Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Clathrin and the -adaptin subunit of the AP-1 clathrin
adaptor have been previously identified on H-K-ATPase-rich
tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring.
Am. J. Physiol. 274 (Cell Physiol. 43):
C1017-C1029]. We further characterized this AP-1 adaptor
from rabbit and hog tubulovesicles biochemically and immunologically.
Clathrin coat proteins were stripped from purified tubulovesicular
membranes and fractionated by hydroxyapatite chromatography. The AP-1
adaptor appears to elute at 200 mM sodium phosphate, based on the
presence of proteins in this fraction that are immunoreactive with
antibodies against three of the four subunits of this heterotetrameric
complex: the
-, µ1-, and
1-adaptin subunits. Although
the putative
-adaptin subunit in this fraction is not immunoreactive
with the anti-
-adaptin monoclonal antibody (MAb), this
-adaptin
is immunoreactive with polyclonal antibodies (PAbs) directed against
the peptide sequence
Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val640,
a region conserved between
1-
and
2-adaptins that is thought to be involved in the binding of clathrin heavy chain.
Immunoprecipitation of the AP-1 adaptor complex from this fraction with
anti-
-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the
-adaptin that did not react with the anti-
-adaptin MAb but did
react with the anti-
-adaptin PAbs. In contrast, immunoprecipitation
of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a
-adaptin that was
recognized by both the anti-
-adaptin MAb and PAbs. These results
suggest that the tubulovesicular AP-1 adaptor complex may be distinct
from that found in the trans-Golgi network and may contain an
immunologically distinct
-adaptin. This immunologically distinct
-adaptin may be diagnostic of apical tubulovesicular endosomes of
epithelial cells.
hydrogen-potassium-adenosine 5'-triphosphatase; trafficking; apical membrane recycling
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INTRODUCTION |
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IN THE GASTRIC OXYNTIC CELL, which secretes HCl,
tubulovesicles are the intracellular pool of the H-K-ATPase that are
recruited to the apical membrane upon secretagogue stimulation (11).
Concomitant with the cessation of HCl secretion are the uptake of
H-K-ATPase from the apical membrane and the reestablishment of the
tubulovesicular compartment. Given the volume of protein and membrane
trafficking associated with this particular secretory process,
vesicular coat proteins might be predicted to play a role in the
regulation of this pathway. In fact, proteins belonging to families of
proteins involved in the regulation of membrane trafficking and protein sorting have been identified on tubulovesicles of the gastric oxyntic
cell. Clathrin and the -adaptin subunit of the AP-1 clathrin adaptor
(21), rab11 (14), rab25 (13), and receptors for soluble
N-ethylmaleimide-sensitive factor (5,
24) are present on tubulovesicles. Clathrin and the AP-1 clathrin
adaptor may regulate the trafficking of the H-K-ATPase-rich
tubulovesicles during the gastric acid secretory cycle by a regulated
interaction of the AP-1 adaptor with the gastric proton pump. A role
for the AP-1 adaptor in the trafficking of the H-K-ATPase was suggested by the ability to copurify from isolated tubulovesicles a complex that
contained the H-K-ATPase and
-adaptin and to cross-link with a
chemical cross-linker a complex that contained the H-K-ATPase and
-adaptin (21). The putative interaction between the H-K-ATPase and
the AP-1 clathrin adaptor would be analogous to that in which a
tyrosine-dependent putative internalization motif in the
-subunit of
the H-K-ATPase has been hypothesized to interact with endocytotic machinery at the apical membrane (6). Moreover, the regulation of
trafficking of the gastric H-K-ATPase by clathrin and clathrin adaptors
is likely to share features with other well-characterized clathrin-dependent protein sorting pathways.
To contribute to the understanding of the mechanism by which the
tubulovesicular AP-1 adaptor may be involved in the regulation of the
trafficking of the H-K-ATPase, we sought to characterize further the
tubulovesicular AP-1 adaptor. The data from these studies suggest that
the tubulovesicular AP-1 adaptor complex contains an immunologically
distinct -adaptin subunit.
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METHODS |
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Antibodies, SDS-PAGE, and Western blotting.
Monoclonal antibodies (MAbs) 100/1 (anti--adaptin), 100/2
(anti-
-adaptin), and 100/3 (anti-
-adaptin) (1) were purchased from Sigma Chemical (St. Louis, MO). Anti-
-adaptin MAb 74 was purchased from Transduction Laboratories (Lexington, KY).
Affinity-purified polyclonal antibodies (PAbs) against the
- (AE/1),
- (GD/2), µ1- (RY/1), and
1- (DE/1) adaptin subunits were
all kind gifts of Dr. Linton Traub (Washington University, St. Louis,
MO) (33). SDS-PAGE was performed according to Laemmli (19). For Western blots, blocking of nitrocellulose was done in a 5% solution of nonfat
milk in PBS-Tween 20 (0.05%). Dilutions of primary antibodies were
typically 1:1,000 in PBS-Tween, except for MAb 100/1 (1:500), MAb 100/3
(1:5,000), and PAbs AE/1, GD/2, RY/1, and DE/1 (all 1:5,000). Secondary
antibodies conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA)
were used at a dilution of 1:20,000 to 1:50,000. Positive signals were
detected by enhanced chemiluminescence (Lumiglo, KPL Laboratories,
Gaithersburg, MD; SuperSignal, Pierce Chemical) and recorded onto Kodak
Bio-Max X-ray film. For successive reprobing of blots with different
antibodies, nitrocellulose membranes were stripped in a solution of 100 mM
-mercaptoethanol, 2% SDS, and 62.5 mM
Tris · HCl (pH 6.7) for 30 min at 50°C. After
stripping, membranes were washed and blocked as normal. All other
reagents were reagent grade.
Isolation of membrane fractions.
Hog and rabbit tubulovesicles were purified from gastric mucosal
homogenates as gastric microsomes on a discontinuous sucrose density
gradient according to established protocols (35). A crude preparation
of clathrin-coated vesicles (CCVs) from hog brain was prepared
according to Pearse and Robinson (23). A crude preparation of membranes
from rabbit adrenal gland was obtained by homogenization of the adrenal
gland in a buffer (MSEP) containing (in mM) 125 mannitol, 40 sucrose, 1 EDTA-Tris, and 5 PIPES-Tris. The postnuclear supernatant was spun at
330,000 g for 20 min in a Sorvall RC
mini-ultracentrifuge. The high-speed pellet was resuspended in a buffer
containing 300 mM sucrose and 5 mM Tris · HCl (pH 7.4). All membranes were stored at 80°C until use.
Hydroxyapatite chromatography of vesicular coat proteins. Membranes (4-10 mg of membrane protein) were stripped of coat proteins with 0.5 M Tris · HCl, 2 mM EDTA, and 1 mM dithiothreitol according to the protocol of Keen et al. (17) The coat proteins were then dialyzed overnight against three changes of MSEP. The dialysate was then applied to a 5-ml hydroxyapatite column equilibrated with MSEP. The nonbinding fraction was collected, and the column was washed with MSEP. Proteins were eluted with a stepwise gradient of sodium phosphate of 10, 100, 200, and 400 mM salt (12 ml for each fraction). In the original protocol of purification of clathrin and clathrin adaptors on hydroxyapatite columns, proteins were dialyzed against a solution of NaCl and sodium phosphate and applied to the column equilibrated in the same buffer. The elution pattern of proteins from the hydroxyapatite column was not found to differ when either buffer was used. Eluted proteins were concentrated by precipitation with cold 10% TCA or 50% ammonium sulfate before SDS-PAGE. Typically, one-tenth to one-twentieth of each fraction was processed in this manner. The remainder of these fractions could be stored up to several weeks with no visible signs of degradation at 4°C after addition of NaN3 to 0.02%.
Immunoprecipitation of tubulovesicular AP-1 adaptors. An aliquot of the AP-1 adaptor eluting at 200 mM sodium phosphate was diluted with an equal volume of immunoprecipitation buffer containing 2.5% 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]. As a control, 200 µg of crude CCVs isolated from hog brain were diluted into half-strength immunoprecipitation buffer. The samples were immunoprecipitated overnight at 4°C with MAb 100/3 and protein G-Sepharose (Pharmacia Amersham Biotech). The immunoprecipitates were washed as described previously (21) and analyzed on silver-stained SDS gels (15) and by Western blots.
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RESULTS |
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Immunoreactivity of -adaptin on isolated
tubulovesicles.
Two fractions of H-K-ATPase-rich tubulovesicles can be routinely
purified from rabbit or hog gastric mucosa. Tubulovesicles sediment at
the 27 and 32% sucrose boundaries of a discontinuous sucrose gradient.
In these purified membrane preparations, the
-subunit of the
H-K-ATPase is the major membrane protein (see Fig. 2). Both fractions
of purified tubulovesicles were previously shown to react with
anti-clathrin and anti-
-adaptin MAbs, with the 32% fraction
typically having a higher specific content of clathrin and
-adaptin
(
-adaptin is shown in Fig.
1A,
lanes
1 and
2; Ref. 21). Because
-adaptin is
diagnostic of the AP-1 clathrin adaptor, we expected to find that the
-adaptin would be immunoreactive against the well-characterized
anti-
-adaptin MAb 100/1 (1), which reacts well with both
1-adaptin from the AP-1 adaptor
(typically Golgi-associated) complex and
2-adaptin from the AP-2 adaptor
(typically plasma membrane-associated) complex. However, the
-adaptin on tubulovesicles from rabbit gastric mucosa appears to be
unusual in that it reacts poorly, if at all, with the
well-characterized anti-
-adaptin MAb 100/1 (Fig.
1B,
lanes 1 and
2). On the other hand,
tubulovesicular
-adaptin shows significant reactivity with MAb 74 (Fig. 1C,
lanes
1 and
2), a MAb developed against an
NH2-terminal fragment composed of
amino acids 75 through 245 that is very well conserved between human
1- and
2-adaptins (10 conservative
amino acid substitutions in this region). This difference in
immunoreactivity of the tubulovesicular
-adaptin to the two MAbs
does not appear to be due to species-specific immunoreactivity. Both
MAbs react readily with
-adaptin from rabbit adrenal gland membranes
(Fig. 1, B and
C,
lane
3). Thus, in light of this unusual
profile of immunoreactivity of the tubulovesicular
-adaptin, we
sought to characterize the tubulovesicular AP-1 adaptor further.
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Fractionation of tubulovesicular coat proteins by chromatography on hydroxyapatite. Hydroxyapatite chromatography has been shown to be a useful technique in the purification of clathrin and adaptors (1, 23). Coat proteins were stripped from rabbit tubulovesicles from the 27% layer and applied to a hydroxyapatite column. Proteins that bound to the column were eluted with a stepwise gradient of sodium phosphate. Shown in Fig. 2 is a Coomassie blue-stained gel of proteins eluted from the column at various phosphate concentrations. Clathrin is eluted with 200 mM phosphate (C. T. Okamoto, K. V. Tyagarajan, Y. Y. Jeng, J. L. McKinney, T. M. Forte, and J. G. Forte, unpublished data). Clathrin heavy chain is clearly visible in the 200 mM fraction in Coomassie blue-stained gels (Fig. 2, lane 6). When these fractions are probed for immunoreactivity against adaptor subunits (Fig. 3), essentially all immunoreactivity is observed in the 200 and 400 mM fractions; thus, for simplicity, the images of the Western blots show only these two fractions.
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Immunoprecipitation of the AP-1 complex from the 200 mM phosphate
fraction and from crude CCVs from brain.
To confirm that the immunologically distinct -adaptin is actually a
part of the tubulovesicular AP-1 adaptor complex, we immunoprecipitated
the AP-1 adaptor complex from the 200 mM phosphate fraction obtained
from hog tubulovesicles (Fig. 5). As a
control, crude CCVs from hog brain were solubilized and also subjected to immunoprecipitation. The conditions under which the
immunoprecipitation was performed are conditions that were previously
shown to immunoprecipitate a protein complex composed of subunits of
the appropriate apparent molecular masses for an AP-1 adaptor complex
(21). Silver-stained SDS gels of the immunoprecipitates were used to
assess the relative amounts of AP-1 adaptors immunoprecipitated from
both membrane preparations (Fig.
5A); slightly more adaptor proteins
appeared to be immunoprecipitated from the 200 mM fraction than from
the crude CCVs. Western blot analyses of the immunoprecipitates to determine the relative levels of
-adaptin in each sample were consistent with the results from the silver-stained gels (Fig. 5B).
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DISCUSSION |
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An AP-1 adaptor complex from oxyntic cell tubulovesicles has been
preliminarily characterized. It appears to be composed of -,
-,
µ-, and
-subunits. The
-adaptin appears to be a
-adaptin that is immunologically distinct from
1- and
2-adaptins of the Golgi-associated AP-1 adaptor and the plasma membrane-associated AP-2
adaptor, respectively. To our knowledge, this adaptor is the first AP-1
adaptor to be identified whose composition is demonstrably different
from that of the Golgi-associated AP-1 adaptor, with the only
apparently different subunit being the
-adaptin subunit. The nature
of the heterogeneity of the tubulovesicular
-adaptin has yet to be
characterized. One possible cause for this heterogeneity is that the
tubulovesicular
-adaptin may be an isoform of the
1- and
2-adaptins. The other
possibility is that some posttranslational modification, such as
phosphorylation, may fortuitously mask the site recognized by the
anti-
-adaptin MAb. In support of this latter hypothesis,
-adaptins have been shown to be phosphorylated in vivo (34). Further
characterization of the tubulovesicular
-adaptin should provide
information regarding the basis of its differing immunologic
reactivities.
The putative difference between the tubulovesicular and Golgi AP-1
adaptors appears to reside only in the -adaptin. Consistent with the
hypotheses that distinct vesicular coat proteins may mediate distinct
membrane trafficking pathways (4, 20, 27), the difference in
-adaptins of the tubulovesicular AP-1 adaptor and the
Golgi-associated AP-1 adaptor is consistent with the previous suggestion that this AP-1 complex is associated with a membrane compartment distinct from the Golgi (i.e., oxyntic cell
tubulovesicles), that is involved in an apical recycling pathway (1).
This situation is unlike that for the newly discovered
Golgi/endosome/lysosome-associated AP-3 adaptor in which all of the
AP-3 subunits are quite distinct from (but obviously related to) the
AP-1 and AP-2 adaptor subunits (7, 9, 31, 32). However, with the recent
demonstration of the interaction of AP-3 with clathrin (8), all of
these adaptors, including the tubulovesicular AP-1 adaptor, appear to be associated with clathrin-dependent vesicular trafficking pathways.
The difference in its -adaptin may allow the tubulovesicular AP-1
adaptor to function differently from the Golgi AP-1 adaptor. Some of
the functions ascribed to
-adaptins may be relevant to the function
of the tubulovesicular AP-1
-adaptin in this particular membrane
trafficking pathway, a regulated apical recycling pathway, in oxyntic
cells.
-Adaptins possess a binding site for clathrin heavy chain (2,
8, 12). Because the tubulovesicular AP-1
-adaptin is immunoreactive
with antibodies raised against this region, this
-adaptin would be
expected to interact with clathrin heavy chain. Previous studies have
identified clathrin on purified tubulovesicles, and immunofluorescent
staining patterns of clathrin heavy chain are consistent with
tubulovesicular localization (21). However, the putative clathrin coat
on tubulovesicles is morphologically indistinct at the electron
microscopic level (3, 10, 16, 28, 29). Thus it is possible that
interaction of clathrin heavy chain with the tubulovesicular AP-1
-adaptin may result in an arrangement of clathrin on tubulovesicles
such that the clathrin coat is translucent by standard electron
microscopic preparatory techniques. Intriguingly, the same situation
may exist for AP-3: the
3-adaptin is distinctly
different from
1- and
2-adaptin, although it
apparently contains a motif for interaction with clathrin, and AP-3 can
be colocalized with clathrin by immunofluorescence and by
immuno-electron microscopy (8), although earlier studies could not find
microscopic evidence for an AP-3-clathrin interaction (9, 31).
Although more highly speculative, other functions of the
tubulovesicular AP-1 -adaptin may include a role in determining targeting to the tubulovesicular compartment, although it is not thought that
-adaptins play such a role, given that the
-adaptins of AP-1 and AP-2 are extremely well conserved (18, 22, 25, 26). Another
function may relate to the role of the adaptor complex in the gastric
acid secretory cycle. In the resting cell, clathrin and the AP-1
complex appear to be bound to tubulovesicles. When the cell is
stimulated, tubulovesicles fuse with the apical membrane. Presumably,
tubulovesicles must be uncoated for fusion to occur. It is tempting to
speculate that the
-adaptin may play a role in conferring a
secretagogue-sensitive uncoating of clathrin and AP-1 adaptors from
tubulovesicles. With respect to this possible function, the
phosphorylation of
1- and
2-adaptin has been shown to
regulate its interaction with clathrin (34); it would be of interest to
determine whether the tubulovesicular
-adaptin is phosphorylated in
a secretagogue-dependent manner.
The extent to which this tubulovesicular AP-1 -adaptin differs from
the other characterized
-adaptins must await its molecular characterization. Because tubulovesicles represent a type of regulated apical recycling compartment, once the tubulovesicular AP-1
-adaptin is characterized at the molecular level, it would be of interest to
determine the expression levels of this AP-1
-adaptin in epithelial cells. We would predict that its expression may be relatively high in
epithelial cells with such an elaborated apical membrane trafficking
pathway. Besides the brain and adrenal gland, the oxyntic cells of the
gastric mucosa may represent a relatively rich source for clathrin and
clathrin adaptors. The ability to harvest biochemical amounts of coat
proteins from oxyntic cell tubulovesicles should aid greatly in their
subsequent characterization at the biochemical, molecular, and
functional levels.
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ACKNOWLEDGEMENTS |
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We thank Dr. Linton Traub for the generous gift of anti-AP-1 adaptor antibodies and the laboratory of Dr. Vincent H. L. Lee for rabbit stomachs.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-51885 and grants from the University of Southern California Gastrointestinal and Liver Diseases Center and the National American Heart Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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 25 June 1998; accepted in final form 27 July 1998.
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