicle fractionation in OptiPrep gradients (fractions 1-5) in two
steps.
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Preparation o f iodixanol-density (iso-osmotic) gradients
To obtain the desired iodixanol concentration, OptiPrep (60% w/v iodixanol stock in
HiO) (Nycomed, Norway) was diluted with 1 x MSEP. OptiPrep density gradients
were pre-formed by centrifugation o f a 20% OptiPrep solution diluted from the 60%
stock solution with MSEP in an RP55S-485 Sorvall swinging bucket rotor in a
Sorvall RC M120EX miniultracentrifuge with the brake-off during both acceleration
and deceleration (300,000 x g, 2hr, 4°C). Membrane suspension (PlOO diluted with 2
times volume of MSEP or a sample prepared from the budding assay) was replaced
with the top 100 pi portion of the gradient solution followed by centrifugation
(150,000 x g, 4°C. 40 min) to fractionate the membranes on the self-generating
gradient. Beginning from the top of the gradient, five fractions were collected. Each
fraction was resuspended in MSEP to the final volume of 1 ml followed by
centrifugation (300.000 x g, 41 > C, 20min). The final membrane pellets were
resuspended in 1 x SDS sample buffer for SDS-PAGE analysis.
Budding assay of the gastric microsomal fractionation
A crude H,K-ATPase-enriched microsomal fraction (PlOO; 0.2mg of protein) was
added together with cytosol (Sl00) and budding assay buffer (25 mM HEPES. pH
6.7, 25 mM KCL. 1.5 mM magnesium acetate) followed by addition of an ATP
regenerating system solution ( I mM ATP, 200 U .M GTP or 200 pM GTPyS, 8.0 mM
creatine phosphate, 0.043 mg/ml creatine phosphokinase). The resulting mixture
(100 pl/tube) was incubated (30 min, 37°C), and the reaction was stopped by placing
22
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it on ice (10 min). The mixture was loaded onto iodixanol gradients as described
above.
SDS-PAGE, Coomassie blue-staining, and Western blotting
The SDS-PAGE protocol was described by Laemmli (33). Sample-boiling time was
minimized to 2 min to minimize boiling induced H,K-ATPase loss from the sample.
The protein composition in the five collected fractions were qualitatively analyzed
by Coomassie blue staining. Immunoreactivity of membrane-associated proteins to
the antibodies (described above) was detected by Western blotting. Non-specific
antibody binding sites were blocked with 5% nonfat milk in PBS-Tween 20 (0.05%)
or TBS (for MAb TD. 1). Primary antibodies were diluted as follows: MAb 100/3,
1:5,000: MAb 2/2E6. 1:200 (cell culture supernatant): MAb TD. 1, 1:1.000: Anti-
adaptin (3 , anti-adaptin a , anti-adaptin y, anti-p50, anti-pl50G lu cd . and anti-dynamin
MAbs, all 1:1.000. Proteins were transferred to nitrocellulose. Immunoreactions
were visualized on Kodak Bio-Max-X-ray film by the detection of HRP-conjugated
goat anti-mouse IgG (1:20,000) or goat-rabbit IgG (1:50,000). For the sequential
analysis of different antibody-induced immunoreactivity of a blot, the nitrocellulose
was stripped in 100 mM P-mercaptpmethanol, 2% SDS. and 62.5 mM Tris-HCl (pH
6.7) before proceeding to primary antibody binding.
23
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Immunoprecipitation of rabbit PlOO
Following OptiPrep gradient separation of rabbit PlOO, fractions I and 4 (400 jil
each) in each microcentrifuge tube were diluted with immunoprecipitation buffer
(2.5% TDB [2.5% Triton X-100, 100 mM triethanolamine HCl, pH 8.6, 100 mM
NaCl, 5 mM Na-EDTA, 0.02% NaN^l, and protease inhibitor cocktail [pepstatin,
chymostatin, leupeptin, benzamidinel, phenylmethylsulfonyl floride, 5 mM NaF,
ImM NaV04) (600 pi for each fraction) to the final volume of I ml with MSEP. The
resultant mixtures were incubated overnight at 4°C with anti-Muncl8 and protein A-
Sepharose. Beads with immune complexes were washed in mixed micelle buffer
(MMB) [4 M NaCl. I M triethanolamine HCl. pH 8.6. 65% w/v sucrose. 10% NaN-*,
20% Trion X-100, 10% SDS] (three times) and final wash buffer (FWB) [same
ingredients as MMB except that detergent and sucrose are excluded] (once). The
final pelleted beads were reusupended in I x SDS SB for Western blot analysis with
anti-100/3.
Uncoating investigation o f clathrin-coat proteins
Budding assay samples (PlOO; budding buffer; ATP. ATP regenerating system;
GTP) were incubated in the presence (PS) or absence (P) of S 100 (37°C for 30 min),
and were subsequently diluted with two volumes of MSEP. The resulting samples
were centrifuged (100.000 rpm. 20 min). by which supernatant (containing potential
uncoated proteins) and pellets were generated. The supernatant and pellets were
analyzed by immunoblotting.
24
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2.4. Results and Discussion
Determination of starting iodixanol concentration for the optimal vesicle
separation
Microsomal proteins in the budding assay were separated into five fractions on
iodixanol gradients (fractions 1-5). With 12.5% or 15% iodixanol, a white band was
observed at the interface of fractions 4 and 5. Materials present in the band were
collected mostly in fraction 5. Protein separation by SDS-PAGE followed by
Coomassie blue-staining showed that fraction 5 contained the highest amount of
proteins, indicating that the majority of vesicles fractionated at a density that could
not be resolved by the gradient formed in 12.5% iodixanol (Figure IOAB). Thus,
iodixanol concentration was further increased to try to resolve distinct membrane
vesicle populations better. At 20% iodixanol, most of the white interface band
materials were collected in fraction 4 and less in fraction 5. On Coomassie blue-
stained gels, proteins in fraction 5 were shifted towards the upper part o f the gradient
(fractions 1-4) (Figure IOC), especially to the fraction 4, which indicated that more
different vesicle types were resolved (more pellets floating to the gradients). Thus,
vesicle fractionation was optimized by the increased iodixanol concentration;
determination of optimal iodixanol concentration was made at 20% for the desired
vesicle resolution in an assay.
25
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Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
A 12.5% B 15% C 20%
Figure 10. Protein distributions through five budding assay fractions (fractions 1-5) at three different iodixanol
concentrations. The crude gastric membrane (PlOO) and soluble cytosolic protein (S100) samples were assayed in the
presence ATP, an ATP regenerating system, and GTP. The fractions were separated on SDS-PAGE gels and stained with
Coomassie blue. A: iodixanol 12,5%, B: iodixanol 15%, C: iodixanol 20%, 95 kDa HKawas enriched throughout the five
fractions. 160 kDa CHC was observed more clearly at the lower Iodixanol concentrations (i.e., 12,5% and 15%).
Vesicle sorting in cell-free budding assay
(i) Modification of the original budding assay model
Our primary objective with a use of budding assay was to investigate the regulation
of vesicle formation in vitro under differing conditions. Our hypothesis was that
specific cytosolic proteins and ATP and/or GTP hydrolysis facilitated vesicle
budding from tubulovesicular membranes at physiological temperature (37°C): these
budded vesicles would then fractionate in low-density iodixanol fractions, whereas
relatively large dense tubulovesicular bulk would remain in the lower portion of the
gradient (i.e., higher density) (Figure 12A).
The budding process should be sensitive to temperature and should not occur at 0°C,
therefore the difference was expected to observe in the amounts of produced vesicles
and protein composition. However, no significant difference in protein distribution
was observed between the samples incubated under two different temperatures (37°C
or 0°C), particularly with respect to the protein content in the low density fractions
(e.g.. fractions 1-3) where newly-formed vesicles were expected to be localized (data
not shown). These results suggested that the starting assay samples had contained
pre-formed vesicles, and no further significant vesicle budding was occurred or
detectable under the conditions of the assay (Figure 12B). These original vesicles
may have been derived from early endosomes. some other unidentified intracellular
membrane organelle, or the plasma membrane (39). Therefore, vesicles observed in
the upper fractions were unlikely to be pinched off from high-density membranes.
27
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Thus, reconstitution of budding mechanisms could not be effected within this assay.
It would be of interest if vesicular components in each fraction were morphologically
examined in the electron microscope, which may provide evidence of the presence o f
different vesicle types by differences in morphology.
(ii) Membrane-associated protein recruitment by P100-S100 factor interactions
Due to the possible presence of pre-formed vesicles in the PlOO membrane fraction
and the lack of qualitative evidence of newly formed vesicles, we focused on specific
membrane-associated proteins present in iodixanol fractions and their recruitment to
membrane vesicles. Thus, incubations were performed under three conditions: 1)
PlOO alone, 2) S I00 alone, and 3) PlOO and S I00 mixture. Based on the observation
that SlOO-alone sample contained a small amount of proteins in the upper two
fractions on a Coomassie blue-stained gel (Figure 16), protein distributions of these
three types of samples were compared for the upper two gradient fractions, fractions
1 and 2.
Simultaneous incubation of PlOO (membranous) and S100 (soluble) proteins
appeared to result in more protein recruitment than with individual incubation of
each pool (Figure 11). Upon inspection, the combined staining intensities of proteins
in fractions I and 2 resulting from separately incubated PlOO and S100 were clearly
less than those observed when PlOO and S 100 were incubated together. These results
suggest that in the presence of S 100. more specific cytosolic elements from S 100
28
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were associated with membranes fractionating in PlOO in the upper fractions. This
observation led to the speculation that the recruitment of cytosolic proteins to the
upper fractionated vesicles occurred during PIOO-SIOO incubation. Neither
prolonged incubation time nor the addition of potential stimulatory factors such as
Ca2 + and cAMP resulted in any qualitative difference (data not shown) in protein
“recruitment” to membranes, relative to controls.
Fraction 1 Fraction 2
kDa
PS P S PS P S
Figure 11. Cytosolic protein recruitment under three conditions: PlOO and S100
mixture (PS), PlOO alone (P), S100 alone (S). All samples were assayed in the
presence of ATP, an ATP regenerating system, and GTP at 37H C. The comparison
was made in the upper two iodixanol gradients.
29
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apical
basolateral
CCV
~^ Differential centrifugation
▼
M icrosom e
P100
Cytosol
S100
37°C
incubation
N ucleotide
Iodixanol
gradient
Figure 12. Schematic cell-free budding assay protocol. Original model (A): the
new vesicle formation was induced by the incubation. Modified models (B): PlOO
contained the mixture of pre-formed vesicles and vesicular intermediates which were
associated with undefined proteins (* ); it appears that additional factor recruitment
or mechanisms are required which drive this intermediate state towards the final
vesicle budding step.
30
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Distribution of CCV components in crude gastric microsomal fractions
Based on the qualitative observation that specific proteins were recruited onto
membranes in the presence of S100, specific proteins associated with membranes
were directly identified by immunoblotting analysis.
(i) Enrichment of H,K-ATPase and clathrin AP-l y-adaptins in Iodixanol*
fractionated vesicles
By Coomassie blue staining, the H.K-ATPase was the most enriched protein through
five fractions as indicated by heavily stained 95 kDa (Figure 10, A-C) confirming
that the H,K-ATPase was the major membrane cargo protein in the parietal cell. Of
the five fractions, fraction I contained the highest amount of H.K-ATPase, which
suggested that fraction 1 was rich in tubulovesicles (Figure 10C). In addition.
Western blotting showed strong HKf3 immunoreactivity through five fractions with
MAb 2/2E6 (Figure 1 3A). It appeared that HK(3 was enriched in the upper four
fractions whereas it was relatively decreased in the high-density fraction 5 (Figure
13A). Parallel y— adaptin enrichment throughout the five fractions (Figure 13B) was
observed indicating that vesicles at all density levels were relatively rich in both
H.K-ATPase and AP-l y-adaptins.
31
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Fraction 1
122-
80-
51-
R
U t t l
HKp
B
122-
80-
51-
AP-1 y-adaptin
Figure 13. CCV protein distributions through five budding assay fractions
(fractions 1-5) in 20% Iodixanol gradient. The crude gastric membrane (PlOO) and
soluble-cytosolic proteins (S100) samples were assayed in the presence ATP. an
ATP regenerating system, and GTP. The fractions were separated on SDS-PAGE
gels, and analyzed by Western blotting with antibodies against. A: HK(3(MAb
2/2E6), B: AP-l yadaptin (MAb 100/3).
(ii) Clathrin-mediated vesicle budding: coating and uncoating mechanisms
In contrast to the H.K-ATPase and adaptor distributions, the immunoblot with the
anti-clathrin heavy chain MAb (TD.l) showed clathrin enrichment in the high-
density fraction either in the presence or absence of S 100 (fractions 4 and 5) (Figure
14). It could be postulated that vesicles in the low-density H.K-ATPase-rich fractions
(fractions 1-3) might represent vesicles with less clathrin coating. Alternatively,
clathrin-coated membranes may have been uncoated during the assay process,
resulting in low-density membranes reflected in the mixture of uncoated (fractions I
and 2) and coated (fractions 3-5) vesicles derived from PlOO. Or possibly, clathrin
32
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may have been detached from vesicles during membrane fractionation, which would
indicate that clathrin was relatively labile with respect to membrane association. The
relatively equal yadaptin distribution throughout the gradient fractions (Figure 13B)
suggests that uncoating of all CCV proteins was not completed during 45-minute
assay incubation and may have been halted after CHC disassociation.
Figure 14. CHC distribution in five budding assay fractions (fractions 1-5) in
20% Iodixanol gradients. The crude gastric membrane (PlOO) in the presence (PS)
or absence (P) of soluble cytosolic proteins (S 100) samples was assayed with ATP. a
ATP regenerating system, and GTP. The fractions were analyzed by Western
blotting with anti-CHC (MAb TD. I).
33
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To determine whether clathrin uncoating occurred under the conditions o f the assay,
CHCs and y-adaptins were qualitatively measured both in supernatant and pellets of
the two PlOO samples in the presence or absence of S100 (to both samples were
added ATP, an ATP regenerating system, and GTP). In either the presence or
absence of S100, both sample supernatants were positive to CHCs and y-adaptins
(Figure 15). However. CHCs in the supernatant of the S 100-added sample was
greatly diminished in comparison with that of the SlOO-free sample (Figure 15A,
lanes 1 and 2). This result was unexpected because the CHC distribution through five
gradient fractions was qualitatively similar for both experimental conditions (Figure
14). The simple interpretation is that direct comparison of PlOO membranes
incubated in the presence or absence of S100 revealed that CHC association with
PlOO remains stable in the presence of S100, but is more labile in the absence of
S100. This observation is further supported by the stronger CHC immunoreactivity
of the pellet from PlOO + S 100 samples (Figure 15, lane 3), compared to that of PlOO
alone (Figure 15. lane 4). Accordingly, it might be speculated that S100 contained
specific cytosolic factors that play regulatory roles in clathrin uncoating.
Similar levels of y-adaptin immunoreactivity were observed in the supernatant when
PlOO was incubated either in the presence or absence of S100, and similar levels of
y-adaptin immunoreactivity were observed in the membrane pellets (Figure 15B).
Thus, S100 did not appear to contain regulatory proteins that significantly affected
the association o f y-adaptins with the membrane.
34
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S u p e rn ata n t Pellets
PS P PS P
y-adaptin
Figure 15. Uncoating of clathrin-coat proteins: CHC (A) and y-adaptin (B).
PlOO samples in the presence (PS) or absence of SLOO (S) were incubated (37°C, 30
min), which were subsequently resuspended in MSEP. Both samples were added
with ATP, an ATP regenerating system, and GTP. High speed centrifugation ( 100K
rpm. 20min) separated the samples into supernatant and pellets; the presence of coat
proteins were analyzed by Western blotting. Lane I: supernatant PS, Lane2:
supernatant S, Lane 3: pellet PS, and Lane 4: Pellet S.
Because vesicles appeared to be formed at some point prior to assay incubation, it
would be more appropriate to view the process as uncoating rather than coating
(budding) steps. It would be interesting to compare the uncoating velocities among
clathrin chains and other membrane factors to determine their affinity to a particular
membrane. Such an effect may depend upon the composition of vesicle membrane
lipids such as PI (4,5) P2. Takei et al (55) performed in vitro budding assays using
liposomes to investigate the effect of lipid content to vesicle budding, in which
35
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quantitatively different vesicle formation was observed with differing lipid
composition.
The heat-shock protein hsc 70 (“uncoating ATPase”) has been implicated in clathrin
uncoating (20, 27, 36): however, specific factors for AP release have not been
identified. Hsc 70 may regulate uncoating of CCVs in vitro as well as in the parietal
cell.
(iii) Cytosolic components in S100
In the budding assay, all the soluble cytosolic proteins in S100 (i.e., high-speed
supernatant of gastric mucosal homogenates) were assumed to be pelleted only in
association with a membrane (PlOO). Accordingly, cytosolic elements with no
binding membranous partners in a S100 alone sample were not likely to be found
associated with iodixanol gradient fractions, resulting in no protein trace on SDS-
PAGE gels of the fractions. Unexpectedly, weak protein signals were observed in
fractions I and 5 in the S 100-alone budding assay sample (Figure 16). Because o f the
unique protein distribution patterns (i.e., limited and specific protein bands in
fractions I and 5) (Figure 16), it was unlikely that the protein collected from
iodixanol gradient of S100 was derived from spontaneous protein aggregation, or
minor amounts of membrane vesicles contaminating the S 100 fraction. The latter
case could be further examined by solubilizing any membrane vesicles present in
36
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SlOO with a nonionic detergent such as Trion X-100 prior to iodixanol gradient
fractionation which membranous proteins are excluded by digestion.
Thus, the simple interpretation for the S 100 complex formation could be that each of
unidentified proteins in fraction 1 is in a complex that can be fractionated on
iodixanol gradients. Western blot analysis implies that the SlOO complex contains
motor protein pl50Glual, vesicle targeting protein Munc-18, and vesicle fission
protein dynamin (Figure 17), but undetectable amounts of clathrin and adaptors (data
not shown). These results suggest that the majority o f clathrin and adaptors from
soluble pools (42) were able to associate with membranes in the assay condition
being associated exclusively with a membrane, whereas dynamin, Munc-18, and
p l 5 0 clucd from soluble pools may become a constituent of either a membrane-bound
protein complex or a soluble, cytoplasmic complex. The identification,
characterization, and functional significance of this protein complex formation
remain to be elucidated.
Distinct cytosolic protein recruitment to a membrane fo r vesicular trafficking
To further analyze the recruitment of cytosolic proteins to a membrane, the
distributions of accessory proteins including GTPase dynamin and microtubule-
dependent motor proteins were investigated in vesicles incubated in the budding
assay.
37
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kDa
200-
116-
94-
67-
45-
Figure 16. Protein complex
formation in SlOO. Weak protein
signals (SlOO factor complex)
were observed in fractions 1 (lane
1) and 5 (lane 5).
32-
Dynamin
.Glued
Figure 17. Western blot analysis of proteins in budding assay SlOO complex
formed in the absence of membrane: 100 kDa dynamin, 67 kDa Munc-18, and
150 dKa pl50clucd. Western blots with negative CHC and AP-l yadaptin were not
shown.
38
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(i) Dynamin involvement in vesicle formation
Dynamin was distributed through five fractions. Its presence throughout the gradient
may represent dynamin-coated vesicular intermediates formed in budding steps,
which failed to complete the entire budding process (i.e., vesicle release) for
unknown reasons (Figure 18). Decreased dynamin immunoreactivity was observed
in low-density fractions 1 and 2 of SlOO-free samples (Figure 19, lanes 2 and 3)
(Figure 20BC). The result suggested that SlOO contained dynamin or dynamin-
related factors, and that recruitment to a membrane was achieved as a result of
interactions between membranous and cytosolic factors.
The GTPy-S effect on dynamin function in the budding assay would be predicted to
be an inhibition of membrane budding resulting from the arrest of dynamin on
clathrin-coated pits due to the dynamin’s blocked GTP hydrolysis (Figure 18).
budding-vesicie
dynamin
Figure 18. Dynamin’s role in vesicle fission by GTP hydrolysis.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
However, Western blotting of samples of P 100 + S100 incubated in the presence of
GTPy-S as well as ATP did not reveal any significant difference in dynamin
distribution or content in the iodixanol gradient fractions (Figure 20AD). Therefore,
in our in vitro system, the inhibitory effect of GTPy-S was not apparent, and
dynamin in 5 fractions were quantitatively unaltered. In our budding assay therefore,
levels of dynamin activity may have been limited due to saturation o f dynamin-
binding sites on pre-formed vesicles before the in vitro incubation and the lack of
formation of new vesicles. Alternatively, the assay may not have been sufficiently
sensitive to detect the effect of GTPy-S.
Fraction 1 Fraction 2
PS P S PS P S
122-
80-
51-
P100 + + - + + -
S100 + + + +
Figure 19. Dynamin as a potential membranous factor for SI00 interaction.
Upper two iodixanol (20%) gradient fractions (fractions I and 2) were compared
among samples containing: P100 and S100 (PS): P100 alone (P): S100 alone (S). All
the samples were added with ATP, an ATP regenerating system, and GTP.
40
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P100 S100 ATP GTP GTPy-S 1 2 3 4 5
+ + + +
A
+ - + +
B
+ + - - +
D
Figure 20. Dynamin immunoreactivity in five budding assay fractions under
various conditions as indicated.
41
* * * * *
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(ii) Microtubule motor proteins: dynein-dynactin complex and kinesin
A role of dynein has been implicated in vesicular membrane transport to the apical
surface as a microtubule-motor in epithelial cells (18). However, molecular details
about dynein-dynactin association and dynactin functions for dynein-driven motility
in H,K-ATPase trafficking have not been characterized. In addition, the role of the
other major family o f microtubule-based motors, kinesin, in H.K-ATPase trafficking
has not been characterized. Here, the presence and distribution of dynein, dynactin,
and kinesin in iodixanol gradient fractions were evaluated on immunoblots after
incubating P100 and/or S 100 under various assay conditions.
The analysis of 5 fractions by Western blots indicates that dynactin subunits
pl50clucd (Figure 22) and p50 (Figure 2 IB), and plus-end motor protein kinesin
(Figure 21 A) were most enriched in fractions I and 4. Thus, microtubule-motor
proteins were abundant in the tubulovesicle-derived fractions. Relative to the ratio of
motor proteins in fractions 1 to the total amount of protein in fraction 1 on
Coomassie blue-stained gels, it could be deduced that fraction I was more enriched
in motor proteins than fraction 4. Thus, plus-end and minus-end motor proteins were
simultaneously present in all five vesicle fractions, of which the highest amount was
present in fraction 1 membranes. These results suggest that functional dynein-
dynactin complexes and kinesins may be switched on/off (functionally
discontinuous) depending on the direction of vesicular transport (i.e., when a parietal
cell is resting or stimulated). Alternatively, these directionally opposite motor
42
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proteins may be both continuously functional in vesicle trafficking by interacting
with each other to optimize polarized vesicle motility.
A
B
C
P100 S100 ATP 1 2 3 4 5
+ + + 9
+ + +
• — —
+ + +
v . . . . . . . . . . . . . . . . . . . .
-211
-122
-80
-51
-211
-122
•80
Figure 21. Kinesin heavy chain (A), p50 (B) and dynein (C) distributions
through 5 fractions. A successive western blotting of a single blot was performed
with : anti-kinesin heavy chain antibody DK410.4.1 (A), anti-p50 (B), and anti-
dynein (C).
43
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B
P100 S100 ATP 1 2 3 4 5
+ + +
J M r ~ m i BBS
rn m m
+
w
I I
II
M
1
+ +
f l M B W
w t * ^ -
+ +
* * $ ' ■ i& t
iK S T ' * .J • • > - \ -
211
122
80
Figure 22. Western blot of pl50G I U L ' d distribution through five fractions in
various conditions.
Their association with other related motor proteins including CLIP-170 (57) remains
for further elucidation to provide protein-protein interaction involvement in vesicular
motility. Ultimately, characterization of these motor proteins is important to
44
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investigate with respect to their involvement in H, K-ATPase trafficking, especially
in regards to their polarized localization and regulatory roles in epithelial cells.
Immunofluorescence localization of pl50G lu cd and p50 dynactin complex on parietal
cells, especially pl50clual on apical sites (32) suggests dynactin’s involvement in
H,K-ATPase insertion and retrieval at the apical membrane; however, the polarity of
dynein-dynactin motility in parietal cells remains unclear.
The amount of motor proteins associated with membranes in SlOO-free samples was
slightly less (Figure 22BC) than in the presence of S100. This result indicated that
motor proteins or related proteins were more likely to be in association with
membranes rather than to be soluble in the cytosol. The dynactin-vesicle association
may have been already established before in vitro incubation because when P100
membranes were incubated in the absence of ATP (Figure 22D), the levels of
pl50ciucd associated with the membranes were greater than those observed in the
presence of ATP (Figure 22A). It would be of interest to determine the motility of
these vesicular membranes to investigate further the role of these motor-associated
proteins in the regulation of H.K-ATPase-rich vesicle motility.
Mutation analysis showed that mutants defective either in a dynein chain or a
dynactin subunit were incapable of binding to a membrane whereas mixture of both
mutants restored membrane binding (32). suggesting co-dependence of dynein and
dynactin for membrane association. Therefore, dynein and dynactin interaction were
45
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expected to co-localize in iodixanol gradient fractions and to be present at similar
relative levels. However, Western blotting showed significantly low dynein
immunoreactivity (Figure C) in comparison to abundant dynactin subunit (pl50G lucd,
p50) immunoreactivity (Figure 21AB). This result may be attributed to dynein being
easily destroyed by boiling, or by the presence of KC1 in the budding buffer, since
dynein-dynactin association was disrupted by the addition of KC1 (100 mM) (32).
Overall, abundant dynactin subunits in S100 - free samples suggest that dynein-
dynactin interaction had already been recruited, possibly from a soluble pool onto a
membrane (Figures 21AB, 22AD).
Munc-18 in the H,K-ATPase targetting
(i) Munc-18 distribution
Immunoblot analysis showed that Munc-18 (Sec-1 protein, 67 kDa) was enriched in
fractions 1 and 4 (Figure 23). This Munc-18 enrichment correlates with the H.K-
ATPase distribution on Coomassie blue-stained gels (Figure 10), suggesting that
Munc-18 is present in tubulovesicle-enriched fractions.
Relative to the total protein content in each of the five fractions (most dense protein
profile in fraction 5). it appears that H.K-ATPase and Munc-18 were most enriched
in fraction I. The enrichment of these two proteins suggests that the H,K-ATPase
may associate with Munc-18. Alternatively, since syntaxin 3 and VAMP co-localize
with the H.K-ATPase and co-purify with H.K-ATPase-rich vesicles (44), Munc-18
46
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may also be a component of this SNARE complex. Thus, co-immunoprecipitation of
Munc-18 was performed with the H.K-ATPase, and also with y-adaptin to detect
possible Munc-18 interaction with coat proteins (see below). Co-
immunoprecipitation with SNAREs was not performed due to the lack of sensitive
anti-syntaxin antibodies.
Figure 23. Immnoblot analysis of Munc-18 in the five fractions (20% iodixanol
gradients) in the presence (A), or absence (B) of S 100.
47
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(ii) Immunoprecipitation of Munc-18 with clathrin y-adaptin
Despite the strong correlation in the distribution of both H,K-ATPase and Munc-18,
H,K-ATPase was not detectable in immunoprecipitates of Munc-18 (data not
shown), suggesting that a stable interaction does not occur between the two proteins.
On the other hand, Munc-18 was co-immunoprecipitated with y-adaptin from
fraction 4 at higher level than fraction I (Figure 24). This result was unexpected
because relatively abundant amounts of both Munc-18 and y-adaptin were observed
on immunoblots (fractions 1-5) of budding assay samples (Figure 23). Accordingly,
there may be a docking molecule which associate Munc-18 with y-adaptin which
fraction 4 contained at higher level than fraction I.
The Munc-18 co-immunoprecipitation with y-adaptin implies that Munc-18 is one of
the CCV constituents, leading to the conclusion that y-adaptin may mediate the
transport of the H,K-ATPase in association with both Munc-18 and clathrin. Thus.
Munc-18 may lead CCVs to a membranous destination by associating with SNARE
proteins on a target membrane. In this respect, Munc-18 is one of the molecules
which are responsible for adaptin localization; the AP-l molecules need to bind both
Munc-18 and H.K-ATPase to determine a membranous destination and complete
vesicle fusion for cargo delivery.
48
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+ + ' GTP7S
+ + dH20
Fractions 1 4 1 4
y— adaptin
Figure 24. Immunoprecipitation of Munc-18 with clathrin y— adaptin.
Immunoprecipitates in fractions I and 4 that contained relatively high amount of
proteins (on Coomassie blue-stained gel [Figure 10C|) were analyzed by Western
blot in the presence or absence of GTPyS.
49
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Conclusion
The H,K-ATPase-packaged CCVs are believed to functionally connect distinct
membranes, tubulovesicular and apical membranes, to effect H,K-ATPase transport
to the apical surface (exocytosis) or the internalization of H,K-ATPase to
tubulovesicules (cytoplasmic membranous tubules and vesicles) (endocytosis) (11).
The regulation of CCV formation is achieved at distinct vesicle formation steps by a
variety of membrane-bound and cytosolic proteins. To characterize the mechanism
of the CCV-mediated trafficking of H.K-ATPase-packaged vesicles, reconstitution
of parietal cell CCV formation was attempted in a cell-free budding assay.
The use of both self-generating and pre-formed properties of iodixanol (OptiPrep)
gradients provided rapid vesicle separation based on vesicular protein densities in the
gastric microsomal membrane fractions. The optimal separation of different types of
vesicles was achieved by the altered density gradient profiles by increasing the
starting iodixanol concentration from 12.5% to 20% (w/v), by which a better vesicle
distribution throughout five density gradient fractions became available. This
achievement suggests that a vesicular protein of interest can be enriched for
purification purpose by designing each gradient profile. In this respect, use of an
iodixanol gradient is an efficient way to isolate the H,K-ATPase from tubulovesicle-
rich fractions (most concentrated in fractions I and 4) for further investigation and
characterization.
50
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A cell-free budding assay was performed for the direct measurement o f CCV
formation under various conditions. No significant change between two different
temperature conditions (i.e., physiological temperature 37°C, and 4°C) suggested that
certain vesicles were already formed prior to the assay, resulting in the mixed
presence of pre-formed vesicles and vesicular intermediates in vitro. This led to the
conclusion that assay conditions were unfavorable for new vesicle formation because
no further vesicle formation was facilitated by the addition of nucleotides and
cytosolic proteins (S 100), which are supposed to enhance recruitment of membrane-
associated factors onto a budding membrane. Rather, the assay procedure
emphasized the presence of different vesicular types in each of five iodixanol
gradient fractions: the presence of membrane-associated proteins on gastric
microsomal vesicles including H.K-ATPase, coat proteins, and motor proteins which
were confirmed on the immunoblots. Unique distribution of CHC through five
fractions suggests that two different types of vesicles can be separated (i.e. vesicles
with/without clathrin). Further modification of current budding assay in vitro
condition will be required to increase the interaction of these membrane-associated
proteins in gastric-vesicular trafficking. For example, the vesicle formation may be
enhanced if more concentrated cytosolic factors are used instead o f S 100, resulting in
an increased recruitment of specific proteins to a membrane. A large body of
investigation in vesicular formation has utilized brain cytosol as a source of
concentrated cytosolic factors; however, some epithelial cell-dependent aspects may
be missed since brain cytosol would reflect synaptic-related cytosolic properties.
51
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Thus, an efficient way to concentrate parietal cytosolic factors needs to be
established to characterize possible mechanisms unique to the parietal cell. In
addition, the morphological comparison of membranes among the five gradient
fractions (e.g., EM examination) would provide the qualitative visualization o f the
different vesicle populations that may have been caused by interactions among
budding enhancers (e.g., cytosol, an ATP-regenerating system) on a membrane.
The existence in both membrane-bound and cytosolic-soluble forms was confirmed
for dynamin and microtubule-dependent motor proteins, whereas, under the
conditions employed here, the exclusive presence on a membrane (P100) was
observed for the coat proteins (clathrin and adaptins). The former result suggested
that the dynamics of parietal cell compartments, where the proteins are motile from
one station to another, are facilitated through protein-protein interactions from both
sources. Furthermore, these protein interactions may be significant in the
determination of motor-mediated vesicular motion polarity which result in the
sorting of different vesicular types in the H.K-ATPase insertion and retrieval. The
putative complexes of proteins in S100 in the membrane-free condition may indicate
that specific soluble protein-protein associations may occur prior to their interactions
with membranes or membrane-bound proteins
The apparently exclusive clathrin-membrane association was further investigated
from a viewpoint of their uncoating from a membrane, in which both uncoated
52
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clathrin and y-adaptin were observed in the assay sample supernatant. Further
investigation of clathrin uncoating mechanisms can be conducted by arresting assay
mixtures at different time-course for possible quantitative protein change. In our
assay, the significance of incubation time is unknown in respect to clathrin; however,
the significantly lower amount of clathrin in less-dense vesicles in contrast to the
relative abundance of GTPases and motors throughout the gradients, suggested that
the uncoating process was interrupted by the cessation of incubation.
A growing list of components involved in CCV formation suggests that protein-
protein interactions may play significant roles in the regulation of budding steps. The
characterization of these proteins in the interactions will lead to the elucidation of the
mechanisms for specific protein recruitment onto a membrane. The real challenge is
to investigate how these protein networks ultimately contribute to ion-pumping
functions of H.K-ATPase to effect HC1 secretion, and to characterize the
mechanisms that regulate macromolecule-trafficking model in epithelial cells.
53
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Asset Metadata

Creator Taniguchi, Michiko (author)

Core Title Biochemical characterization of hydrogen,potassium-ATPase-rich membranes from the gastric parietal cell

School Graduate School

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Ou, Jing-Hsiung James (committee chair), Dennert, Gunther (committee member), Okamoto, Curtis T. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-296423

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