INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE REGULATION OF THE TRAFFICKING of membrane transporters is becoming widely recognized as a basic mechanism by which cells, epithelial cells in particular, regulate solute transport (8). The gastric parietal (oxyntic) cell represents a model system in which to study the means by which solute transport can be regulated by the vesicular trafficking of a membrane transporter, the H-K-ATPase (13, 21). In the resting parietal cell, the gastric H-K-ATPase is sequestered in an intracellular system of tubulovesicular membranes. When cells are stimulated to secrete HCl, the tubulovesicular membranes fuse with the canalicular (apical) membrane, thus delivering the H-K-ATPase to the apical membrane. When the stimulus is removed, the H-K-ATPase is retrieved from the canalicular membrane, and the tubulovesicular compartment is reestablished (22).

The tubulovesicular compartment of the parietal cell has recently been demonstrated to contain key components of the essential machinery to regulate the trafficking of the H-K-ATPase, such as the small GTPases rab11 (10) and rab25 (26), and proteins implicated in vesicular docking/fusion, such as syntaxin 3 and vesicle-associated membrane protein (VAMP) (10, 41). Some of these components, including rab11, show secretagogue-stimulated changes in subcellular membrane localization (11), and additional functional studies suggest that rab11 is a key regulator of H-K-ATPase trafficking (20). In addition, a tyrosine-containing motif in the cytoplasmic domain of the -subunit of the heterodimeric H-K-ATPase has been implicated to serve as a sorting signal for the reinternalization of the H-K-ATPase from the apical membrane on cessation of acid secretion (17). The motif in the -subunit is strikingly similar to the internalization signal in the transferrin receptor that allows the transferrin receptor to interact with clathrin adaptor protein-2 (AP-2) at the plasma membrane. Clathrin and an AP-1 clathrin adaptor have been recently identified on tubulovesicles (39). Moreover, the AP-1 adaptor and the H-K-ATPase appear to interact, as shown by their copurification from tubulovesicles solubilized with a nondenaturing detergent (39). Thus significant progress in recent years has been made in cataloging components of the molecular machinery ostensibly involved in the regulation of H-K-ATPase trafficking.

In the present study the role of clathrin in the regulation of trafficking of the H-K-ATPase in gastric parietal cells was investigated by biochemical and morphological approaches. Clathrin from H-K-ATPase-rich membranes was further characterized immunologically and by mass spectrometry. Clathrin and adaptors formed baskets in vitro. Immunofluorescent labeling and electron-microscopic localization of clathrin in resting parietal cells suggest a role for clathrin in the regulation of membrane traffic between tubulovesicular membranes and the canalicular membrane. Immunofluorescent labeling of clathrin in secretagogue-stimulated parietal cells suggests that clathrin is not translocated to the apical membrane with the H-K-ATPase and may therefore play a direct role in H-K-ATPase trafficking at another stage of the secretory cycle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Anti-clathrin heavy chain monoclonal antibodies (MAbs) X-22 (36) and TD.1 (36); the anti-clathrin light chain MAbs X-16 (1), LCB.1 (1), and CON.1 (36); and anti--adaptin MAb AP.6 (15) were obtained from two sources: as kind gifts of Dr. Frances Brodsky (University of California, San Francisco) and from X-22, TD.1, CON.1, and AP.6 hybridoma cell culture supernatants. The hybridomas were purchased from the American Type Culture Collection. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-FL)-phallacidin and rhodamine-phalloidin were purchased from Molecular Probes (Eugene, OR) and were used according to the vendor's instructions. Anti-dynamin MAb and anti-clathrin heavy chain MAb 23 were purchased from Transduction Labs (Lexington, KY). Anti--adaptin MAb 100/3 was purchased from Sigma Chemical (St. Louis, MO). Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody was purchased from Bio-Rad (Hercules, CA). Enhanced chemiluminescence (ECL) reagents were purchased from Pierce Chemical (Rockford, IL). Cy3-conjugated and FITC goat anti-mouse antibodies were purchased from Jackson ImmunoResearch Labs (West Grove, PA). ProLong antifade mounting medium was purchased from Molecular Probes. Vectashield antifade mounting medium was purchased from Vector Laboratories (Burlingame, CA). Digitonin was purchased from Boehringer (Indianapolis, IN). Cimetidine, 8-bromo-cAMP, and collagenase were purchased from Sigma Chemical. SCH-28080 was a gift from Dr. J. Kaminsky (Schering, Kenilworth, NJ). All other materials were reagent grade. Purified gastric microsomes were prepared as described previously (53). Crude clathrin-coated vesicles (CCV) from brain were purified according to the protocol of Pearse and Robinson (40).

SDS-PAGE and related procedures. Protein determinations were made using the bicinchoninic acid protein assay (Pierce Chemical). SDS-PAGE was performed according to Laemmli (32). Because of the sensitivity of the H-K-ATPase to extended boiling, samples containing H-K-ATPase were boiled for only 2 min in sample buffer before they were loaded on gel. Samples without H-K-ATPase were boiled for 5 min.

Hydroxyapatite chromatography of coat proteins from purified gastric microsomes. Clathrin coat proteins and other peripheral membrane proteins were stripped from purified gastric microsomes (starting with 3-30 mg of microsomal membrane proteins) essentially according to the protocol of Keen et al. (29). The stripped proteins were dialyzed overnight against three changes of a solution of 125 mM mannitol, 40 mM sucrose, 1 mM EDTA, and 5 mM PIPES buffer, pH 6.7 (MSEP). The dialysate was applied to a 5-ml hydroxyapatite column equilibrated with MSEP, and proteins were eluted by a stepwise gradient of 12 ml each of 10, 100, 200, and 400 mM sodium phosphate, pH 7.0. NaN₃ was added to the eluted fractions, and these fractions were stored at 4°C. For SDS gels and Western blots, proteins in 0.3- to 1.2-ml aliquots were precipitated with 10% TCA and resuspended in SDS gel buffer before electrophoresis.

In-gel digestion. A slightly modified in-gel digestion method from Rosenfeld et al. (44) was performed. Protein bands (~5 µg of protein) were minced, and the gel slices were destained with three washes of 50% acetonitrile-25 mM NH₄HCO₃ (~10 min each). The destained gel pieces were dried in a Speedvac (Savant, Farmingdale, NY) for 30 min and then rehydrated in 50 µl of 25 mM NH₄HCO₃ (pH 8.0) with 0.01 µg/µl trypsin. The slices were overlaid with 50 µl of 25 mM NH₄HCO₃ and incubated for 15 h at 37°C. Peptides were recovered by three extractions of the digestion mixture with 50% acetonitrile-5% trifluoroacetic acid. All supernatants were pooled, concentrated to 5 µl in a Speedvac, and brought back up to 25 µl in 50% acetonitrile-5% trifluoroacetic acid. The peptide mix was stored at 20°C until further analysis.

Matrix-assisted laser desorption delayed extraction reflection time of flight mass spectrometry of clathrin peptides. Aliquots () of unseparated tryptic digests were cocrystallized with -cyano-4-hydroxycinnamic acid and analyzed using a matrix-assisted laser desorption delayed extraction reflection (MALDI) time of flight (TOF) mass spectrometry (MS) instrument (Perceptive Biosystems, Voyager Elite mass spectrometer, Framingham, MA) equipped with a nitrogen laser at the University of California, San Francisco, Mass Spectrometry Facility. Measurements were performed in a positive ionization mode. All MALDI spectra were externally calibrated using a standard peptide mixture. For postsource decay (PSD) spectra, tryptic peptides were fractionated by reverse-phase microbore HPLC. PSD spectra were acquired on a TofSpec SE MALDI-TOF MS (Micromass, Manchester, UK) with a nitrogen laser and operated in the reflectron mode.

Database searches for protein identification. Experimentally determined masses were used for database interrogation with use of MS-Fit software (16, 43). PSD data interrogation was performed using MS-Tag. Both software programs were developed at the University of California, San Francisco, Mass Spectrometry Facility and are available on the World Wide Web at http://prospector.ucsf.edu. Protein searches were carried out in the National Center for Biotechnology Information protein database and the SwissProt database by using a protein molecular weight of 150-250 kDa, a peptide mass tolerance of 0.5 Da, and a minimum match of 50% of peptides observed in the total digest.

In vitro polymerization of clathrin and negative-stain electron microscopy. For in vitro polymerization of clathrin coats, 100 µg of purified gastric microsomes were stripped as described above. The stripped proteins were dialyzed overnight against three changes of 20 mM MES, pH 6.5, and either 1 mM sodium EGTA or 2 mM CaCl₂ to analyze the calcium dependency of clathrin and clathrin adaptor polymerization. The dialysate was spun at 300,000 g for 20 min in a miniultracentrifuge (model RC M120EX, Sorvall). The pellet was resuspended directly into SDS sample buffer, and proteins in the supernatant were precipitated with 10% TCA. The entire samples were loaded onto SDS gels for analysis by Coomassie blue staining and Western blot. Alternatively, 1-4 ml of the hydroxyapatite fraction enriched in tubulovesicular clathrin and AP-1 adaptors (i.e., proteins eluted by 200 mM sodium phosphate) were dialyzed overnight against one change of 20 mM MES, pH 6.5, and two changes of 20 mM MES, pH 6.5, and either 1 mM sodium EGTA or 2 mM CaCl₂. The dialysate was spun at 300,000 g for 20 min. The supernatant was removed, and proteins in the supernatant were precipitated in 10% TCA. The precipitated proteins were resuspended in SDS gel buffer to the same volume as the 300,000-g pellet. Equal volumes of fractions were run on gels for Coomassie blue staining or Western blotting. The efficiency of clathrin and clathrin adaptor polymerization was quantitated by scanning Coomassie blue-stained gels or films of immunoblots at 300 dpi with a UMAX Vista 6E color scanner, and the digitized images were processed using the NIH Image version 1.55 program. For Western blots developed by ECL, films exposed for different times were analyzed to ensure that the signals from bands fell within the linear range and were linear relative to each other.

High-pressure freezing of isolated gastric glands for transmission electron microscopy and immunogold electron microscopy. Gastric glands were isolated from rabbit stomach as previously described (54). Isolated gastric glands were incubated at 37°C in a balanced salt solution (54) containing 10⁴ M cimetidine as the H₂ receptor antagonist to the natural secretagogue histamine. Aliquots of glands were sedimented by light centrifugation and transferred to the 100-µm deep well of a type A high-pressure freezing planchette (Ted Pella, Redding, CA) and then frozen in a high-pressure freezing machine (model HPM 010, Bal Tec), as described by McDonald (35). Frozen samples were freeze substituted in 2% osmium tetroxide plus 0.1% uranyl acetate in acetone for morphological studies or in 0.2% glutaraldehyde plus 0.1% uranyl acetate in acetone for immunolocalization studies. Cells were kept in fixative at dry ice temperature (approximately 78°C) for 3 days and then warmed to room temperature over 12 h. After three 10-min rinses in pure acetone, osmium-fixed cells were infiltrated and embedded in Epon-Araldite resin. Glutaraldehyde-fixed cells were also rinsed three times in acetone, infiltrated, and embedded in LR White resin. Thin sections (60-70 nm thick) were cut on a Reichert Ultracut E (Leica, Deerfield, IL) or an MTX (RMC, Tucson, AZ) ultramicrotome, poststained with uranyl acetate and lead citrate, and observed in a JEOL 100CX transmission electron microscope operating at 80 kV.

Immunogold staining protocol. For immunogold labeling, LR White sections were picked up on 100-mesh nickel grids coated with Formvar film and carbon, incubated in blocking buffer (5% BSA, 0.1% fish gelatin, and 0.05% Tween 20 in PBS) for 30 min, and then incubated with primary antibodies diluted in blocking buffer for 1.5-2 h. MAb X-22 was used as undiluted cell culture supernatant; MAb 2G11 cell culture supernatant was diluted 1:5. The commercially procured MAb23 was diluted 1:100. Sections were rinsed in PBS-Tween, and then PBS and incubated for 1 h in secondary antibodies conjugated to 10-nm gold particles [goat anti-mouse IgG F(ab')₂ (H + L); Ted Pella] diluted 1:20 in blocking buffer. Sections were washed as described above, fixed in 0.5% glutaraldehyde in PBS for 5 min, and rinsed in PBS and water. Sections were poststained in 2% uranyl acetate for 5 min and in lead citrate for 3 min. Identically treated samples were stained with secondary antibody only as controls and revealed no labeling pattern.

Fractionation of sucrose density gradient-purified gastric microsomes on discontinuous glycerol velocity gradients. The additional fractionation of sucrose density gradient-purified gastric microsomes on discontinuous glycerol velocity gradients was adapted from Salem et al. (45). Gastric sucrose microsomes (200 µg) sedimenting at the 32% barrier on sucrose density gradients were diluted to 0.4 mg/ml in MSEP. It was layered on top of a discontinuous glycerol gradient comprised of 0.5 ml each of 20, 40, and 80% glycerol, diluted in MSEP. The sample was centrifuged in an RP55S-485 Sorvall swinging bucket rotor at 55,000 rpm for 30 min in a Sorvall RC M120EX miniultracentrifuge. The membranes sedimenting at each interface were collected, diluted to 1.0 ml with MSEP, and recentrifuged at 150,000 g for 40 min. The pellets were resuspended in SDS gel sample buffer and analyzed by SDS-PAGE and Western blot. For the membranes sedimenting at the 20 and 40% glycerol layers, the entire samples were loaded onto the gels; for membranes sedimenting at the 80% layer, one-eighth to one-fourth of the sample was loaded onto the gels.

Immunofluorescent staining of isolated gastric glands and cultured parietal cells. Isolated glands were incubated with 10⁵ M cimetidine (resting) for 30 min at 37°C in a total of 10 ml of a suspension of a 1:10 dilution of glands. In the case of staining with anti-dynamin MAb, glands were fixed in 3.7% formaldehyde in PBS for 20 min and then permeabilized in 0.5% Triton X-100-PBS for 20 min, both at room temperature. After fixation and permeabilization, glands were blocked for 30-60 min at room temperature with a solution of 1% BSA and 0.066% (wt/vol) fish skin gelatin diluted into PBS. Glands were incubated with anti-dynamin MAb (diluted 1:100 in blocking solution) for 2 h at room temperature. Glands were then incubated with Cy3-conjugated secondary antibody diluted 1:500 into PBS-0.05% Tween 20 for 60 min at room temperature. Glands were counterstained with BODIPY FL-phallacidin simultaneously with the secondary antibody. Glands were then washed and mounted in ProLong or Vectashield antifade medium.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Immunodetection of clathrin heavy chain and light chains on Western blots of purified gastric microsomes. The MAb TD.1 was used to confirm the presence of clathrin in density gradient fractions of H-K-ATPase-rich gastric microsomes derived from tubulovesicles of parietal cells. A Coomassie blue-stained gel of the purified microsomal fractions from rabbit gastric mucosa is shown in Fig. 1A (lanes 1 and 2), with the position of the -subunit of the H-K-ATPase indicated. The Western blots in Fig. 1B show that clathrin heavy chain, as recognized by MAb TD.1, is present in microsomes purified from rabbit gastric mucosa, confirming earlier identification of clathrin heavy chain in these H-K-ATPase-rich membranes (39).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Immunodetection of clathrin heavy chain on purified gastric microsomes of parietal cells. A: Coomassie blue-stained SDS gel of sucrose density gradient-purified gastric microsomes from rabbit gastric mucosa (24 µg protein/lane). Position of the H-K-ATPase -subunit (HK) is indicated. Lane 1, microsomes purified at the 27% sucrose layer; lane 2, microsomes purified at the 32% layer. Apparent molecular masses are indicated, and the identity of the markers is as follows: myosin, -galactosidase, phosphorylase B, BSA, ovalbumin, and carbonic anhydrase. B: Western blot of purified gastric microsomes (15 µg protein/lane) with anti-clathrin heavy chain monoclonal antibody (MAb) TD.1. Lane 1, 27% sucrose layer; lane 2, 32% sucrose layer. Immunoreactivity was detected by enhanced chemiluminescence (ECL) with a 2-min exposure to film. Position of prestained molecular mass markers (myosin and -galactosidase) is shown. C: comparison of clathrin content of crude clathrin-coated vesicles (CCVs) from brain and purified gastric microsomes. Coomassie blue-stained SDS gel of crude CCVs from hog brain and purified gastric microsomes (10 µg protein/lane) is shown. Lane 1, crude CCVs from hog brain; lane 2, hog gastric microsomes purified at the 32% layer. D: Western blot with MAb TD.1. Lane 1, CCVs from hog brain (5 µg); lane 2, hog gastric microsomes purified at the 32% layer (5 µg). Signal detected by ECL with a 2-min exposure.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Identification of clathrin light chain isoforms on purified gastric microsomes by immunoblotting. A: Western blot of crude CCVs from hog brain (3 µg) and hog gastric microsomes, 32% layer (10 µg), with anti-light chain A MAb X-16. Signal was detected by ECL with a 2-min exposure. B: Western blot of crude CCVs from hog brain (3 µg) and hog gastric microsomes, 32% layer (10 µg), with anti-light chain B MAb LCB.1. Signal was detected by ECL with a 1-min exposure. C: Western blot of crude CCVs from hog brain (lanes 1 and 2) and hog gastric microsomes (lanes 3 and 4) with anti-light chain (common) MAb CON.1. Lane 1, 3 µg of CCVs; lane 2, 5 µg of CCVs; lane 3, 10 µg of 32% gastric microsomes; lane 4, 20 µg of 27% gastric microsomes. Positions of light chains A and B (LCa and LCb) are indicated. Signal was detected by ECL with a 15-s exposure.

Fractionation of gastric microsomal clathrin by hydroxyapatite chromatography. Purified gastric microsomes were stripped of their clathrin coats (and other peripheral membrane proteins) by incubation with 0.5 M Tris · HCl and 2 mM EDTA. After dialysis in a low-ionic-strength buffer, the stripped proteins were applied to a hydroxyapatite column, and proteins were eluted by a stepwise sodium phosphate gradient. A major protein, eluted by the addition of 0.2 M sodium phosphate and clearly visible on Coomassie blue-stained gels (Fig. 3A, lane 4), was identified as clathrin heavy chain on the basis of its immunoreactivity with anti-clathrin heavy chain MAb 23 (Fig. 3B) and TD.1 (not shown). Clathrin light chains (with approximately the same ratio of LCa to LCb as in isolated membranes) were also detected in Western blots of the 0.2 M sodium phosphate eluate (Fig. 3C). From densitometric analyses of Coomassie blue-stained gels, clathrin heavy chain was determined to comprise 14 ± 3% (SD, n = 10 independent preparations) of the total protein eluted in this fraction. By comparison, in crude CCVs isolated here (Fig. 1C), clathrin comprises ~12% of the total protein. If we assume that the recovery of clathrin heavy chain by hydroxyapatite chromatography is 100%, we estimate that gastric microsomes contain ~17 µg of clathrin heavy chain per milligram of microsomal protein. If one assumes that the H-K-ATPase comprises minimally 50% of the purified gastric microsomal protein, a ratio of 50 copies of H-K-ATPase per copy of clathrin heavy chain can be calculated.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Hydroxyapatite chromatography of gastric microsomal peripheral membrane proteins. Gastric microsomal membranes were stripped of their peripheral membrane proteins in 0.5 M Tris · HCl buffer and fractionated on hydroxyapatite. Each fraction was analyzed by SDS-PAGE and immunoblotting. Each lane contains the proteins from 1/40 of the total volume of each fraction. A: Coomassie blue-stained gel. Lane 1, nonbinding fraction; lane 2, proteins eluted with 10 mM sodium phosphate; lane 3, proteins eluted with 100 mM sodium phosphate; lane 4, proteins eluted with 200 mM sodium phosphate; lane 5, proteins eluted with 400 mM sodium phosphate. Positions of molecular mass markers are shown and are the same as described in Fig. 1. B: Western blot of the same fractions in A with anti-clathrin heavy chain MAb 23. C: Western blot of the same fractions in A with anti-light chain MAb CON.1. D: Western blot of the same fractions in A with anti--adaptin MAb 100/3. Signal was detected by ECL with a 2-min exposure.

Characterization of gastric microsomal clathrin by MS. The 160-kDa gel band immunoreactive with anti-clathrin heavy chain MAbs (Fig. 3, A and B, lane 4) was subjected to in-gel trypsinolysis, and the recovered peptide mixture was analyzed by MALDI-TOF-MS to yield a peptide mass fingerprint (Fig. 4). When a database search was performed with 17 input peptide masses, only conventional human, bovine, and rat clathrin heavy chain (clathrin heavy chain I) matched the input criteria given in MATERIALS AND METHODS. These observed peptides are compared with the predicted masses from tryptic digestion of clathrin heavy chain I in Table 1, all agreeing within a mass accuracy of 0.2 Da.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Matrix-assisted laser desorption delayed extraction reflection (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of peptides generated from in-gel tryptic digestion of gastric microsomal clathrin enriched by hydroxyapatite chromatography. The hydroxyapatite fraction enriched in immunoreactive clathrin heavy chain was resolved by SDS-PAGE. The 160-kDa band immunoreactive with anti-clathrin heavy chain antibodies was excised from the Coomassie blue-stained gel and subjected to in-gel proteolysis with trypsin. Aliquots (1/25) of unseparated tryptic digests were cocrystallized with -cyano-4-hydroxycinnamic acid and analyzed using an MALDI TOF instrument equipped with a nitrogen laser. Measurements were performed in a positive ionization mode. The peptide mass fingerprint is shown with peptides matching clathrin heavy chain I assigned to their respective masses. m/z, Mass-to-charge ratio.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Postsource decay (PSD) spectrum of 1,415.82 m/z peptide purified by reverse-phase microbore HPLC. Sequencing of peptide with m/z of 1,415.82 confirms that gastric microsomal clathrin heavy chain is conventional clathrin heavy chain (clathrin heavy chain I).

In vitro polymerization of gastric microsomal clathrin and adaptors. Proteins stripped from purified gastric microsomes with 0.5 M Tris buffer were dialyzed against a low-ionic-strength buffer in the absence or presence of Ca²⁺. The polymerized proteins were sedimented and compared with the nonpolymerized proteins. The overall polypeptide pattern of the sedimented proteins (Fig. 6A, lane 2) differs from that of the proteins remaining in the supernatant (Fig. 6A, lane 3). The prominent 160-kDa protein in the sedimentable material was confirmed to be clathrin heavy chain by immunoblot analysis (not shown). Also, as shown in the Western blot in Fig. 6B, -adaptin was quantitatively recovered in a sedimentable complex. However, the quantitative polymerization of clathrin and adaptors did not appear to depend on the presence of Ca²⁺ in the dialysis buffer. Of the total recovered clathrin, 69 ± 21% (SE, n = 5 independent experiments) was found in the pellet in the absence of Ca²⁺, whereas 59 ± 30% (SE, n = 11 independent experiments) was found in the pellet in the presence of Ca²⁺.

View larger version (69K): [in this window] [in a new window]   Fig. 6.   In vitro polymerization of gastric microsomal clathrin and adaptors. A: Coomassie blue-stained gel of proteins assembled into a sedimentable complex on dialysis of proteins stripped from purified gastric microsomes. Lane 1, starting material (proteins stripped from purified gastric microsomes by 0.5 M Tris); lane 2, proteins in a pelletable complex after dialysis in low-ionic-strength buffer; lane 3, proteins remaining in the supernatant. For the pellet and the supernatant (lanes 2 and 3), equal proportions of the entire sample were loaded in each lane. B: Western blot with anti--adaptin MAb 100/3 of the same samples in A. Signal was detected by ECL with a 2-min exposure. C: Coomassie blue-stained gel of proteins assembled into a sedimentable complex on dialysis of proteins in the fraction enriched in clathrin obtained from the hydroxyapatite column (200 mM sodium phosphate eluate). Lane 1, starting material; lane 2, proteins in a pelletable complex after dialysis; lane 3, proteins remaining in the supernatant. For the pellet and the supernatant (lanes 2 and 3), equal proportions of the entire sample were loaded in each lane.

View larger version (142K): [in this window] [in a new window]   Fig. 7.   Electron micrographs of gastric microsomal clathrin and AP-1 clathrin adaptors assembled in vitro into baskets. Baskets were visualized by negative staining with 1% uranyl acetate. Clathrin and AP-1 clathrin adaptors were assembled by dialysis of the fraction from hydroxyapatite chromatography that is enriched in clathrin. The pelletable material was resuspended and processed for negative-stain electron microscopy. Scale bar, 100 nm.

Immunofluorescent labeling of clathrin in digitonin-permeabilized resting parietal cells. With most of our previously used immunostaining protocols, the distribution of clathrin immunostaining within parietal cells was suggestive of labeling of the tubulovesicular compartment (39); however, we could not clearly distinguish between labeling of clathrin on tubulovesicles or other intracellular membranes and labeling of a cytoplasmic pool of clathrin. Thus a prefixation step of permeabilization by digitonin was employed to visualize membrane-associated clathrin (33). Figure 8, A and A', shows confocal scanning laser micrographs of digitonin-permeabilized gastric glands doubly stained for F-actin and clathrin, respectively. Canalicular (apical) membranes of resting parietal cells are delineated by intense staining of F-actin with BODIPY FL-phallacidin (Fig. 8A). The canaliculi within parietal cells are clearly identifiable as a network of tubular structures projecting from the gland lumen (Fig. 8, A-C). The lumen of the gland, including the apical membranes of parietal cells as well as all other cell types in the gastric gland, is also prominently stained with BODIPY FL-phallacidin. Abundant intracellular staining for clathrin is evident in parietal cells, with a significant amount of staining associated with the regions peripheral to canalicular membranes; this staining pattern is consistent with clathrin immunoreactivity on tubulovesicular membranes. Interestingly, clathrin immunoreactivity is also readily visible at the apical canalicular membranes of parietal cells (Fig. 8A', arrowheads), as indicated by its colocalization with F-actin staining of canaliculi (Fig. 8A).

View larger version (110K): [in this window] [in a new window]   Fig. 8.   Confocal immunofluorescence microscopy of nonstimulated rabbit gastric glands doubly stained for F-actin (A-C) and clathrin (A'), dynamin (B'), and -adaptin (C'). BODIPY FL-phallacidin was used for F-actin staining. F-actin staining was prominent along the gland lumen, which is made up of the apical membrane of nonparietal and parietal cells. Within parietal cells, F-actin staining is also obvious on the basolateral membrane and the intracellular canaliculi (arrowheads), which are invaginated and branching apical membranes of these cells. Gastric glands were permeabilized with digitonin before fixation and costained for F-actin (A) and anti-clathrin heavy chain MAb X-22 (A'). Immunostaining for clathrin was observed on canalicular membranes (arrowheads) and abundantly throughout the cytoplasm in parietal cells. This latter staining may represent staining of tubulovesicles. Gastric glands were fixed, permeabilized, and costained for F-actin (B) and dynamin by use of anti-dynamin MAb (B'). The most prominent staining for dynamin is on canaliculi of parietal cells (arrowheads). Digitonin-permeabilized gastric glands costained for F-actin (C) and the -adaptin subunit (C') of the AP-2 clathrin adaptor. Canalicular staining for -adaptin is indicated (arrowheads).

Identification and localization of clathrin-coated pits and vesicles in parietal cells by thin-section electron microscopy of isolated rabbit gastric glands prepared by high-pressure rapid freezing and freeze substitution. Previous ultrastructural studies have not been able to demonstrate convincingly the presence of clathrin-coated membranes in parietal cells (7, 23, 28, 47). Yet, the biochemical and immunofluorescence data suggest that clathrin is a significant component of tubulovesicles and apical membranes. Thus we sought to reexamine the identification and localization of clathrin in parietal cells at the ultrastructural level. Isolated rabbit gastric glands were prepared for thin-section electron microscopy by high-pressure rapid freezing and freeze substitution. A low-magnification (×4,000) image of a resting, nonsecreting parietal cell is shown in Fig. 9A; this micrograph demonstrates the excellent morphological preservation of parietal cells by the high-pressure rapid-freezing technique. Prominent features observed previously in electron micrographs of resting cells that are also observed here are the numerous mitochondria, intracellular canaliculi (which are invaginations of the apical membrane), and, barely visible, the elaborate system of tubulovesicles in proximity to intracellular canaliculi. At higher magnification, coated membranes are clearly visible along the intracellular canaliculus, which also includes nicely preserved microvilli and their microfilaments (Fig. 9B). Coated pits are prominent at the canalicular membranes (Fig. 9C, arrows), and most of the coated pits appear at the base of canalicular microvilli. On average, these pits are 60-90 nm diameter. Coated membranous structures in the subapical cytoplasm (Fig. 9C, arrowhead) are occasionally visible. The ability to identify a significant number of coated pits at the canalicular membrane reinforces the conclusion that this membrane in resting parietal cells is an endocytotically active zone. Moreover, these coats possess a morphology that is highly reminiscent of conventional clathrin-coated membranes, suggesting that clathrin is involved in endocytotic processes at the canalicular membrane.

View larger version (202K): [in this window] [in a new window]   Fig. 9.   Transmission electron micrographs of isolated rabbit gastric glands fixed by high-pressure rapid freezing. A: low-power magnification of a parietal cell in a gastric gland. Numerous electron-dense mitochondria (m) and intracellular canaliculi (c) are visible. A glancing section through a nucleus (n) is also seen. Tubulovesicles are barely visible at this magnification in the regions around the intracellular canaliculi. B: high-power magnification of a region surrounding an intracellular canaliculus with short microvilli projecting into the lumen of the canaliculus. Microfilaments are evident in the microvilli lining the lumen of the intracellular canaliculus. Several endocytic structures, some of which appear to possess a coat at the cytosolic face of the membranes, are visible. Mitochondria and numerous tubulovesicular structures are seen in the surrounding cytoplasm. C: higher-power view of a region surrounding an intracellular canaliculus. Several coated endocytic structures are seen invaginating from the canalicular surface (arrows) or as coated vesicles in the subapical cytoplasm (arrowhead). Cytoplasmic space also includes numerous tubulovesicles, several of which appear as indented saccules or C-shaped structures. Scale bars, 1 µm.

Immunogold electron-microscopic localization of clathrin and H-K-ATPase in resting parietal cells. Isolated rabbit gastric glands were processed for immunogold localization of clathrin and H-K-ATPase within resting parietal cells. The immunogold localization of clathrin by use of anti-clathrin heavy chain MAb X-22 is shown in Fig. 10A in a region including and surrounding a canaliculus. Gold particles clearly decorate invaginated membranes or pits at the canalicular surface, usually at the bases of microvilli (arrowheads). These data thus confirm that the coated pits observed in Fig. 9, B and C, are comprised of clathrin. In Fig. 10A and in the more extensive subapical cytoplasmic view in Fig. 10B, densely staining, 80- to 100-nm-diameter vesicular profiles are also decorated with gold particles, as are the ends of the tubulovesicles (arrows). Thus, although a distinctive coat was not observed on these membranes by standard electron-microscopic staining protocols, clathrin can be localized to these sites by immunogold labeling. Similar results were obtained with another anti-clathrin heavy chain antibody, MAb 23 (not shown). This identification of clathrin at the ultrastructural level on tubulovesicles of parietal cells very likely corresponds to the clathrin identified biochemically on purified gastric microsomes.

View larger version (114K): [in this window] [in a new window]   Fig. 10.   Immunogold labeling of clathrin with anti-clathrin heavy chain MAb X-22 in parietal cells. Nonstimulated gastric glands were prepared for immunoelectron microscopy by high-pressure rapid freezing. A: section showing canaliculus and subcanalicular space. B: section deeper within the cytoplasm of parietal cell. Small clusters of gold particles indicated that clathrin at the apical canalicular membrane was predominantly associated with the membrane at the base of microvilli (arrowheads), usually concentrated in apical pits or invaginations, or subapical vesicles. Within the cytoplasm, accumulations of clathrin-associated gold particles were frequently found on the ends of tubulovesicles and on small dense structures that appeared to be near, or sometimes budding from, tubulovesicles (arrows). Scale bars, 0.5 µm.

View larger version (179K): [in this window] [in a new window]   Fig. 11.   Immunogold labeling of H-K-ATPase in parietal cells. Immunoelectron microscopy was carried out on the same preparations used for clathrin staining in Fig. 10, but with MAb 2G11 used for H-K-ATPase. Gold particles were abundant in the cytoplasm in close association with tubulovesicles. The apical canalicular membrane was also decorated with gold particles, usually more obvious along the lengths of the microvilli and less concentrated at the microvillar bases. Virtually no gold particles were associated with mitochondria or other intracellular organelles. Scale bar, 0.5 µm.

Subfractionation of gastric microsomes by glycerol gradient centrifugation. The immunogold labeling of clathrin in parietal cells suggests that at least two different types of intracellular membranes may possess a clathrin coat: the ends of tubulovesicles and the densely staining vesicles. Such membranes may cofractionate with "conventionally" purified H-K-ATPase-rich gastric microsomes. Thus we subjected purified gastric microsomes to additional fractionation on a discontinuous glycerol gradient. Figure 12 shows Coomassie blue-stained gels and Western blots of purified gastric microsomes subfractionated on a discontinuous glycerol gradient. By this approach, a population of clathrin-rich and H-K-ATPase-poor membranes was identified at the 40% glycerol boundary, whereas the majority of H-K-ATPase was found in membranes sedimenting at the 80% glycerol boundary (Fig. 12A). It is clear from the immunoblots that clathrin is also present in the 80% glycerol fraction (Fig. 12B), and after correction for the total amount of protein, this fraction still contained the majority of the total microsomal clathrin. Interestingly, the 80% fraction is also enriched in the -adaptin subunit of the AP-1 clathrin adaptor (Fig. 12C). Thus the glycerol gradient can effect the separation of two types of clathrin-coated membranes, those poor in H-K-ATPase and those rich in H-K-ATPase, and may correspond to two (or more) intracellular populations of clathrin-coated membranes identified by immunogold electron microscopy.

View larger version (53K): [in this window] [in a new window]   Fig. 12.   Fractionation of purified gastric microsomes on discontinuous glycerol velocity gradients. Gastric microsomes purified on a 32% sucrose density gradient were further fractionated on discontinuous glycerol velocity gradients. A: Coomassie blue-stained gel of glycerol gradient fractions. The position of the migration of the H-K-ATPase -subunit is shown. B: Western blot with anti-clathrin heavy chain MAb TD.1. C: Western blot with anti--adaptin MAb 100/3. Signals on Western blots were detected by ECL with films exposed for 2 min. Lane 1, 20 µg of 32% sucrose density gradient-purified gastric microsomes; lane 2, membranes fractionating at the 20% glycerol layer; lane 3, membranes fractionating at the 40% glycerol layer; lane 4, membranes fractionating at the 80% glycerol layer, representing of total protein fractionating at this layer. The migration of molecular markers is shown.

Immunofluorescent staining of clathrin, AP-1 clathrin adaptors, and H-K-ATPase in resting and stimulated primary cultures of rabbit parietal cells. With the characterization of the steady-state localization of clathrin in resting cells, we sought to characterize the role of clathrin in the dynamic membrane trafficking processes occurring during functional resting-to-stimulated transition of parietal cells. Primary cultures of parietal cells have proven to be a good system with which to evaluate membrane recruitment and structural rearrangement associated with stimulation (2).

View larger version (86K): [in this window] [in a new window]   Fig. 13.   Immunofluorescent labeling of clathrin heavy chain and F-actin or H-K-ATPase in cultured parietal cells under various secretory conditions. Primary parietal cell cultures were held in a resting state through the addition of 100 µM cimetidine as an H2 receptor antagonist (resting) or maximally stimulated through addition of 100 µM histamine and 30 µM IBMX (stimulated). In some cases, cells were treated with the proton pump inhibitor SCH-28080 (5 µM) in addition to histamine plus IBMX (stimulated + SCH-28080). After 25 min of incubation with drugs at 37°C, cells were fixed and processed for immunofluorescence. Some cells were doubly stained for F-actin (Phall) and clathrin (X-22) or for F-actin and H-K-ATPase (HK). In those cases where cells were doubly stained for clathrin and H-K-ATPase, the MAb 2G11 antibody for H-K-ATPase was previously biotinylated, and rhodamine-conjugated avidin was used as the fluorescent probe. In the resting state, F-actin can be seen predominantly at the apical membrane vacuoles within the parietal cells and somewhat less intensely at the surrounding plasma (basolateral) membrane. In these resting cells, clathrin heavy chain and H-K-ATPase are distributed in a similar, although not identical, pattern, i.e., some staining at the apical membrane vacuoles and punctate staining throughout most of the cytoplasmic space. In stimulated parietal cells, the apical membrane vacuoles have expanded to enormous size because of accumulation of HCl and water. There appears to be a great deal of overlap in the staining for F-actin, clathrin, and H-K-ATPase, possibly because of the diminution of cytoplasmic space as the vacuoles have expanded. For cells stimulated in the presence of the proton pump inhibitor (stimulated + SCH-28080), the apical membrane vacuoles are not expanded or are only slightly enlarged, and in most cases there have been some peripheral extensions in the form of lamellipodia. Even though HCl secretion does not occur, H-K-ATPase has been recruited to the apical membrane vacuoles and has been virtually cleared from the cytoplasm (tubulovesicles), essentially colocalizing with F-actin at the apical membrane vacuoles. On the other hand, much of the clathrin is still distributed throughout the cytoplasm. Scale bar, 10 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 14.   Immunofluorescent double labeling of F-actin and clathrin heavy chain, clathrin light chain, or -adaptin in cultured parietal cells under various secretory conditions. Primary parietal cell cultures were held in a resting state through the addition of 100 µM cimetidine (resting), maximally stimulated by the addition of 100 µM histamine and 30 µM IBMX (stimulated), or treated with the proton pump inhibitor SCH-28080 (5 µM) in addition to histamine plus IBMX (Stim + SCH-28080). After 25 min of incubation with the drugs, cells were fixed, permeabilized, and probed for F-actin (A, B, and C) and clathrin heavy chain by use of MAb 23 (A', B', and C'), for F-actin (D, E, and F) and clathrin light chain by use of CON.1 MAb (D', E', and F') or for F-actin (G and H) and -adaptin by use of MAb 100/3 (G' and H'). Morphological responses to stimulants plus inhibitor were the same as for Fig. 13; i.e., vacuoles expanded when stimulated and the enlarged swelling was prevented by the pump inhibitor. A large portion of clathrin, when probed for heavy chain (C') or light chain (F'), remained distributed throughout the cytoplasm in parietal cells stimulated in the presence of SCH-28080. Similarly, much of the signal for -adaptin (H') remained distributed throughout the cytoplasm in parietal cells stimulated in the presence of SCH-28080. In parallel tests, H-K-ATPase was found to be cleared from the cytoplasm and recruited to the apical membrane vacuoles when the cells were stimulated in the presence of SCH-28080 (not shown). Scale bars, 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Biochemical characterization of gastric microsomal clathrin. Previously, clathrin and an AP-1 clathrin adaptor were identified on gastric microsomes from parietal cells and preliminarily characterized (39). In this study, gastric microsomal clathrin was characterized biochemically as a first step in the elucidation of its function in the regulation of membrane trafficking in the gastric parietal cell. Clathrin appears to constitute a significant fraction of the total peripheral membrane proteins of purified gastric microsomes. The enrichment of clathrin from gastric microsomes on hydroxyapatite columns reported here should serve as a convenient preliminary step in the purification of clathrin from gastric microsomes. Gastric mucosal tissue may represent an easily obtainable source of clathrin from epithelial cells and should therefore facilitate the biochemical analysis of clathrin and associated proteins from a secretory epithelial cell.

Ultrastructural localization of clathrin on tubulovesicles. With the high-pressure rapid-freezing technique for preservation of gastric glands for immunoelectron microscopy, we have finally successfully identified clathrin on intracellular membranes resembling tubulovesicles in parietal cells. The amount of anti-clathrin labeling we observe on tubulovesicles at the ultrastructural level relative to the amount of H-K-ATPase immunoreactivity appears to be consistent with our biochemical estimation of the amount of clathrin relative to H-K-ATPase. An intriguing feature of the distribution of clathrin is its localization to specific sites on tubulovesicular membranes, i.e., at their ends. The implication of these findings is that clathrin may be involved in the formation of vesicles budding from tubulovesicles in resting parietal cells, with the canalicular membrane as a potential target membrane domain (see below). For example, clathrin on tubulovesicles may be involved in recycling of apical membrane components from tubulovesicles to the apical membrane, such as receptors for soluble N-ethylmaleimide-sensitive factor attachment protein (t-SNARES) like syntaxin 3 (41).

Localization of clathrin and associated proteins at the canalicular membrane. In addition to clathrin, -adaptin and a member of the dynamin family of large GTPases were also immunolocalized to canalicular membranes. Using the high-pressure rapid-freezing protocol and standard staining techniques or immunogold labeling, we have also been able to visualize coated pits and membranes at the canalicular surface that are morphologically very similar to conventional clathrin-coated pits. Taken together, these results provide the first evidence that the canalicular membrane of the resting parietal cell is endocytotically active in a process involving clathrin, the AP-2 clathrin adaptor, and a dynamin. It would be of interest to identify the endocytic cargo in resting parietal cells to characterize further the role of clathrin in the physiology of resting cells; in this pathway, a possible candidate for endocytotic cargo might be a v-SNARE, e.g., VAMP (10, 41). Interaction of v-SNARE with endocytotic machinery has been shown in the case of synaptotagmin-AP-2 adaptor interactions at neuronal synapses (46, 56).

Clathrin and membrane trafficking in the parietal cell secretory cycle. Morphological data presented here suggest that the canalicular membrane of resting parietal cells is endocytotically active, and this process is mediated by clathrin, the AP-2 clathrin adaptor, and dynamin. The endocytotic cargo is likely to be destined for tubulovesicles, early endosomes, or some yet uncharacterized intracellular membrane compartment. To maintain the steady-state tubulovesicular or intracellular membrane surface area, membranes need to be recycled from these intracellular compartments to the apical membrane. This process also appears to be mediated by clathrin. Three models may account for the steady-state localization of clathrin on canalicular and intracellular membranes in resting parietal cells. The first model is one in which continuous endocytosis and recycling is occurring in resting cells, as occurs in most cells. Thus one would predict that the population of intracellular membranes would be comprised of a set of clathrin-coated early endosomal membranes and a distinct set of regulated secretory H-K-ATPase-rich tubulovesicular membranes that might not participate in the constitutive endocytotic-recycling pathway and might not be clathrin coated. However, the clathrin-coated early endosomes may copurify with the H-K-ATPase-rich membranes on sucrose density gradients, thereby giving the impression at the biochemical level that clathrin resides on H-K-ATPase-rich membranes.