An immunologically distinct beta -adaptin on tubulovesicles of gastric oxyntic cells

Curtis T. Okamoto and Young Y. Jeng

Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Clathrin and the gamma -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 gamma -, µ1-, and sigma 1-adaptin subunits. Although the putative beta -adaptin subunit in this fraction is not immunoreactive with the anti-beta -adaptin monoclonal antibody (MAb), this beta -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 beta 1- and beta 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-gamma -adaptin MAb 100/3 resulted in the coimmunoprecipitation of the beta -adaptin that did not react with the anti-beta -adaptin MAb but did react with the anti-beta -adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a beta -adaptin that was recognized by both the anti-beta -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 beta -adaptin. This immunologically distinct beta -adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.

hydrogen-potassium-adenosine 5'-triphosphatase; trafficking; apical membrane recycling

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 gamma -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 gamma -adaptin and to cross-link with a chemical cross-linker a complex that contained the H-K-ATPase and gamma -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 beta -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 beta -adaptin subunit.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Antibodies, SDS-PAGE, and Western blotting. Monoclonal antibodies (MAbs) 100/1 (anti-beta -adaptin), 100/2 (anti-alpha -adaptin), and 100/3 (anti-gamma -adaptin) (1) were purchased from Sigma Chemical (St. Louis, MO). Anti-beta -adaptin MAb 74 was purchased from Transduction Laboratories (Lexington, KY). Affinity-purified polyclonal antibodies (PAbs) against the gamma - (AE/1), beta - (GD/2), µ1- (RY/1), and sigma 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 beta -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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Immunoreactivity of beta -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 alpha -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-gamma -adaptin MAbs, with the 32% fraction typically having a higher specific content of clathrin and gamma -adaptin (gamma -adaptin is shown in Fig. 1A, lanes 1 and 2; Ref. 21). Because gamma -adaptin is diagnostic of the AP-1 clathrin adaptor, we expected to find that the beta -adaptin would be immunoreactive against the well-characterized anti-beta -adaptin MAb 100/1 (1), which reacts well with both beta 1-adaptin from the AP-1 adaptor (typically Golgi-associated) complex and beta 2-adaptin from the AP-2 adaptor (typically plasma membrane-associated) complex. However, the beta -adaptin on tubulovesicles from rabbit gastric mucosa appears to be unusual in that it reacts poorly, if at all, with the well-characterized anti-beta -adaptin MAb 100/1 (Fig. 1B, lanes 1 and 2). On the other hand, tubulovesicular beta -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 beta 1- and beta 2-adaptins (10 conservative amino acid substitutions in this region). This difference in immunoreactivity of the tubulovesicular beta -adaptin to the two MAbs does not appear to be due to species-specific immunoreactivity. Both MAbs react readily with beta -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 beta -adaptin, we sought to characterize the tubulovesicular AP-1 adaptor further.


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Fig. 1.   Western blots of purified gastric tubulovesicular and crude adrenal gland membranes from rabbit probed with anti-gamma -adaptin MAb 100/3 (A), anti-beta -adaptin MAb 100/1 (B), or anti-beta -adaptin MAb 74 (C). Lane 1: 27% tubulovesicular layer (7 µg). Lane 2: 32% tubulovesicular layer (9 µg). Lane 3: crude adrenal gland membrane fraction (60 µg). All immunoreactive signals from blots were detected by enhanced chemiluminescence (ECL), and films were exposed for 2 min. Blot showing difference in beta -adaptin immunoreactivity is representative of 6 analyses from 4 rabbits. Comparison of tubulovesicular beta -adaptin with adrenal gland beta -adaptin was performed in duplicate. Migration of a prestained molecular mass marker is indicated.

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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Fig. 2.   Coomassie blue-stained gel of tubulovesicular coat proteins (stripped from 27% layer rabbit tubulovesicles) fractionated on hydroxyapatite. Lane 1: 27% layer tubulovesicular membrane preparation (24 µg). Position of alpha -subunit of H-K-ATPase is indicated (HKalpha ). Lane 2: proteins stripped from tubulovesicles by 0.5 M Tris · HCl (24 µg). Lanes 3-7: nonbinding fraction and 10, 100, 200, and 400 mM sodium phosphate eluates, respectively. One-tenth of total volume of each recovered fraction (lanes 3-7) was precipitated with 10% TCA before loading onto gel. * Position of clathrin heavy chain in 200 mM fraction. Migration of molecular mass markers is indicated. Gels are representative of 9 preparations from 5 rabbit stomachs.


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Fig. 3.   Western blots of adaptor subunits in hydroxyapatite fractions. A single blot was blotted successively with anti-gamma -adaptin MAb 100/3 (A), anti-gamma -adaptin polyclonal antibody (PAb) AE/1 (B), anti-beta -adaptin MAb 74 (C), anti-AP-1 adaptor µ-subunit PAb RY/1 (D), anti-AP-1 adaptor sigma -subunit PAb DE/1 (E), and anti-beta -adaptin PAb GD/2 (F). Lane 1: proteins eluted with 200 mM sodium phosphate. Lane 2: proteins eluted with 400 mM phosphate. For both fractions, one-twentieth of total volume of each fraction was loaded onto each lane. All immunoreactive signals from blots were detected by ECL, and films were exposed for 2 min, except for blots in D and E, for which exposure times were 20 min. Blots are representative of 4 blots from membrane preparations from 3 rabbit stomachs. Migration of prestained molecular weight markers is indicated. TD, tracking dye.

When these fractions are probed by Western blot for gamma -adaptin with anti-gamma -adaptin antibodies MAb 100/3 or AE/1, essentially all of the immunoreactivity is observed in the 200 mM phosphate fraction (Fig. 3, A and B, lane 1). The lower band of the doublet of anti-gamma -adaptin MAb 100/3 immunoreactivity is suspected to be a degradation product of gamma -adaptin that is truncated near the COOH terminus and is therefore not recognized by AE/1 (which was raised against the last 12 amino acids of gamma -adaptin). When the same fractions are probed with anti-beta -adaptin MAb 74 (Fig. 3C), essentially all of the immunoreactivity is observed in the 400 mM phosphate fraction. The absence of anti-beta -adaptin MAb 74 immunoreactivity in the 200 mM fraction does not appear to be a result of extensive proteolysis, as immunoreative degradation products are not observed in the rest of the gel. Thus there appears to be a dissociation between gamma -adaptin and beta -adaptin immunoreactivity in these fractions. Interestingly, the 400 mM fraction, although relatively abundant in other 100-kDa proteins (Fig. 2A, lane 7), demonstrates poor reactivity to anti-gamma -adaptin antibodies (Fig. 3, A and B, lane 2). In addition, although anti-alpha -adaptin MAbs react exclusively with the 400 mM fraction, the reactivity is relatively weak (not shown). The basis for strong anti-beta -adaptin immunoreactivity and relatively weak anti-gamma -adaptin and anti-alpha -adaptin reactivity in the 400 mM fraction needs to be investigated.

A possibility for the dissociation of anti-gamma - and anti-beta -adaptin immunoreactivity in the two hydroxyapatite fractions may be a consequence of an artifactual dissociation of the AP-1 adaptor complex into its component subunits before or during the chromatographic step. To determine whether the conditions employed for chromatography resulted in the dissociation of adaptor subunits, we probed the hydroxyapatite fractions with affinity-purified anti-peptide PAbs, RY/1 and DE/1, raised against the other subunits of the AP-1 adaptor complex, µ1 and sigma 1, respectively (33). Both anti-peptide antibodies were reactive with the 200 mM fraction (Figs. 3, D and E), a result consistent with the AP-1 adaptor remaining intact under the conditions employed for chromatography.

To find the "missing" beta -adaptin for the AP-1 adaptor eluted at 200 mM phosphate, these fractions were probed with anti-beta -adaptin anti-peptide PAbs (GD/2) raised against a region absolutely conserved in beta 1- and beta 2-adaptins, Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val640 (33). This region is thought to be involved in the binding of clathrin heavy chains to beta -adaptin (12, 30, 33). As shown in Fig. 3F (compared with Fig. 3C), these anti-peptide antibodies react nearly equally well with both the 200 and 400 mM phosphate fractions, with the lower band of the reactive doublet presumably representing a degradation product of the beta -adaptins (33). Given the profile of immunoreactivity with all of the anti-beta -adaptin antibodies, these data are consistent with the conclusion that there are at least two distinct beta -adaptins on tubulovesicles. In addition, given that all of the anti-alpha -adaptin (AP-2) immunoreactivity is observed in the 400 mM fraction (not shown), these data together suggest that the tubulovesicular AP-1 adaptor complex consists of an immunologically distinct beta -adaptin.

To provide further evidence that the tubulovesicular AP-1 adaptor may possess a beta -adaptin distinct from that in the Golgi AP-1 adaptor, tubulovesicular coat proteins from membranes from the 32% layer from a different species (hog) were also fractionated on hydroxyapatite columns. As shown in Fig. 4, when tubulovesicles from the 32% layer are used as the starting material, hydroxyapatite column fractions derived from these membranes are qualitatively quite similar to those derived from the 27% layer (Fig. 4A). Also, as shown in Fig. 4, the distribution of immunoreactivity for the anti-gamma -adaptin subunit is strongly similar to that observed for the fractions from the 27% layer, with the difference being that some gamma -adaptin immunoreactivity is observed to elute at 400 mM sodium phosphate (Fig. 4B). Otherwise, the distribution of immunoreactivity for the beta -adaptin subunit is identical to that observed for the fractions from the 27% layer: anti-beta -adaptin MAb immunoreactivity is strong in the 400 mM sodium phosphate fraction and absent in the 200 mM fraction (Fig. 4C), and GD/2 immunoreactivity is strong in both fractions (Fig. 4D). Thus, from tubulovesicles from two different mammalian species, an immunologically distinct beta -adaptin is found in hydroxyapatite fractions enriched in gamma -adaptin.


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Fig. 4.   Fractionation of hog tubulovesicular adaptors on hydroxyapatite. A: Coomassie blue-stained gel. Lane 1: tubulovesicles from 32% layer (24 µg). Position of alpha -subunit of H-K-ATPase is indicated. Lane 2: proteins stripped from tubulovesicles (24 µg). Lane 3: nonbinding fraction. Lanes 4-7: proteins eluted with 10, 100, 200, and 400 mM sodium phosphate, respectively. One-twentieth of total volume of each fraction was loaded onto each lane (lanes 3-7). B-D: Western blot of 200 (lane 1) and 400 (lane 2) mM sodium phosphate fractions was successively probed with anti-gamma -adaptin MAb 100/3 (B), anti-beta -adaptin MAb 74 (C), and anti-beta -adaptin PAb GD/2 (D). Immunoreactive signals from each blot were detected by ECL with a 2-min exposure for each blot. Gels and blots are representative of 3 preparations from 2 hog stomachs. Migration of molecular mass markers is indicated.

Immunoprecipitation of the AP-1 complex from the 200 mM phosphate fraction and from crude CCVs from brain. To confirm that the immunologically distinct beta -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 gamma -adaptin in each sample were consistent with the results from the silver-stained gels (Fig. 5B).


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Fig. 5.   Immunoprecipitation of AP-1 adaptors from 200 mM sodium phosphate fraction (lane 1) and from crude clathrin-coated vesicles (CCVs) from brain (lane 2) with anti-gamma -adaptin MAb 100/3. Immunoprecipitations were performed as described in METHODS. A: silver-stained SDS gels of immunoprecipitates. B-D: Western blot of immunoprecipitates was successively probed with anti-gamma -adaptin MAb 100/3 (B), anti-beta -adaptin MAb 74 (C), and anti-beta -adaptin PAb GD/2 (D). Immunoreactive signals were detected with ECL with a 2-min exposure for each blot. Results are representative of 2 independent immunoprecipitations. Migration of molecular mass markers is indicated.

When these immunoprecipitates were probed with anti-beta -adaptin MAb 74, reactivity was observed in the brain CCV fraction but not in the 200 mM phosphate fraction (Fig. 5C). However, both immunoprecipitates were immunoreactive with GD/2 (Fig. 5D). Thus the tubulovesicular AP-1 adaptor complex appears to contain a beta -adaptin that is immunologically distinct not only from another tubulovesicular beta -adaptin (compare Fig. 3, C and F) but also from the beta -adaptin of Golgi-associated AP-1 adaptors (Fig. 5, C and D). Moreover, these data suggest that this immunologically distinct beta -adaptin in the tubulovesicular AP-1 complex may be conserved among species and is relatively abundantly expressed in mammalian oxyntic cells.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

An AP-1 adaptor complex from oxyntic cell tubulovesicles has been preliminarily characterized. It appears to be composed of gamma -, beta -, µ-, and sigma -subunits. The beta -adaptin appears to be a beta -adaptin that is immunologically distinct from beta 1- and beta 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 beta -adaptin subunit. The nature of the heterogeneity of the tubulovesicular beta -adaptin has yet to be characterized. One possible cause for this heterogeneity is that the tubulovesicular beta -adaptin may be an isoform of the beta 1- and beta 2-adaptins. The other possibility is that some posttranslational modification, such as phosphorylation, may fortuitously mask the site recognized by the anti-beta -adaptin MAb. In support of this latter hypothesis, beta -adaptins have been shown to be phosphorylated in vivo (34). Further characterization of the tubulovesicular beta -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 beta -adaptin. Consistent with the hypotheses that distinct vesicular coat proteins may mediate distinct membrane trafficking pathways (4, 20, 27), the difference in beta -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 beta -adaptin may allow the tubulovesicular AP-1 adaptor to function differently from the Golgi AP-1 adaptor. Some of the functions ascribed to beta -adaptins may be relevant to the function of the tubulovesicular AP-1 beta -adaptin in this particular membrane trafficking pathway, a regulated apical recycling pathway, in oxyntic cells. beta -Adaptins possess a binding site for clathrin heavy chain (2, 8, 12). Because the tubulovesicular AP-1 beta -adaptin is immunoreactive with antibodies raised against this region, this beta -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 beta -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 beta 3-adaptin is distinctly different from beta 1- and beta 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 beta -adaptin may include a role in determining targeting to the tubulovesicular compartment, although it is not thought that beta -adaptins play such a role, given that the beta -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 beta -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 beta 1- and beta 2-adaptin has been shown to regulate its interaction with clathrin (34); it would be of interest to determine whether the tubulovesicular beta -adaptin is phosphorylated in a secretagogue-dependent manner.

The extent to which this tubulovesicular AP-1 beta -adaptin differs from the other characterized beta -adaptins must await its molecular characterization. Because tubulovesicles represent a type of regulated apical recycling compartment, once the tubulovesicular AP-1 beta -adaptin is characterized at the molecular level, it would be of interest to determine the expression levels of this AP-1 beta -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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