1 Department of Pharmaceutical
Sciences,
hydrogen-potassium-adenosine 5'-triphosphatase trafficking; apical membrane recycling; internalization motif
VESICULAR TRAFFICKING AND protein sorting by transport
vesicles are mediated by coat proteins (7, 38, 48, 50). The regulation
of the trafficking of solute and water transporters in epithelial cells
represents a growing area of investigation (6). HCl secretion by the
gastric oxyntic cell is a model system to study the regulation of the
trafficking of ion transporters (21). Two major protein sorting steps
are required for gastric HCl secretion by the oxyntic cell. In the
nonsecreting, resting oxyntic cell, the gastric proton pump, the
H-K-ATPase, resides in an intracellular tubulovesicular compartment
lying below the apical membrane. On stimulation of acid secretion,
these tubulovesicles fuse with the apical membrane (or invaginations
thereof known as intracellular canaliculi). The H-K-ATPase is thus
inserted into the apical membrane where it can function to acidify the lumen of the stomach. On the cessation of gastric acid secretion, the
H-K-ATPase is retrieved from the apical membrane, and the tubulovesicular compartment is reestablished. Thus the regulation of
the gastric HCl secretory cycle involves the regulated recycling of the
H-K-ATPase to and from the apical membrane of the oxyntic cell. As has
been characterized for many other vesicular trafficking and processes,
the trafficking of tubulovesicles and the sorting of the H-K-ATPase are
likely to be mediated by coat proteins.
Given that the H-K-ATPase is the major membrane protein in
tubulovesicles, the H-K-ATPase should represent the major cargo protein
for putative tubulovesicular coat proteins. The gastric H-K-ATPase
belongs to the growing family of heterodimeric P-type ATPases, to which
the ubiquitous Na-K-ATPase also belongs. The minimal functional unit of
these ATPases is a 100-kDa catalytic Other than two members of the Rab family of small GTPases, Rab11 and
Rab25, (8, 26, 27), the proteins involved in the regulated apical
recycling of the H-K-ATPase have not been characterized. Despite the
potential for the H-K-ATPase to interact with clathrin adaptors, a
classical clathrin coat on tubulovesicles has not been morphologically
identified at the electron microscopic level (4, 20, 23, 33, 51).
However, tubulovesicles are apparently derived from an elaboration of
the Golgi apparatus during the development of the acid secretory
machinery in oxyntic cells (19). Thus our hypothesis is that the
putative tubulovesicular coat may be related to other previously
characterized Golgi-associated coats, the clathrin adaptors AP-1 (47)
or AP-3 (13, 55) or the nonclathrin coat COPI (15, 53).
In this study, we have identified components of a tubulovesicular coat
complex. Despite the lack of morphological evidence by electron
microscopy for clathrin on tubulovesicles, we find that the
tubulovesicular coat contains immunoreactive clathrin and the
Golgi-associated AP-1 clathrin adaptor subunits, Materials.
Anti-HK Purification of gastric microsomes.
Gastric mucosal subcellular membrane fractions and H-K-ATPase-rich
microsomes were prepared from rabbit, rat, or hog gastric mucosae by
differential centrifugation and discontinuous sucrose density gradient
centrifugation according to established protocols (63). Purified
gastric microsomes were collected in the density gradient media in
aliquots of 300 µl and stored at SDS-PAGE and related procedures.
Protein determinations were made using the bicinchoninic acid protein
assay (Pierce Chemical). SDS-PAGE was performed according to the
protocol of Laemmli (35). Due to the sensitivity of the H-K-ATPase to
extended boiling, samples containing H-K-ATPase were boiled for 2 min
in sample buffer before being loaded onto gel. In the absence of
H-K-ATPase, samples were boiled for 5 min. For silver staining of SDS
gels, the protocol of Heukeshoven and Dernick (32) was
used. Urea SDS-PAGE gels were run according to the protocol of Ahle et
al. (1).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-Adaptin and clathrin heavy chain were identified on
tubulovesicles of gastric oxyntic cells with the anti-
-adaptin
monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb
(MAb 23), respectively. In Western blots, crude gastric microsomes from
rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for
-adaptin and clathrin. In immunofluorescent labeling of isolated
rabbit gastric glands, anti-
-adaptin and anti-clathrin heavy chain
immunoreactivity appeared to be concentrated in oxyntic cells. In
primary cultures of rabbit oxyntic cells, the immunocytochemical
distribution of
-adaptin immunoreactivity was similar to that of the
tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase.
Further biochemical characterization of the tubulovesicular
-adaptin-containing complex suggested that it has a subunit
composition that is typical of that for a clathrin adaptor: in addition
to the
-adaptin subunit, it contains a
-adaptin subunit and other
subunits of apparent molecular masses of 50 kDa and 19 kDa. From
solubilized gastric microsomes from rabbit,
-adaptin could be
copurified with the major cargo protein of tubulovesicles, the
H-K-ATPase. Thus this tubulovesicular coat may bind directly to the
H-K-ATPase and may thereby mediate the regulated trafficking of the
H-K-ATPase at the apical membrane of the oxyntic cell during the
gastric acid secretory cycle. Given the similarities of the regulated
trafficking of the H-K-ATPase with recycling of cargo through the
apical recycling endosome of many epithelial cells, we propose that
tubulovesicular clathrin and adaptors may regulate some part of an
apical recycling pathway in other epithelial cells.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-subunit and a noncovalently
associated, glycosylated
-subunit. The
-subunit of the P-type
ATPases is polytopic (has multiple transmembrane domains), and it
contains a site for the binding and hydrolysis of ATP and the binding
sites for cation transport. The
-subunit is a type II transmembrane
protein (NH2 terminus is
cytoplasmic), and it can apparently modulate the ion transport capabilities of the associated
-subunit (16, 25). Although the ion
transport functions of the gastric H-K-ATPase have been extensively
studied (46), specific sorting signals responsible for the regulated
recycling of the H-K-ATPase at the apical membrane remain obscure.
However, when expressed in a heterologous epithelial cell system, each
subunit of the H-K-ATPase is apically targeted (28). In the case of the
-subunit of the gastric H-K-ATPase (HK
), the putative apical
targeting signal apparently resides in the
NH2-terminal half of the protein.
In addition, intriguingly, all gastric H-K-ATPase
-subunits (HK
)
cloned thus far contain a tetrapeptide motif in their cytoplasmic
domain, FR(or Q)XY (where F = Phe, R = Arg, Q = Gln, X = any amino
acid, and Y = Tyr); this motif is highly reminiscent of the
internalization signal found in the transferrin receptor (28) and
conforms to the consensus motif for binding to the µ-subunits of the
AP-1, AP-2, and AP-3 clathrin adaptors (5, 13, 40, 41). Thus both
subunits of the H-K-ATPase may have the potential to interact with
tubulovesicular coat proteins via these putative sorting signals. In
fact, supportive evidence for a functional role for the motif in HK
has been recently provided in which the Tyr in this motif appears to be
involved in the targeting of the H-K-ATPase to a regulated compartment and also appears to be required for the cessation of gastric acid secretion, presumably by forming part of an internalization motif (11).
- and
-adaptin. The localization of clathrin and the
AP-1-related adaptor to tubulovesicular membranes may represent a novel
localization for clathrin-coated membranes in epithelial cells. In
addition, the H-K-ATPase, the major cargo protein of tubulovesicles,
may interact directly with the tubulovesicular adaptor. Thus clathrin and the AP-1-related clathrin adaptor may be involved in regulating the
trafficking of the H-K-ATPase during the HCl secretory cycle.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
monoclonal antibody (MAb) (56) was a kind gift from Dr. Adam
Smolka (Medical University of South Carolina, Charleston, SC).
Anti-
-adaptin MAb 100/3 and anti-
-adaptin MAb 100/1 (1), fish
skin gelatin, poly-D-lysine
hydrobromide and BSA were purchased from Sigma (St. Louis, MO).
Anti-
-adaptin MAb 88 (mouse
-adaptin fragment corresponding to
COOH-terminal amino acids 642-821 used as immunogen),
anti-
-adaptin MAb 74 (human
-adaptin fragment corresponding to
NH2-terminal amino acids
75-245 used as immunogen), and anti-clathrin heavy chain MAb 23 (rat clathrin heavy chain fragment corresponding to
NH2-terminal amino acids
4-171 used as immunogen) were purchased from Transduction
Laboratories (Lexington, KY).
6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)hexanoyl]sphingosine (NBD-C6-ceramide) was purchased
from Molecular Probes (Eugene, OR). Fast flow protein G-Sepharose was
purchased from Pharmacia. Goat anti-mouse IgG coupled to horseradish
peroxidase (HRP) and prestained molecular mass standards for SDS-PAGE
were purchased from Bio-Rad (Hercules, CA). Goat anti-mouse IgG
conjugated to rhodamine was purchased from Jackson Immunological
Laboratories (Bar Harbor, ME). Wheat germ agglutinin (WGA)- and
Ricinus
communis agglutinin I (RCA
I)-Sepharose were purchased from E-Y Laboratories (San Mateo, CA).
3,3'-Dithiobis(sulfosuccinimidyl propionate) (DTSSP) was
purchased from Pierce Chemical (Rockford, IL). Protease inhibitors
phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)benzenesulfonyl fluoride HCl were purchased from Calbiochem (San Diego, CA). Protease inhibitors antipain, leupeptin, and pepstatin A were purchased from
Chemicon (Temecula, CA). Lumi-Glo enhanced chemiluminescence (ECL)
detection reagent was purchased from Kirkegaard & Perry Laboratories
(Gaithersburg, MD). All other biochemical reagents were reagent grade.
80°C. Gastric microsomes
from rat gastric mucosa were purified on 10-40% continuous sucrose gradients according to the protocol of Crothers et al. (12).
Purified gastric microsomes are virtually all oriented with the
cytoplasmic membrane leaflet facing outward.
-adaptin MAb 74, 1:1,000; anti-
-adaptin MAb
88, 1:1,000; and MAb 2/2E6, 1:200 (cell culture supernatant). Goat
anti-mouse-HRP secondary antibody was used at 1:20,000 dilution. Blocking of nitrocellulose was done in 5% nonfat milk in PBS-Tween 20. HRP was detected by ECL, and the signal was visualized
on Kodak Bio-Max X-ray film.
Immunofluorescent labeling of isolated rabbit gastric glands.
Isolated rabbit gastric glands (3) were either fixed in 3.7%
formaldehyde in PBS and subsequently permeabilized in 0.1% Triton
X-100 in PBS or fixed and permeabilized in cold (20°C) methanol. After blocking in either 0.66% fish skin gelatin or 0.1%
BSA in PBS, glands were stained in suspension. All primary antibodies
were used at 1:100 dilution in PBS-0.05% Tween 20-0.66% fish skin
gelatin or 0.1% BSA, and all secondary antibodies were used at 1:500
dilution in the same buffer. Glands were immobilized on
polylysine-coated coverslips before viewing with a Zeiss Axioskop epifluorescence microscope.
Labeling of isolated rabbit gastric glands with NBD-C6-ceramide. Isolated glands were washed several times with sterile 10 mM HEPES-buffered minimal essential medium and incubated with NBD-C6-ceramide (5 µM) for 20 min at 4°C. Labeled glands were washed in HEPES-buffered minimal essential medium and then incubated in the same medium for 30 min at 37°C. Labeled glands were examined under the microscope and immediately photographed. The images were digitized and processed with Adobe Photoshop.
Immunofluorescent labeling and scanning confocal microscopy of
cultured oxyntic cells.
Primary cultures of rabbit oxyntic cells were prepared as previously
described (9, 58). Cells maintained in culture for 48 h were fixed in
4% paraformaldehyde for 15 min at 4°C. Cells were permeabilized
with 0.3% Triton X-100 in 15% donkey serum for 30 min
and then incubated with either MAb 100/3 (1:1,000) or anti-HK
(1:2,000) for 2 h at 22°C. Specific labeling was localized with
Cy5-donkey anti-mouse IgG. All cells were double labeled with BODIPY FL
phallacidin (Molecular Probes). Cells were visualized using scanning
confocal microscopy (Molecular Dynamics, Sunnyvale, CA).
Stripping of gastric microsomal coat proteins.
Two hundred micrograms of purified gastric microsomes (27 or 32%
layer) from rabbit were stripped of coat proteins by two washes in 0.5 M Tris · HCl, pH 7.0, 2 mM Na-EDTA, and 0.2 mM
dithiothreitol, according to the protocol of Keen et al. (34). Samples
were separated into high-speed supernatants and pellets. The proteins in the supernatants were concentrated by precipitation in ice-cold 10%
trichloroacetic acid. Samples were analyzed by Coomassie blue staining
for total protein and by Western blot for the distribution of
-adaptin and HK
between the supernatants and pellets.
Buffers for immunoprecipitation. Triton dilution buffer (TDB) consists of 2.5% (or 5%) Triton X-100, 100 mM triethanolamine HCl, pH 8.6, 100 mM NaCl, 5 mM Na-EDTA, 0.02% NaN3, and protease inhibitors [4-(2-aminoethyl)benzenesulfonyl fluoride HCl, phenylmethylsulfonyl fluoride, leupeptin, antipain, and pepstatin].
Mixed micelle buffer (MMB) consists of 1% Triton X-100, 0.2% SDS, 150 mM NaCl, 20 mM triethanolamine HCl, pH 8.6, 5 mM Na-EDTA, 5% sucrose, 0.2% NaN3, and protease inhibitors. Final wash buffer (FWB) is the same as MMB, except that detergents and sucrose are omitted. Triton X-100, triethanolamine, and glycerol (TTG) consists of 1% Triton X-100, 100 mM triethanolamine HCl, pH 8.6, 10% glycerol, 5 mM Na-EDTA, 1 mM Na3VO4, and protease inhibitors. In the absence of glycerol, this buffer is referred to as TT. Triton X-100, glycerol, and HEPES (TGH) consists of 1% Triton X-100, 10% glycerol, 50 mM Na-HEPES, pH 7.3, 1 mM Na3VO4, and protease inhibitors. This buffer was adapted from the protocol of Sorkin and Carpenter (57).Immunoprecipitation of gastric microsomal coat complex with MAb 100/3. Purified gastric microsomes from rabbit were solubilized in 2.5% TDB and incubated overnight with MAb 100/3 and protein G-Sepharose. Immune complexes were washed three times in MMB and once in FWB. Immunoprecipitates were analyzed by silver staining of SDS gels.
Copurification of -adaptin with solubilized
H-K-ATPase.
Purified gastric microsomes (100 µg) from rabbit were solubilized in
either TTG, TT, or TGH. The solubilized samples were incubated with 40 µl WGA- or RCA I-Sepharose bead suspension (equivalent to
100-160 µg of lectin) for 2 h at 4°C. Lectin precipitates
were washed with MMB and FWB before SDS-PAGE. Samples were analyzed on
Western blots for
-adaptin and HK
. There was no difference in the
amount of
-adaptin coimmunoprecipitated with the H-K-ATPase when the
detergent-treated samples were centrifuged at 100,000 g to
remove unsolubilized material; thus the high-speed centrifugation step
was normally omitted. Also, as a control, microsomes were solubilized
in 1% SDS before dilution with TGH and lectin affinity chromatography.
Cross-linking of gastric microsomal coat proteins to H-K-ATPase.
Gastric microsomal proteins from rabbit were cross-linked with DTSSP
according to a protocol adapted from Simpson et al. (54). Purified
gastric microsomes (200 µg) were diluted into an equal volume of 50 mM Na-HEPES-2 mM MgCl2, to which
DTSSP was added to 2 mM from a freshly prepared stock solution of 20 mM
in dimethylformamide. The samples were cross-linked for 30 min at room
temperature, after which unreacted DTSSP was quenched with 150 mM
glycine. SDS was added to 1%, and the samples were boiled for 2 min.
The samples were then diluted with TDB and subjected to WGA affinity chromatography overnight at 4°C as described above. The isolated, cross-linked sample was analyzed by Western blots for -adaptin. This
experiment was performed twice.
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RESULTS |
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Distribution of clathrin and -adaptin in crude
gastric mucosal membrane fractions and in density gradient-purified
gastric microsomes.
To test the hypothesis that oxyntic cell tubulovesicles interact with
Golgi-related vesicular coat proteins, the distribution of clathrin
heavy chain and the
-adaptin subunit of the
Golgi-associated AP-1 clathrin adaptor was determined
by Western blot analysis of gastric mucosal subcellular membrane
fractions (Fig. 1). Figure 1A shows Coomassie
blue-stained electrophorograms of rabbit gastric homogenates
fractionated by differential centrifugation
(lanes 1-4)
and of subsequent purification of H-K-ATPase-rich membrane vesicles
from the crude microsomal pellet on discontinuous sucrose density
gradients (lanes
5-7).
Microsomes sedimenting at the 27 and 32% sucrose interfaces are highly
enriched in the H-K-ATPase; HK
is the most prominent protein band in
Coomassie blue-stained gels (Fig.
1A,
lanes
5 and
6). The amount of HK
band
correlates well with the amount of H-K-ATPase enzymatic activity in
these membrane preparations. The greatest amounts of HK
protein and highest specific ATPase activity (not shown) were found in the 27%
layer (Fig. 1A,
lane
5). By virtue of the enrichment of
the H-K-ATPase in the 27 and 32% layers of density gradient-purified microsomes, these membrane fractions represent fractions enriched in
oxyntic cell tubulovesicles.
|
|
Immunofluorescent labeling of clathrin and -adaptin
in oxyntic cells.
The functional secretory unit of the gastric mucosa is the gastric
gland (21). In vivo, the gland is mainly composed of four types of
epithelial cells: surface mucous cells, mucous neck cells, zymogen
(pepsinogen)-secreting chief cells, and HCl-secreting oxyntic
(parietal) cells. Gastric glands can be isolated by collagenase digestion of gastric mucosa. These isolated rabbit gastric glands are
primarily composed of larger, bulging HCl-secreting oxyntic cells
interspersed with the smaller mucous neck cells and chief cells, as
shown in the phase-contrast micrograph in Fig.
3A. In isolated glands, the apical membranes of the oxyntic and nonoxyntic cells form a central lumen that can be delineated by staining apical
membranes with the lectin Helix
pomatia agglutinin conjugated with
FITC (Fig. 3B). A typical
distribution of oxyntic cells within an isolated gland is shown in Fig.
3C, in which oxyntic cells have been
stained with a MAb against HK
(10).
|
|
Immunofluorescent labeling of -adaptin in primary
cultures of oxyntic cells.
Isolated oxyntic cells maintained in primary culture assume a
morphological arrangement distinct from their tissue and glandular form
(9, 58). During the isolation of oxyntic cells, the intracellular
canaliculi are pinched off at the lumen, resulting in the formation of
intracellular canalicular vacuoles (representing the apical membrane).
Thus cultured oxyntic cells tend to acquire a more simplified
morphology, thereby facilitating the subcellular localization of
proteins. The apical membrane and subapical tubulovesicular compartment
may be more easily identified by staining for F-actin (Fig.
5, b and
e) and H-K-ATPase (Fig.
5d), respectively.
Soluble pools of clathrin coat proteins and stripping of
-adaptin from gastric microsomal membranes.
The gastric microsomal (tubulovesicular) clathrin coat proteins were
further characterized biochemically. Coat proteins exist in
membrane-bound and soluble pools (50). As expected, besides the
membrane-bound pool of clathrin and
-adaptin on gastric microsomes, the high-speed supernatant of gastric mucosal homogenates also contains
clathrin and
-adaptin (Fig. 1, B
and C,
lane
4). However, this soluble pool of
clathrin and
-adaptin may also be derived from sources other than
tubulovesicles, such as from Golgi membranes of oxyntic and chief
cells.
|
|
Characterization of the other subunits of the clathrin adaptor
complex on gastric microsomes.
Although immunoreactivity with the anti--adaptin MAb 74 was robust
in density gradient-purified gastric microsomes (Figs. 1D and
2D), we were surprised to find that
little, if any, reactivity to the well-characterized anti-
-adaptin
MAb 100/1 (1) was observed in gastric microsomes, although such
reactivity was robust in control membranes from rabbit adrenal gland
(not shown). This dichotomy in reactivity to the anti-
-adaptin MAbs
was also observed in gastric microsomes from rat and hog (not shown),
suggesting that the
-adaptin of gastric microsomal membranes may be
distinct from that of Golgi-associated AP-1 and/or may possess
some unique features. The basis for the difference in immunoreactivity
of the gastric microsomal
-adaptin with one MAb (MAb 74) and not another (MAb 100/1) is currently being investigated.
|
Copurification of -adaptin with solubilized
H-K-ATPase.
The H-K-ATPase is the major membrane protein of purified gastric
microsomes. Thus this enzyme would represent the major cargo protein
for a tubulovesicular coat complex.
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DISCUSSION |
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On secretagogue-induced stimulation of gastric acid secretion in the oxyntic cell, the gastric proton pump, the H-K-ATPase, is recruited to the apical membrane from subapical H-K-ATPase-rich tubulovesicles. With cessation of gastric acid secretion, the H-K-ATPase is retrieved from the apical membrane as the tubulovesicular compartment is reestablished. Given the extensive, relatively synchronous vesicular trafficking steps associated with this process, the oxyntic cell may represent a good model system in which to study the regulation of membrane trafficking and protein sorting by vesicular coat proteins in epithelial cells.
We have identified clathrin and an AP-1 adaptor complex on oxyntic cell
tubulovesicles. This report is the first report of clathrin on oxyntic
cell tubulovesicles; these tubulovesicles may represent a novel
compartment to which clathrin and AP-1 adaptors are localized. The
presence of clathrin on tubulovesicles is intriguing, given that
morphologically distinct clathrin coats have not been reported in any
of the numerous electron microscopic analyses of the exocytic and
endocytic trafficking of the H-K-ATPase associated with HCl secretion
by the oxyntic cell (4, 20, 23, 33, 51). This incongruence suggests
that tubulovesicular clathrin may be functionally and/or
structurally different from previously characterized conventional
clathrin. Relevant to this speculation, a novel clathrin heavy chain
gene was recently cloned and characterized; this gene is selectively
expressed in skeletal muscle in adults but ubiquitously in all of the
limited number of fetal tissues that were assayed (36). Alternatively,
the clathrin light chains (7) or -adaptin (2, 24), the other
proteins thought to influence the polymerization of clathrin and the
structure of clathrin coats, may regulate an alternative mode of
polymerization of clathrin in oxyntic cells such that morphologically
distinct clathrin baskets are not visible by electron microscopy.
Molecular characterization of this clathrin heavy chain and its
accompanying light chains will be required to determine the
relationship of the tubulovesicular clathrin to the other previously
cloned isoforms and may account for its novel morphology in oxyntic
cells. Indeed, with respect to the structure of clathrin coats, a
similar situation may exist for tubulovesicular elements in early
endosomal compartments; the presence of conventional clathrin on
endosomes had gone unnoticed until recent critical
immuno-electron-microscopic analyses were performed (59).
The tubulovesicular compartment of the oxyntic cell may represent a gross elaboration of apical recycling endosomes found in other epithelial cells. Several similarities exist between membrane trafficking during gastric acid secretion and apical recycling in other epithelial cells. First, as with regulation of trafficking associated with the apical recycling endosome (17, 30, 45), gastric acid secretion is stimulated by increasing intracellular cAMP (62). Second, as with apical trafficking (19, 29, 37), gastric acid secretion is highly dependent on intact microfilaments and associated proteins such as ezrin (31, 39, 64, 65). Finally, Rab11, a marker of the tubulovesicular compartment (8, 27), also regulates trafficking in another recycling endosomal compartment, the pericentriolar (perinuclear) recycling endosome (61). Thus this tubulovesicular coat complex may represent an adaptor complex that is common to many epithelial cells and is involved in the regulation of membrane traffic to and/or from an apical recycling endosome.
For this tubulovesicular adaptor coat, the cargo protein appears to be
the H-K-ATPase. Although direct binding of the tubulovesicular coat to
the H-K-ATPase has not been definitively demonstrated, the H-K-ATPase
appears to reside in a complex with the tubulovesicular coat. The
simplest explanation is that the tubulovesicular coat binds to the -
and/or
-subunit of the H-K-ATPase, similar to the manner in
which clathrin adaptors bind to motifs present in the cytoplasmic
domains of other membrane proteins (5, 38, 40, 41, 60). In this regard,
the cytoplasmic domain of HK
contains a tetrapeptide sequence
[FR(or Q)XY] highly reminiscent of the internalization
signal of the transferrin receptor (28); binding of adaptors to the
H-K-ATPase may be mediated by this putative sorting signal. Indeed,
recent work has shown that the motif YXX
(where Y = Tyr, X = any
amino acid, and
= bulky aromatic amino acid) is an optimal one for
interaction with the medium chains of AP-1 and AP-2 adaptors (5, 40).
However, as tempting as this speculation may be, identification of the
binding site on HK
and/or HK
for the tubulovesicular
adaptor will rely on in vitro binding assays. Recently, evidence has
been presented suggesting that the Tyr in the putative motif in HK
serves as a signal to target HK
to a regulated compartment and is
required for the cessation of acid secretion (11), implying that this Tyr also serves as an internalization motif. However, because of the
uncertainty of the physiological evidence presented in Ref. 11, we must
be circumspect in our interpretation. Alternatively, binding of coat
complexes to tubulovesicles may involve another non-ATPase
tubulovesicular membrane protein, such as a receptor for soluble
N-ethylmaleimide-sensitive factor (SNARE) (8,
38, 43). However, because the coat complex is coimmunoprecipitated with
the H-K-ATPase, any other putative docking receptor for the adaptor
would also have to be noncovalently associated with the H-K-ATPase. The
final alternative is that the coat complex recognizes both the
H-K-ATPase and another docking protein.
The relative abundance of clathrin and adaptors on oxyntic cell tubulovesicles, together with the previous identification of SNARE proteins on tubulovesicles (8, 43), supports the hypothesis that the mechanism of HCl secretion involves membrane translocation and fusion events (22) rather than an osmotically regulated expansion (secreting state) and collapse (nonsecreting state) of preexisting tubules that are contiguous with the apical membrane (44). Thus coincident on tubulovesicles are key components of the cellular machinery necessary for both sorting of the H-K-ATPase (clathrin coat) and vesicular fusion (syntaxins and VAMPs).
In conclusion, a clathrin and an AP-1 adaptor coat complex are
associated with the tubulovesicular compartment of the gastric oxyntic
cell. This finding appears to represent another distinct non-Golgi
localization of -adaptin (14). The step at which the coat proteins
regulate the recycling of its cargo, the H-K-ATPase, is unknown. In a
study by Schofield et al. (52), coated vesicles, although not
distinctly clathrin-coated vesicles, were observed during the return of
oxyntic cells from the stimulated (secreting) state to the resting
(nonsecreting) state; thus clathrin and adaptors may regulate the
reuptake of the H-K-ATPase from the apical membrane with the cessation
of HCl secretion. The function of clathrin and AP-1 adaptors in the
gastric acid secretory cycle may be tested by determining the
sensitivity of particular steps of the cycle to reagents [e.g.,
brefeldin A, guanosine
5'-O-(3-thiotriphosphate), and
aluminum fluoride] that have been used to modify AP-1 adaptor function in other cell types (49, 64). Further biochemical and
molecular characterization of the tubulovesicular coat and its
interaction with its cargo should help provide important details with
respect to the role of clathrin and adaptors in gastric acid secretion.
Moreover, characterization of the role of adaptor coat proteins in the
regulation of gastric acid secretion may consequently provide important
clues regarding the regulation of apical recycling in many other
epithelial cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Catherine Chew for providing cultured oxyntic cells and Dr. Sarah Hamm-Alvarez for a critical reading of this manuscript.
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FOOTNOTES |
---|
This work was supported by a Zumberge Award from the University of Southern California, American Heart Association National Grant-in-Aid 96-1205, a New Investigator Award from the American Association of Colleges of Pharmacy, a Pilot Project Grant from the University of Southern California Gastrointestinal and Liver Diseases Center (to C. T. Okamoto), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-10141 (to J. G. Forte), DK-38063 (to J. R. Goldenring), and DK-4370 (to J. R. Goldenring), and the Kuwait Foundation for the Advancement of Sciences KFAS-95-07-02 (to S. M. Karam).
Address for reprint requests: C. T. Okamoto, Dept. of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033.
Received 3 October 1997; accepted in final form 6 January 1998.
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REFERENCES |
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