Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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[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 ![]() |
RESULTS |
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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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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
-subunit). A representative SDS-PAGE profile of microsomal material
is shown in the inset to Fig. 1. Clearly, the
-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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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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