Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase

Joseph G. Duman, Kamala Tyagarajan, Michelle S. Kolsi, Hsiao-Ping H. Moore, and John G. Forte

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 ± 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 ± 0.9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 ± 3.1 and 9.6 ± 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

acid secretion; dominant negatives; membrane recruitment; membrane recycling; rab isoforms; small GTPases; matrix-assisted laser desorption mass spectrometry


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TEMPORAL AND SPATIAL CONTROL of the events of vesicular traffic is of tantamount importance to the function of eukaryotic cells. Much research has been directed at identifying the factors that mediate these functions as well as elucidating their mechanisms of action. The rab GTPases are an intriguing set of mammalian protein factors that have been implicated in these processes (26, 47). Rabs are members of the ras superfamily of small GTPases. Like other members of this superfamily, they are small (usually 20-25 kDa), share conserved guanine nucleotide binding and hydrolysis motifs, and are isoprenylated on sites at or near their COOH termini. Rabs share ~40% homology with each other (and their yeast protein transport homologs, YPT) and have features that set them apart from other ras-related proteins. Rabs are ubiquitous in eukaryotic cells, and each of the >30 members of the YPT/rab subfamily localizes to a distinct intracellular region.

Evidence for the involvement of YPT/rabs in vesicular traffic is voluminous and convincing. Early observations with temperature-sensitive yeast mutant strains for Sec4p or Ypt1p showed that, at nonpermissive temperatures, vesicles accumulated at target membranes but failed to fuse there (34). Studies in mammalian systems using constitutively active and dominant negative point mutants of specific rabs have likewise produced telling results. Among the results of these studies are the assignment of rab3 to regulated exocytic fusion events in neurons (3), rab5 to budding (27) and homotypic fusion (5) of early endosomes, rab4 to recycling from the early endosome to the plasma membrane (42), and many others. These results have led to the hypothesis that a specific rab is required for each of the vesicular docking and fusion events that characterize a eukaryotic cell. Spatial control of these processes is thought to come from the distinct localization of individual rabs and temporal control to come from effectors that determine the phosphorylation state of the bound guanine nucleotide; like other small GTPases, rabs appear to be active in the GTP-bound state and inactive in the GDP-bound state.

Effectors that modulate rab location and the phosphorylation state of the rab-bound nucleotide, such as rab escort proteins (REP), guanine nucleotide exchange factors (GEF), GTPase-activating proteins (GAP), and GDP-dissociation inhibitors (GDI), are generally believed to exert their effects in a manner paralleling their counterparts specific to other members of the ras superfamily (29, 32), although experimental data to this conclusion are not unequivocal. These modulations, however, are critical for rab function. Many effectors, both upstream and downstream, associate in a manner dependent on the phosphorylation state of the bound nucleotide. Even if upstream effectors of rab prove to mirror those of other ras-related proteins, identification of downstream effectors has complicated our view of rab function. These include Zn2+ finger proteins, such as rabphilin 3A (29) and Rim (45) (both of which also contain phospholipid-binding C2 domains), novel proteins capable of interactions with multiple rabs, such as rabaptin-5 (43), proteins with GEF activity, such as rabex-5 (22), and proteins with motor domains, such as rabkinesin-6 (13). Genetic studies have implied that the ubiquitous soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) may also be rab effectors, although the interactions between rabs and SNAREs are likely transient (24, 25, 39). These observations make it difficult to conceive of a mechanism that explains the action of every rab.

Rab11 is a member of the rab subfamily that shows a perinuclear localization in most cells examined. In Chinese hamster ovary cells, functional rab11 is required for traffic of the transferrin receptor through the recycling endosome on its way back to the plasma membrane (41). Rab11 and its homologs have also been implicated in basolateral protein trafficking in polarized cells (8) and fruit ripening (46). The gene product originally designated rab11 shows 100% identity among mammalian species thus far examined and is very similar to a recently cloned homolog, rab11b (23).

Our model system for the study of vesicular traffic events involving rab11a is the rabbit gastric parietal cell, which is responsible for secreting hydrochloric acid into the stomach lumen. The H+-K+-ATPase acts as the proton pump in this process (15). In the resting parietal cell the H+-K+-ATPase is sequestered in a compartment of cytoplasmic tubulovesicles; the low K+ permeability of these membranes prevents enzyme turnover. On stimulation, the H+-K+-ATPase-rich tubulovesicles move to the deeply invaginated apical surface of the cell in a massive vesicular traffic event (16, 17). The high K+ permeability of the apical plasma membrane permits H+-K+-pump turnover, generating a transmembrane proton gradient >1,000,000-fold. Goldenring et al. (18) noted colocalization of rab11a with H+-K+-ATPase in resting parietal cells and observed a shift in the distribution of rab11a to the plasma membrane on stimulation of the cells, prompting their speculation that rab11a is responsible for the stimulus-dependent membrane translocation (18). More recently, this same laboratory has provided evidence that the mechanism of action of rab11a-mediated vesicular fusion is distinct from rab3-mediated fusion in neuroendocrine cells (7).

Despite this body of work, direct evidence for the involvement of rab11a in parietal cell stimulation has yet to be produced. In the present study, we confirmed the presence of rab11 in parietal cell tubulovesicles, showing that rab11 is stoichiometric with H+-K+-ATPase and that it exists in multiple isoforms. An adenoviral system was used to express the dominant negative point mutant of rab11a, in which asparagine-124 is mutated to isoleucine (N124I), in primary cultures of parietal cells. Functional and morphological assays demonstrated that secretion was inhibited by the mutant rab11a N124I, directly implicating rab11a in the parietal cell activation pathway.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of recombinant adenovirus expressing rab11 mutant. Human rab11a was isolated by PCR amplification from a human Hep G2 library (Chiron, Emeryville, CA) using primers based on the Madin-Darby canine kidney clone. The human rab11a was then cloned into Xho I/Not I sites of a pCDM8-derived vector that contains the influenza hemagglutinin (HA) epitope tag sequence at the NH2 terminus (9). Mutant HA-tagged rab11a N124I was generated by PCR mutagenesis of pCDM8-HA-rab11a and verified by DNA sequencing analysis. The HA-rab11a N124I sequence was then placed under the control of a tetracycline-regulatable transcription system (20) and packaged into adenovirus (courtesy of Dr. Steve Hardy). Recombinant adenovirus expressing tTA was a generous gift of Dr. Steve Hardy at Somatix.

Preparation of H+-K+-ATPase-enriched microsomal vesicles. H+-K+-ATPase-containing gastric microsomal vesicles were isolated from unstimulated rabbit stomach as previously described (35). Briefly, the gastric mucosa was homogenized and crude microsomes were isolated as the postmitochondrial membrane pellet that sedimented at 100,000 g for 1 h. The pellet was resuspended in 40% sucrose (9 ml) and overlaid with successive layers of 30% sucrose (11 ml), 10% sucrose (16 ml) containing 5 mM Tris, and 0.2 mM EDTA, pH 7.4, in a 37-ml tube. After centrifugation at 80,000 g for 4 h, the purified gastric microsomal vesicles were collected from the interface between 10% and 30% sucrose.

Trypsinization of vesicles and reverse-phase HPLC separation of tryptic digest. Tubulovesicles (~100 mg of protein) were treated with trypsin (5 mg) in 20 mM Tris · HCl (pH 7.5) at 37°C for 1 h. The material was diluted fourfold and centrifuged at 100,000 g for 1 h. The supernatant was carefully separated from the pellet, boiled for 5 min, and stored at -20°C. The tryptic supernatant was separated on an Aquapore OD-300 C18 reverse-phase column (Applied Biosystems) on a Michrom UMA model 600 HPLC system and monitored at 214 nm. The first 5 min of the gradient were isocratic at 95% eluent A [2% acetonitrile (Acn)-0.1% trifluoroacetic acid (TFA)] and 5% eluent B (98% Acn-0.1% TFA). This was followed by a linear gradient of 5-15% eluent B in 15 min, 15-50% eluent B at 75 min, and 50-75% eluent B at 90 min. The flow rate was 50 ml/min. Individual fractions were collected and subjected to mass analysis.

Gel electrophoresis and stoichiometric measurements. Purified gastric microsomes were subjected to SDS-PAGE on either 10% or 4-20% gradient gels. Various amounts of microsomes were run, ranging from 8 to 80 µg of total protein. The gel was stained with Coomassie blue R-250, destained, and dried. The alpha -subunit of the H+-K+-ATPase, which runs as a prominent band at ~95 kDa, was quantitated with NIH Image 1.61 at a very low level of total protein, because the signal saturates at high protein content. The much weaker signal at ~24 kDa, presumably rab11, was quantitated at much higher levels of total protein, because it does not saturate in the range tested. Measurements were corrected for the difference in total protein and for the molecular size difference of the two proteins: the alpha -subunit of the H+-K+-ATPase is roughly four times larger than rab11.

For two-dimensional electrophoresis, Immobiline DryStrips (18 cm, pH 3-10, nonlinear) were used for the first-dimension isoelectric focusing on a Multiphor II (Amersham Pharmacia Biotech). Purified microsomes (150 µg) were dissolved in the rehydration buffer {7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), pharmalyte, pH 3-10, and a trace of bromphenol blue} and incubated with the Immobiline DryStrips overnight in a rehydration tray. Proteins were focused at 20°C, using an EPS 3500 XL electrophoresis power supply, after which the strips were stored at -80°C. The strips were subjected to a reduction-alkylation step (3 mM DTT, followed by 0.1 mM iodoacetamide) and then to SDS-PAGE on a 15% Duracryl gel. After electrophoresis the gel was fixed with 10% acetic acid-45% methanol for 30 min and then was stained in FAST stain (ZOION Research, Worcester, MA) for 1 h. The gel was rinsed in 10% acetic acid for 20 min, and, after scanning, bands of interest were excised and subjected to in-gel digestion.

In-gel digestion. In-gel digestion was a modification of the method of Rosenfeld et al. (36). Protein bands (~5 µg) were excised and minced using a new razor blade, and the pieces were destained with three washes of 50% Acn-25 mM NH4HCO3 (~10 min each). The destained gel pieces were dried in a Speedvac vacuum, followed by rehydration in 50 µl of 25 mM NH4HCO3 (pH 8.0) that included 0.01 µg/ml trypsin. The pieces were overlayed 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% Acn-5% TFA. All supernatants were pooled and concentrated to 5 µl in a Speedvac and brought back up to 25 µl in 50% Acn-5% TFA. The peptide mix was stored at -20°C until further analysis.

MALDI-TOF mass spectrometry of rab11 peptides. Aliquots (1/25th) of unseparated tryptic digests were cocrystallized with alpha -cyano-4-hydroxycinnamic acid and analyzed using a matrix-assisted laser desorption (MALDI) delayed-extraction reflectron time of flight (TOF) instrument (Voyager Elite mass spectrometer; Perseptive Biosystems, Framingham, MA) equipped with a nitrogen laser. Measurements were performed in a positive ionization mode. All MALDI spectra were externally calibrated using a standard peptide mixture. Some post-source-decay (PSD) spectra were acquired on a TofSpec SE MALDI-TOF mass spectrometer (Micromass, Manchester, UK) with a nitrogen laser and operated in the reflectron mode.

Database searches for protein identification. Database interrogations based on experimentally determined peptide masses were carried out using mass spectrometry (MS)-Fit; PSD data interrogation was performed using MS-Tag; both software were developed in the University of California San Francisco MS Facility and are available at http://prospector.ucsf.edu. Both the National Center for Biotechnology Information protein database and Swiss Prot database were searched. Search parameters included the putative protein molecular weight and a peptide mass tolerance of 100-200 parts/million.

Primary culture of gastric parietal cells from rabbit. New Zealand White rabbits were sedated with a subcutaneous cocktail of 100 mg/ml ketamine, 20 mg/ml xylazine, and 10 mg/ml acepromazine maleate. Pentobarbital sodium (Nembutal; Abbott) was administered intravenously to achieve surgical anesthesia, and a midline abdominal incision was made. The aorta was then exposed and clamped, and a cannula was inserted for perfusion with PBS (in mM: 150 NaCl, 3.64 phosphate, 1 CaCl2, 1 mM MgSO4, pH 7.4) at high pressure. After the stomach was cleared of blood, it was removed from the animal, opened, and washed several times with PBS. The animal was killed by a lethal dose of Nembutal. The gastric mucosa was scraped from the smooth muscle layer and finely minced. The minced mucosa was washed three times with PBS and twice with MEM (GIBCO) containing 20 mM HEPES, pH 7.4 (MEM-HEPES). Cultures enriched in parietal cells were obtained either by the method of Chew et al. (10) or by the following method. The minced mucosa was digested in MEM-HEPES containing 0.125 mg/ml collagenase (Sigma) and 0.25 mg/ml BSA at 37°C for ~30 min. The reaction was stopped by threefold dilution of the collagenase solution with MEM-HEPES. Because of their large size, relatively intact gastric glands settled out in 10-15 min, leaving individual cells suspended in the medium. The suspended cells were strained and washed three times with MEM-HEPES. Cells were next incubated for 30 min in medium B [DMEM/F-12 (GIBCO) supplemented with 20 mM HEPES, 0.2% BSA, 10 mM glucose, 8 nM epidermal growth factor, 1× SITE (selenite, insulin, and transferrin) medium (Sigma), 1 mM glutamine, 100 U/ml penicillin-streptomycin, 400 µg/ml gentamicin sulfate, 25 µg/ml amphotericin B, and 15 µg/l geneticin or 20 µg/ml novobiocin, pH 7.4] to prevent yeast infection. Plating onto Matrigel (Collaborative Biomedical)-coated coverslips followed, and cells were thereafter incubated at 37°C in culture medium A (i.e., medium B less amphotericin B).

Adenoviral infection of gastric parietal cells. Infections were performed 3-5 h postplating. Cells were either infected with the tTA-expressing virus alone or with this virus in addition to a tTA-dependent virus, either a construct containing HA-tagged rab11a N124I or a construct of actin tagged with green fluorescent protein (GFP-actin, generated by the method in Ref. 21). The tTA protein is a fusion of the Escherichia coli tetracycline repressor protein and the transcription-activating domain from herpes simplex virus protein 16 (VP16). Transcription of tTA-dependent genes is dependent on the binding of tTA to the E. coli tetracycline promoter, which is placed immediately upstream of the gene of interest, thus forming an inducible system. Because infection of the cultures in the presence of tetracycline prevents the interaction of tTA with the tetracycline promoter, it is possible to precisely regulate the expression level of the gene of interest. The tTA gene itself is placed downstream of the human cytomegalovirus (CMV) promoter, which is constitutively active. Infection was executed by addition of ~6 × 106 particles/ml of the tTA activator virus, with or without ~3 × 106 particles/ml of one of the expression viruses, to the medium surrounding the cultured cells. Cultures were incubated at 37°C for 12 h and then washed with PBS and given fresh medium. Tetracycline was added to some cultures at a concentration of 1 µg/ml and replaced at the same concentration after washing. Various concentrations of each of the viruses were tested with our culture system: for each virus, up to 5-fold more and 10-fold less virus was tested. We chose our experimental conditions based on the level of expression of rab11a N124I as determined by Western blot (see Fig. 3) and the general appearance of the cells. In addition, we examined infected cultures by immunostaining to ascertain the efficiency of expression. Cell counting indicated that 75% of positively identified parietal cells (159 of 210 cells) stained strongly with the anti-HA monoclonal antibody 16B12 (see Fig. 4), and another 6% expressed weaker levels of staining that were clearly above background.

[14C]AP uptake assays. Stimulation of parietal cells was quantified using the AP uptake assay. This assay measures the accumulation of AP in acidic spaces caused by the action of the proton-pumping enzyme H+-K+-ATPase. In its neutral state, AP freely equilibrates across biological membranes, but protonation of this weak base in acidic spaces gives it a positive charge and traps it (4). [14C]AP (46 nCi/ml) was added to the medium surrounding each coverslip. Cells were either held in a resting state, through addition of the H2 blocker cimetidine to a final concentration of 100 µM, or stimulated, through addition of histamine and IBMX to final concentrations of 100 and 30 µM, respectively. Cultures were gently shaken for 25 min at 37°C. At the end of the incubation, the coverslips were removed from the medium, quickly dipped in PBS to remove external counts, blotted, and transferred to two times sample buffer (125 mM Tris · HCl, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol, pH 6.8), where they remained for at least 1 h at room temperature. Cells were then scraped from the coverslip. Samples of the supernatant and the scrapings were assayed for [14C]AP by liquid scintillation counting, and the protein content was assayed using the filter paper blot method (28). The protein data allowed us to normalize the [14C]AP uptake based on the relative number of cells per coverslip (4, 10). These data were used to calculate the ratio of AP concentration inside to AP concentration outside the cell space for each sample, giving the AP uptake ratio. To normalize AP uptake values among our various culture preparations, the data are expressed as the AP index, meaning that the AP uptake ratio of the uninfected resting cells in each experiment was set to 1.0, and all other values were adjusted accordingly. Raw AP uptake ratios varied somewhat from preparation to preparation, but averages ranged from 20 to 30 for resting, cimetidine-treated cells, and 100 to 300 for stimulated cells. Within each experiment the duplicate or triplicate measurements were in good agreement.

Western blotting of cell lysates. A portion of the cell scrapings was used for Western blots as an assay for the expression of the HA-tagged rab11a N124I. Equal amounts of protein were loaded onto 4-20% acrylamide gradient gels. After running, protein was transferred to polyvinylidene difluoride membranes by a wet-transfer apparatus (Idea Scientific), and the blot was blocked in 5% milk in PBS. Blots were probed with mouse monoclonal anti-HA 16B12 (BAbCo) at a 1:2,000 dilution in milk, followed by secondary probing with horseradish peroxidase-tagged goat anti-mouse IgG (Jackson). Bands were developed using the Renaissance kit (NEN). Expression of GFP-actin was assayed by fluorescence microscopy.

Immunofluorescence and confocal microscopy. After infection, cells were stimulated or held resting as described under [14C]AP uptake assays. Some cells were treated with 5 µM Sch-28080. Cells were fixed by treatment with 3.7% formaldehyde in PBS for 20 min, followed by permeabilization in 0.3% Triton X-100 in PBS for 15 min and blocking in 2% BSA in PBS for 15 min. H+-K+-ATPase was detected by 1-h-long incubation with a 1:3 dilution of culture supernatant of monoclonal antibody 2G11, a mouse monoclonal antibody against its beta -subunit (Affinity Bioreagents), and HA-rab11a N124I was detected with a 1:2,000 dilution of the 16B12 antibody. F-actin was detected by coincident incubation with 80 nM rhodamine-labeled phalloidin (Molecular Probes). 2G11 and 16B12 were detected by FITC-labeled goat anti-mouse IgG (Jackson) in a 30-min subsequent incubation. All antibody dilutions were made in PBS containing 2% BSA. Coverslips were supported on slides by grease pencil markings and mounted in Gel/Mount (Biomeda). Cells were visualized by conventional fluorescence microscopy with a Nikon Microphot FX-2 using Inovision software to collect images. Confocal images of glands were obtained with a Bio-Rad MRC-1024 (Richmond, CA) equipped with a krypton/argon laser using a Zeiss Axioplan microscope and a ×60 plan-Apo numerical aperture 1.4 oil immersion objective.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study grew out of efforts to explore the topology of the gastric H+-K+-ATPase. Our strategy was to isolate H+-K+-ATPase-containing tubulovesicles from gastric mucosa, subject them to trypsinolysis, and assess the protein topology from released peptides identified by MALDI-MS. In so doing, we found that the supernatant from these tryptic digests contained a number of peaks whose masses did not match those expected for H+-K+-ATPase. For some of the more prominent peaks we employed PSD analysis to obtain sequence data and identify the peptides using the MS-Tag program. For example, the PSD spectrum of the peptide, corresponding to a mass per unit charge (m/z) of 1274.6, uniquely identified its sequence as AQIWDTAGQER, amino acids 62-72 of rab11. As summarized in Table 1, six other peptides in the microsomal tryptic supernatant were identified as tryptic fragments of rab11 on the basis of MALDI-MS and PSD analysis. These peptides account for ~45% of the sequence of rab11 and span the NH2-terminal half of the protein. The PSD data also identified a signal of m/z 1563.6 to be (Ac)GTRDDEYDYLIFK, an acetylated (Ac) form of the NH2-terminal peptide from rab11. This was reconfirmed by PSD analysis of a chymotryptic peptide of m/z 1435.5 that identified it to be the NH2-terminal peptide (Ac)GTRDDEYDYLF. Thus the NH2-terminal glycine of rab11 from rabbit gastric microsomes is acetylated.

                              
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Table 1.   Rab11-derived peptides recovered from tryptic digests of various fractions of microsomal protein

To confirm the identification of rab11 in the total tubulovesicular digest, SDS-PAGE was performed to purify rab11 before mass spectroscopy (Fig. 1, inset). The band at ~24 kDa was excised and subjected to in-gel trypsinolysis. MALDI-MS analysis, as exemplified in Fig. 1, revealed prominent signals at m/z 689.3, 858.4, 944.4, 1080.5, 1160.4, 1274.5, and 1563.5, all of which were identified with the MS-Fit program as having originated from rab11 (Table 1). In fact, several of these peaks were the same as those identified from the digest of whole microsomes. The peptides identified from the 24-kDa band include peptides nearer the COOH terminus than those identified from the supernatant of total microsomal digest. Because the signals from rab11 clearly dominated the signals from other proteins that may be contained in the 24-kDa band, we endeavored to determine the stoichiometry of rab11 to H+-K+-ATPase by quantifying the Coomassie blue-stained protein in the 24-kDa band (presumed rab11) and the 95-kDa band (H+-K+-ATPase alpha -subunit). A representative SDS-PAGE profile of microsomal material is shown in the inset to Fig. 1. Clearly, the alpha -subunit of the H+-K+-ATPase is the most prominent protein in the density-purified microsomal vesicles. However, accounting for their difference in molecular size and assuming that the stained bands predominantly contained the proteins in question, we estimated that there was one copy of rab11 for every six copies of the H+-K+-ATPase in rabbit gastric microsomes. Although potential sources of error preclude this as a precise stoichiometry, it does demonstrate the abundance of rab11 in gastric tubulovesicles.


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Fig. 1.   MALDI-MS spectrum of rab11-derived peptides identified in the ~24-kDa protein band from purified gastric microsomes. Microsomal proteins were separated by SDS-PAGE; protein band in 24-kDa region was subjected to in-gel trypsinolysis, and resultant peptides were analyzed by MALDI-MS. Mass values (m/z) and putative sequence assignments for rab11 tryptic fragments are indicated above peaks; assignments were made using MS-Fit function of the Protein Prospector package. A typical fragment of trypsin at m/z 842.4 is also indicated. Several unassigned peaks at m/z 614, 659.3, 802.3, and 825.0 did not match masses expected from tryptic digests of rab11; they could belong to other proteins, be modified rab11 peptides, or come from matrix ions used for analysis. PSD analysis identified the signal at 1316.5 to be LQIWDTAGQER, which is close to the rab11 peptide 72AQIWDTAGQER82 but may more likely belong to some other rab protein, such as rab1b, 3, 4a, 4b, 8, 10, 12, 14, 15, or 16. Inset: representative lane from SDS-PAGE separation of gastric microsomes. Positions of alpha -subunit of H+-K+-ATPase (HKalpha ) and rab11 are indicated.

We next performed two-dimensional gel electrophoresis to provide a detailed portrait of constituents in the 24-kDa band. Coomassie blue staining revealed three major spots in the 24-kDa region of the gel, as well as two less prominent spots (Fig. 2). Each of these spots was excised and subjected to in-gel trypsinolysis and MALDI-MS, and the protein was identified by MS-Fit. For the three most prominent spots, we found strong signals that could be matched to predicted tryptic fragments of rab11 (Table 1), indicating that the gastric microsomal rab11 occurred in several isoforms. Two of the three spots, 4 and 5, contained a peak at m/z near 3203.6, which is a predicted tryptic fragment unique to rab11a but not to rab11b. On the other hand, spot 6 contained a peak at m/z of 1021.3, closely corresponding to peptide 167Asn-Arg174, which is unique to the rab11b isoform. The minor spot at 4b in Fig. 2 gave peaks that were distinctly not rab11. This was also the case for spot 6b, although PSD analysis of one peak at m/z 1316.5 suggests that it may belong to another rab protein (see legend to Fig. 1).


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Fig. 2.   Microsomal proteins in region of 24 kDa separated by 2-dimensional gel electrophoresis. Approximately 150 µg purified microsomes were solubilized in 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol, and pH 3-10 pharmalyte and were subjected to isoelectric focusing in a pH 3-10 nonlinear gradient on Immobiline DryStrips, as described in MATERIALS AND METHODS. For the second dimension, strips were subjected to SDS-PAGE on a 15% gel. After electrophoresis the gel was fixed and stained, and visualized spots were assigned a tracking number before being excised and subjected to in-gel digestion and analysis of peptides by MALDI-MS. Only spots numbered 4, 4b, 5, 6, and 6b, in the vicinity of 24 kDa and pH 5-7, are shown.

Functional studies of rab11a were conducted using the mutant rab11a N124I. This mutant is predicted to be incapable of nucleotide binding and therefore to serve as a dominant negative variant. Adenoviral infections of primary cultures of parietal cells were performed to introduce this mutant. The rab11a N124I-encoding construct places the mutant gene under the control of the tetracycline-transactivator protein (tTA), so coinfection of the cells with an adenovirus encoding this protein was required. Because the mutant rab11a was tagged with HA, we were able to assess infection of these cultures by Western blot analysis of the cell lysates and by probing with anti-HA antibodies (Fig. 3). Uninfected cells and cells infected with the tTA virus alone showed no immunoreactivity with 16B12, a mouse monoclonal directed against the HA tag of the mutant rab11a. Cells infected with both tTA and the rab11a N124I virus, on the other hand, exhibited a strong 16B12 signal at ~25 kDa, the expected size of the mutant protein. Expression of the mutant was either abrogated or attenuated if the cultures were grown in the presence of tetracycline, which binds the tTA protein and makes it unable to interact with the tetracycline-sensitive promoter. We also used 16B12 as an immunocytological probe. As expected, no staining was observed in uninfected cultures, but infected cultures revealed that a large proportion of the parietal cells (~75%) stained positive with anti-HA antibodies (Fig. 4). Rab11a N124I was thus shown to have a diffuse cytoplasmic localization in infected parietal cells. All of these experiments were performed using the infection conditions given in MATERIALS AND METHODS. These results demonstrate that rab11a N124I is expressed in infected parietal cells and that it has a distribution similar to that of H+-K+-ATPase.


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Fig. 3.   Gastric parietal cells express hemagglutinin (HA)-tagged rab11a N124I when coinfected with the rab11a N124I-expressing virus and the tetracycline transactivator (tTA)-expressing virus. Parietal cells were cultured for 12 h without (-) or with (+) rab11a N124I adenoviral infection, tTA adenoviral infection, or 1 µg/ml tetracycline. After measurement of the response to acid secretagogues by aminopyrine uptake, cultures were lysed and separated by SDS-PAGE on 4-20% gradient gels, and Western blots were probed with anti-HA monoclonal antibody 16B12. A-C: preparations originating from different animals.



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Fig. 4.   Expression of HA-tagged rab11a N124I in cultured parietal cells detected by immunofluorescence. Cells coinfected with tTA- and rab11a N124I-expressing viruses were fixed, permeabilized, and probed with rhodamine-phalloidin (F-Act) and anti-HA 16B12 monoclonal antibody (HA), which was detected with FITC-conjugated goat anti-mouse IgG. Most parietal cells from infected cultures clearly expressed HA-tagged rab11 N124I throughout the cell, although expression levels were low in a few cells (*). Uninfected cells showed no staining above autofluorescence (data not shown). Bar = 20 µm.

The [14C]AP uptake assay was used to compare the stimulation of parietal cells in their uninfected state to those infected with tTA and rab11a N124I. This assay measures the accumulation of a weak base into acid spaces created by the activity of the H+-K+-ATPase on stimulation of the parietal cell cultures. Our results are reported in Fig. 5 as the normalized AP index, which is the times stimulation over the resting uninfected cells in each experiment. In our initial experiments, stimulation of uninfected cells led to a large accumulation of AP, averaging 5.5 ± 1.6 times that of the resting state. For cells expressing rab11a N124I, the AP index of stimulated cells was significantly reduced to 2.2 ± 0.8 (P < 0.05). These results indicate that expression of the mutant rab11 decreased the secretory response of the cells by ~60%.


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Fig. 5.   Acid secretion is inhibited in parietal cells expressing rab11a N124I. Parietal cells were cultured, and some infected, as described in MATERIALS AND METHODS. [14C]aminopyrine (AP) uptake was used to assay stimulation of cultures in response to secretagogues. Data are expressed as AP index, which is the times stimulation of each sample relative to uninfected resting cells for the same experimental preparation. Stimulation (Stim) was effected with 100 µM histamine plus 30 µM IBMX. Alternate samples were maintained in the resting state (Rest) through treatment with 100 µM cimetidine. Responses by uninfected (Ctrl) and rab11a N124I-expressing (N124I) cultures are given as means ± SE for 6 separate experiments (P < 0.05).

To determine whether this decreased secretory response was a specific effect of the mutant or an artifact of viral infection, another series of experiments was performed. In addition to uninfected cells and rab11a N124I-infected cells, cultures were infected with the tTA virus alone or with tTA and another virus encoding GFP-tagged actin under the control of the CMV promoter. In these experiments, shown in Fig. 6, stimulating uninfected control cells gave an AP index of 10.0 ± 2.9. For cells expressing tTA alone or tTA plus GFP-actin, the values for the AP index after stimulation were 9.0 ± 3.1 and 9.6 ± 0.9, respectively; none of these values is significantly different from that for the uninfected cells. Again, cells expressing rab11a N124I showed markedly decreased degrees of stimulation, with a mean AP index of only 3.2 ± 0.9, ~68% below that of uninfected cells (P < 0.05). These results show that parietal cell stimulation is specifically inhibited by the rab11a N124I mutant and is not due to virus infection. The apparent discrepancy between stimulated AP values between the initial experiments (Fig. 5) and these control experiments (Fig. 6) is probably due to a greater efficiency that we gained as we perfected the coverslip AP uptake assay.


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Fig. 6.   Inhibition of acid secretion by rab11a N124I is not a nonspecific effect of adenoviral infection. Parietal cells were cultured and some infected with various combinations of virus, as described in MATERIALS AND METHODS. [14C]AP was used to assay stimulation (Stim) of cultures by 100 µM histamine and 30 µM IBMX. Controls were maintained in the resting state (Rest) by treatment with 100 µM cimetidine. Data are expressed as AP index, which is the times stimulation of each sample relative to uninfected resting cells for the same experimental preparation. Responses of uninfected cells (Ctrl), cells expressing only tTA, cells expressing green fluorescent protein-actin (Act), and cells expressing rab11a N124I (N124I) are given as means ± SE for 6 separate experiments. Rab11a N124I-expressing cells demonstrated marked inhibition of stimulatory activity (P < 0.05).

Confocal fluorescence microscopy was used as an independent test of the effect of the rab11a N124I mutant on parietal cell stimulation. Cultured cells, including control preparations and those infected with rab11a N124I, were stimulated or kept in the resting state for 25 min and then fixed, permeabilized, and stained for F-actin and H+-K+-ATPase (Fig. 7). The uninfected cultures showed predictable staining patterns. In resting cells, F-actin was prominent around the apically derived secretory vacuoles within the cells as well as being evident on the surrounding plasma membrane; H+-K+-ATPase was distributed throughout the cells in a punctate manner (Fig. 7A). On stimulation, the vacuoles of uninfected cells swelled with the activated accumulation of HCl and water, and the distribution of H+-K+-ATPase changed to coincide with F-actin on these swollen apical vacuolar surfaces (Fig. 7B). These are the typical morphological responses of stimulated parietal cells, consistent with the regulated recruitment of H+-K+-ATPase from the cytoplasmic tubulovesicles into the apical plasma membrane. For the cultures expressing rab11a N124I, resting cells were quite comparable to uninfected cells (Fig. 7C). However, on stimulation, swelling of vacuoles was much less evident and H+-K+-ATPase remained largely distributed throughout the cytoplasm in a punctate manner, i.e., the cells expressing rab11a N124I remained in the resting morphology (Fig. 7D). Cells infected in the presence of tetracycline behaved much like uninfected cells (Fig. 7E).


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Fig. 7.   Rab11a N124I prevents recruitment of H+-K+-ATPase from cytoplasm to apical membrane-derived vacuoles in cultured parietal cells. Cells were cultured and left uninfected (A, B) or coinfected with tTA- and rab11a N124I-expressing adenoviruses alone (C, D) or in presence of 1 µg/ml tetracycline (E). Cultures were held in a resting state through addition of 100 µM cimetidine (A, C) or stimulated with 100 µM histamine and 30 µM IBMX (B, D, E). After fixation and permeabilization, cells were doubly stained for F-actin (F-act) and H+-K+-ATPase (HK). Cells were visualized by confocal microscopy. Bar = 20 µm.

To more closely examine the morphological response to stimulation, we repeated the experiment in the presence of Sch-28080, which is a specific H+-K+-ATPase-inhibitor (44). When uninfected cells were stimulated with histamine and IBMX in the presence of Sch-28080, there was minimal vacuolar swelling as the pump was inhibited, but the H+-K+-ATPase was clearly depleted from the cytoplasm and accumulated at the apical vacuoles, consistent with the recruitment step of the secretory process being independent of proton transport per se (cf. Fig. 8, a and b). When rab11a N124I-expressing cells were stimulated, H+-K+-ATPase remained randomly distributed throughout the cells and net translocation of the pump enzyme was very much reduced (Fig. 8, c and d). On the other hand, when tetracycline was included with the rab11a N124I-infected cells, stimulation-associated translocation of the H+-K+-ATPase to the vacuolar membranes appeared normal (Fig. 8e). Thus expression of the N124I mutant of rab11a appears to inhibit acid secretion by reducing the stimulation-associated recruitment of H+-K+-ATPase.


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Fig. 8.   Effects of rab11a N124I on tubulovesicle recruitment are evident in presence of H+-K+-ATPase inhibitor Sch-28080. Parietal cells were cultured and left uninfected (A, B) or coinfected with tTA- and rab11a N124I-expressing adenoviruses in absence (C, D) or in presence of 1 µg/ml tetracycline (E). Cultures were held in a resting state (A, C) through addition of 100 µM cimetidine or stimulated with 100 µM histamine and 30 µM IBMX in presence of 5 µM Sch-28080 (B, D, E). After fixation and permeabilization, cells were doubly stained for F-actin (F-act) and H+-K+-ATPase (HK) and visualized by fluorescence microscopy. Bar = 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Goldenring et al. (18) have shown that rab11a is highly expressed in rabbit parietal cells and that it is associated with the cellular compartments that are known to contain the H+-K+-ATPase. Nevertheless, we were surprised by the mass spectroscopy data obtained from tryptic digests of isolated gastric microsomes. Rab11-derived peaks were obvious even against the considerable background of peptides from the H+-K+-ATPase, which dominates the protein profile of these microsomes. Purification by gel electrophoresis before trypsinolysis established that peptides from rab11a were the predominant signals that came from the molecular mass region where rab proteins are expected. Furthermore, the putative rab11 band was easily visible, permitting us to calculate a stoichiometry of about one copy of rab11 to six copies of H+-K+-ATPase, the major constituent of gastric microsomes. Although these data emphasize the relative abundance of rab11a, the specific number cannot be regarded as more than a careful estimation. This is partially due to the fact that the 24-kDa band, as one might suspect, does not comprise a totally homogeneous protein population, even though the two-dimensional gel data suggest that rab11 proteins are the most prominent. Although rab11-derived peptides are conspicuous in the mass spectrum of tryptic digests of the 24-kDa band (Fig. 1), fragments not corresponding to rab11 also appear. It is difficult to comment on the significance of this estimation to other rab proteins, because measurements like this one have not routinely been performed in other systems.

Three distinct spots seen at ~24 kDa on two-dimensional gels were identified as rab11, suggesting different isoforms on the gastric microsomes. The two most prominent spots were best predicted as rab11a. A closely related rab protein, rab11b, has been cloned (23). This species has high similarity to rab11a, differing chiefly in stretches of its hypervariable COOH-terminal region. Our data indicate that one of the spots is likely to be rab11b, because tryptic digests of this spot yielded a peptide that is unique to this isoform. Moreover, the putative rab11b spot represents the most acidic rab11 spot on the two-dimensional gel, and rab11b has a predicted lower isoelectric point (pI = 5.86) than that for rab11a (6.12). Even if rab11b is present on the gastric microsomes, the rab11a isoform clearly identified here is certainly present in far greater abundance.

The separation of rab11a species on the two-dimensional gel may also reflect posttranslational modifications. For instance, there are also cases in which rab proteins are known to be phosphorylated, most notably in the case of rab4, which is a target for p34/cdc2 at the onset of mitosis (2) and for ERK1 in response to certain extracellular stimuli (11). If the present data do include phosphoisoforms of rab11, then the phosphorylation of rab11 is significantly different from that of rab4, since phosphorylation of the latter results in its inability to associate with membranes (1, 2). We also considered prenyl isoforms. This is probably not the case, as we would not have expected such a large change in pI. Moreover, it has been shown that rab escort proteins (REPs) bind unprenylated rabs, present them to geranylgeranyl transferase (GGTase), and do not allow their membrane association until they are doubly geranylgeranylated (37).

Expression of rab11a N124I in parietal cells leads to reduced AP accumulation, our index of acid secretion in response to cell activation. We were careful to distinguish this phenomenon from the nonspecific effects of adenoviral infection, because we have observed that, even though the rab11a N124I and tTA viruses lack the E1 and E3 genes, which are required for viral replication, the cells can still be killed by a high titer of the virus. Our method of infection did lead to altered morphology of the parietal cells (cells typically assumed a more rounded shape relative to uninfected cells). However, when infected with viruses with comparable promoter numbers leading to the expression of GFP-actin, inhibition of acid secretion did not occur, even though the GFP-actin-encoding vector retained its E3 genes. Cell rounding in this case was comparable to that seen in rab11a N124I-expressing cells. In addition, the introduction of higher titers of the tTA virus alone into the parietal cells did not significantly affect acid secretion, even when we theoretically doubled the number of constitutively active viral promoters in each cell (data not shown).

The immunocytochemical data from fluorescence and confocal microscopy demonstrated that the expression of mutant rab11a blocks the morphological transformation of parietal cells. Cells infected with rab11a N124I failed to show the stimulation-associated redistribution of H+-K+-ATPase to the apical vacuoles and the accompanying vacuolar swelling. The action of tetracycline to restore the morphological response of infected cells confirmed that inhibition was not a nonspecific effect of adenoviral infection. These data indicate that the introduced defect of rab11a N124I expression is specific for the vesicular trafficking events, since only minimal recruitment of H+-K+-ATPase to the apical vacuoles occurred in infected cells.

Our functional data strongly implicate rab11a in the stimulation of the gastric parietal cell. Mutation of an asparagine to an isoleucine in the nucleotide binding pocket is predicted to create a nonnucleotide binding variant. This presumably leads to occupation of the GEF responsible for rab11a activation, which in turn prevents activation of endogenous rab11a. The effect that the rab11a mutant has on the function of rab11b in parietal cells is related to the extent to which the cellular rab11-activating machinery is able to distinguish between these two isoforms. At the present time, we know very little about this machinery. Alternative dominant negative mutants, those with a serine or threonine replaced by asparagine in the GAP effector domain, are perhaps more easily rationalized in this mechanism, because this mutation locks the protein in a GDP-bound state (40). However, due to the general lack of knowledge regarding rab GEFs, one cannot assert that the latter mutant is actually preferable for a study such as this. In addition, there is strong precedent for a dominant negative effect by analogous mutants in other rabs. Rab5 N133I exerts such an effect on early endosomal fusion in mammalian systems (5, 19), and expression of rab7 N125I in BHK cells leads to perturbed cation-independent mannose 6-phosphate receptor localization and impaired processing of cathepsin D (14, 33). That rab11a should participate in a regulated exocytotic event is also not surprising. Rab4 has been implicated in the insulin-dependent delivery of the GLUT-4 transporter from an intracellular microsomal compartment to the plasma membrane in rat adipocytes (12) and skeletal muscle (38). Rab3, however, provides the best-documented instance of this phenomenon through its role in the delivery of synaptic vesicles to the axonal plasma membrane in neurons (reviewed in Ref. 3).

The direct implication of rab11a in the process of stomach acidification fits well with an emerging portrait of the factors and activities required for the stimulation of the gastric parietal cell. Our laboratory has previously described the expression of several target SNAREs in rat parietal cells (30). Further, we found that syntaxin 3 and the vesicle SNARE-associated membrane protein are associated with H+-K+-ATPase-rich tubulovesicles (30). Syntaxin 1 and soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), generally thought to be specifically expressed in neural cells, were also identified in parietal cells (30). Other trafficking molecules, such as rab25 and secretory carrier membrane proteins (SCAMPs) have also been localized to the tubulovesicles of these cells (6). These findings demonstrate that the machinery necessary to conduct a massive trafficking event is present in parietal cells. Elucidation of their mechanisms of action in this system remains an exciting challenge.

The present study confirms the hypothesis that rab11a plays an important role in the regulated traffic of the H+-K+-ATPase from the cytoplasmic tubulovesicular network to the apical plasma membrane. In so doing, we lend further support to the membrane recycling hypothesis of acid secretion by the parietal cell (16). Models that attribute the morphological transformation of the cell to osmotically driven expansion of a contiguous apical membrane (31) fail to explain why expression of a dominant negative variant of a known regulator of membrane traffic should inhibit it. Elucidation of the detailed mechanics for this process will entail the identification of downstream effectors of rab11a in these cells, as well as definitively assigning functional significance to the phosphorylation state of the bound nucleotide. Evidence has been presented elsewhere, which suggests that this process differs in rab11a-mediated tubulovesicle fusion in parietal cells from rab3-mediated neural fusion events (7). Our confirmation of a stimulatory role for rab11a validates this observation, further demonstrating the value of the parietal cell model for the study of regulated membrane traffic.


    ACKNOWLEDGEMENTS

We thank Dr. Daniel Kalman and Aneil Mallavarapu [Dept. of Microbiology, Univ. of California, San Francisco (UCSF)] for their gift of the GFP-actin-encoding adenovirus and Dr. David Ammar (Univ. of California, Berkeley) for his assistance and helpful discussion.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants DK-10141 and DK-38972 (to J. G. Forte) and GM-35239 (to H.-P. H. Moore). Mass spectra were obtained at the UCSF Mass Spectroscopy Facility, which is supported by the Biomedical Research Technology Program of the National Center of Research Resources, National Institutes of Health Grants RR-01614 and RR-08282.

Present address of K. Tyagarajan: Lynx Therapeutics, 25861 Industrial Blvd., Hayward, CA 94545.

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 and other correspondence: J. G. Forte, Dept. of Molecular and Cell Biology, Univ. of California, Berkeley, CA 94720-3200 (E-mail: forte{at}socrates.berkeley.edu).

Received 18 March 1999; accepted in final form 18 May 1999.


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