Institute for Molecular Medicine and Genetics, Departments of Medicine, Surgery, and Cellular Biology and Anatomy, Medical College of Georgia, and Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912
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ABSTRACT |
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Previous investigations in several systems have demonstrated that Rab3 family members redistribute to soluble fractions on fusion of secretory granules with target plasma membranes. Rab proteins are then recycled back onto mature secretory vesicles after reinternalization of the membrane. Although this cycle is well established for Rab3, far less is known about redistribution of other Rab proteins during vesicle fusion and recycling. In the gastric parietal cell, Rab11a is associated with H-K-ATPase-containing tubulovesicles, which fuse with the apical plasma membrane (secretory canaliculus) in response to agonists such as histamine. We have analyzed distribution of Rab11a and other tubulovesicle proteins in resting and histamine-stimulated rabbit parietal cells. Stimulation of isolated gastric glands in the presence of 100 µM histamine and 100 µM 3-isobutyl-1-methylxanthine did not cause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a, Rab25, syntaxin 3, and SCAMPs increased immunoreactivity in stimulus-associated vesicles prepared from rabbits treated with histamine compared with those from ranitidine-treated animals. The large GTPase dynamin was found in both vesicle preparations, but there was no change in amount of immunoreactivity. Immunofluorescence staining of resting and histamine-stimulated primary cultures of parietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate that Rab11a does not cycle off the membrane during the process of tubulovesicle fusion with the secretory canaliculus. Thus Rab11a may remain associated with recycling apical membrane vesicle populations.
tubulovesicle proteins; rabbit; dynamin; syntaxin; SNARE proteins
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INTRODUCTION |
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THE PROCESS OF VESICLE trafficking within cells is critical to the orderly functioning of intracellular systems. This trafficking requires a series of regulated vesicle fusion and membrane budding and retrieval events at all points along the exocytotic and endocytotic pathways. Recent investigations have stressed the importance of highly conserved mechanisms for membrane vesicle docking at points of vesicle processing. These docking complexes are formed from the association of SNARE proteins on vesicles (v-SNARE) and their target fusion membrane surfaces (t-SNARE) (38). In addition, other proteins are required for the orderly assembly and recognition of the docking complex (25). In particular, members of the Rab small GTP-binding protein family appear to be required for assembly of SNARE complexes (40).
The Rab family of small GTP-binding proteins regulates various stages of vesicle trafficking along the endocytotic and exocytotic pathways (3, 34). Present evidence suggests that Rab proteins are necessary for the orderly fusion of specific vesicle populations with target acceptor membranes. This process has been studied most extensively for the Rab3 subfamily members that are present on small synaptic vesicles and packaged secretory granules from endocrine and exocrine cells. Studies in synaptosomes (14, 27), adrenal chromaffin cells (12), and pancreatic acinar cells (26) have demonstrated that Rab3 isoforms cycle off granule and vesicle membranes during the process of fusion. This process appears to be mediated by the guanine nucleotide dissociation inhibitor (GDI) protein (37, 49). After reinternalization of secretory membranes, Rab3 isoforms appear to recycle back to the mature secretory vesicle membranes (14, 26). This pathway of Rab3 recycling has led to the suggestion that the association of Rab3 isoforms with membranes indicates maturation to secretory-competent vesicles.
In contrast to packaged secretory processes, epithelial cells maintain and regulate the repertoire of apical and basolateral membrane ion channels and transporters through the control of their insertion into plasma membrane surfaces. This mechanism is well established for three critical cAMP-dependent processes: 1) insertion of water channels into the apical membranes of cortical collecting duct cells (30, 32, 39), 2) insertion of cystic fibrosis transmembrane conductance regulator into the apical membranes of colonic and other chloride-secreting epithelia (31, 44), and 3) delivery of the H-K-ATPase to the apical surface of gastric parietal cells (15). In the case of proton-pumping gastric parietal cells, H-K-ATPase molecules are sequestered within a class of intracellular tubulovesicles that lie deep to an extensive invagination of the apical membrane referred to as the intracellular canaliculus (6, 16, 41). On stimulation of an increase in intracellular cAMP by histamine, the tubulovesicle membranes fuse with the canaliculus to deliver the H-K-ATPase to the luminal surface (16, 41, 47). This massive fusion event elicits a fivefold increase in apical plasma membrane surface area, the largest reversible membrane fusion event observed in mammalian cells. Nevertheless, on cessation of the stimulus and return of intracellular cAMP to resting levels, the H-K-ATPase is rapidly reincorporated into tubulovesicles that are competent for another round of fusion (6, 16). This dynamic membrane fusion and retrieval process makes the parietal cell an important model for the study of apical membrane recycling.
Little is known concerning the proteins that regulate apical plasma membrane recycling. Recently, a number of investigations have indicated that Rab11a is involved in the process of recycling to the plasma membrane in nonpolarized and polarized cells. Ullrich et al. (46) showed that Rab11a was associated with the pericentrosomal recycling system responsible for recycling of transferrin to the membrane surface in Chinese hamster ovary cells and fibroblasts. Similar results were reported by Green et al. (23) in K-562 cells. We recently noted that Rab11a and Rab25, which shares 68% sequence identity with Rab11a, are associated with the recycling system in polarized Madin-Darbin canine kidney (MDCK) cells (19). We previously noted the enrichment of Rab11a in gastric parietal cells (21), and more recently we demonstrated that Rab11a and Rab25 are present on immunoisolated H-K-ATPase-containing membranes (7). These results have supported the hypothesis that the tubulovesicles represent an elaboration of the apical epithelial recycling system present in a number of epithelial cells.
We have not been able to document the presence of any Rab3 family member in gastric parietal cells. Thus the parietal cell represents an important system to compare the recycling of Rab11a with that observed in other systems for Rab3 family members. In our original investigations we observed that, in concert with stimulation, Rab11a redistributed into heavier membrane fractions in parallel with the H-K-ATPase (21). In these studies no redistribution of Rab11a into a cytosolic fraction was observed. We have now studied in detail the redistribution of Rab11a along with other tubulovesicle proteins in parietal cells during stimulation. These studies, with use of biochemical and immunocytochemical criteria, suggest that Rab11a does not cycle off the membranes into the cytosol during secretory stimulation.
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MATERIALS AND METHODS |
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Materials.
Male New Zealand White rabbits were obtained from Shelton's Bunny
Barn. The monoclonal antibody (MAb) specific for the -subunit of the
gastric H-K-ATPase (12.18) was a generous gift from Dr. Adam Smolka
(Medical University of South Carolina, Charleston, SC).
Affinity-purified anti-syntaxin 3 and anti-syntaxin 4 antibodies were a
gift of Mark Knepper (National Institutes of Health) (29). The
production of Rab11a- and Rab25-specific MAb has been described elsewhere (20). Mouse monoclonal anti-dynamin (clone 41) was obtained
from Transduction Laboratories (Lexington, KY). Anti-mouse IgG-coated
magnetic Dynabeads were obtained from Dynal (Great Neck, NY). Fc
fragment-specific secondary antibodies conjugated with horseradish
peroxidase and Cy5-conjugated secondary antibodies were from Jackson
ImmunoResearch Labs (West Grove, PA). Enhanced chemiluminescence (ECL)
substrate (SuperSignal) was obtained from Pierce (Rockford, IL).
Nonimmune control IgG2b was purchased from Sigma Chemical (St. Louis,
MO). Immobilon-P polyvinylidine difluoride membranes were purchased
from Millipore (Bedford, MA). Bodipy-phallacidin was obtained from
Molecular Probes (Portland, OR). All other reagents were from standard
suppliers and were of the highest purity available.
Tubulovesicle preparation. Gastric tubulovesicles were prepared from resting rabbit gastric mucosa, as described by Crothers et al. (10). Briefly, male New Zealand White rabbits were anesthetized by intravenous administration of a mixture of ketamine and xylazine, and their stomachs were perfused under high pressure with oxygenated PBS and removed. The gastric mucosa was scraped off the serosa with a glass slide, minced with scissors, and homogenized in five volumes of homogenization buffer [in mM: 113 mannitol, 37 sucrose, 0.4 EDTA, 5 MES, pH 6.7, 5 benzamidine, and 0.1 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)] plus a protease inhibitor cocktail (in µg/ml: 1.75 aprotinin, 2.5 soybean trypsin inhibitor, 1 chymostatin, pepstatin A, and leupeptin). The homogenate was centrifuged sequentially at 50, 4,000, 14,000, and 100,000 g. The 100,000-g pellet was resuspended in 10% sucrose buffer (5 mM HEPES-NaOH at pH 7.4, 300 mM sucrose) and fractionated over discontinuous sucrose gradients consisting of layers of 20, 27, and 33% sucrose. The vesicles partitioning at the 10-20% sucrose interface were used for immunoadsorption experiments.
Immunoadsorption of tubulovesicles.
For a single immunoadsorption experiment, 750 µg of Dynabeads were
washed three times in PBS containing 1% BSA, blocked for 30 min at
4°C in PBS-1% BSA, and washed twice in PBS-0.1% BSA. The blocked
beads were incubated overnight at 4°C with MAb specific for the
-subunit of the gastric H-K-ATPase (12.18) or nonimmune IgG2b. The
beads were washed four times for 30 min at room temperature with
PBS-0.1% BSA and incubated with tubulovesicles (20 µg of protein)
for 2 h at room temperature in PBS-0.1% BSA plus a protease inhibitor
cocktail (5 mM benzamidine, 0.1 mM AEBSF, 1.75 µg/ml aprotinin, 2.5 µg/ml soybean trypsin inhibitor, and 1 µg/ml chymostatin, pepstatin
A, and leupeptin). After 2 h the unbound material was removed,
centrifuged at 20 psi for 5 min at 4°C in an Airfuge (Beckman
Instruments, Stanford, CA), and resuspended in SDS sample buffer. The
bound material was eluted from the beads in SDS sample buffer, and all
samples were heated at 65°C for 5 min. The proteins were separated
by SDS-PAGE, electrophoretically transferred to Immobilon-P
polyvinylidine difluoride membranes, and analyzed by immunoblotting.
The proteins of interest were then detected by incubating the blots
sequentially with an Fc fragment-specific secondary antibody conjugated
to horseradish peroxidase (Jackson ImmunoResearch Labs) and an ECL
substrate (SuperSignal). For quantitation, autoradiographs were
digitized using an Alpha Innotech image analyzer and Alpha Ease
software (San Diego, CA), and integrated densities from three identical
experiments were determined. The exposure of the images analyzed by
densitometry was monitored using the Saturation Palette of the Alpha
Ease software (Alpha Innotech, San Leandro,CA). All images quantitated
by densitometry were within the optimal exposure range. Results (means ± SE) are expressed as percent recovery. The statistical
significance of the densitometry data was analyzed using the
nonparametric t-test.
Rab11a and Rab25 redistribution in gastric glands. Isolated gastric glands were prepared from the fundic mucosa of New Zealand White rabbits as previously described (4, 18). Gastric glands (200 µl of packed glands in 8 ml of media) were incubated in the presence of 100 µM ranitidine or 100 µM histamine with 100 µM 3-isobutyl-1-methylxanthine (IBMX) for 10 min at 37°C in a shaking water bath. At the end of the incubation, glands were rapidly separated from media by centrifugation, and the gland pellet was homogenized on ice in 800 µl of homogenization buffer with 30 strokes of a Dounce homogenizer. The membrane and supernate fractions were then separated rapidly through successive centrifugation at 100 g for 1 min, then the supernate was centrifuged for 5 min in an Airfuge at 150,000 g. The high-speed pellet was resuspended in 500 µl of homogenization buffer. For detection of Rab11a, 50 µg of high-speed supernate protein and 25 µg of membrane protein were resolved on SDS-PAGE gels and transferred to Immobilon-P. For detection of Rab25, 100 µg of high-speed supernate protein and 50 µg of membrane protein were resolved on SDS-PAGE gels and transferred to Immobilon-P. Blots were probed with the anti-Rab11a and anti-Rab25 MAb, and specific labeling was determined using ECL, as described above. Autoradiographic images were quantitated digitally as described above, and Rab11a or Rab25 immunoreactivity in membrane and cytosolic fractions was determined as a percentage of total immunoreactive material (n = 3).
Preparation of gastric stimulus-associated vesicles. Stimulus-associated (SA) vesicles were prepared from New Zealand White rabbits by the method of Crothers et al. (11). Briefly, rabbits were pretreated with 3 mg/kg sc chlorpheniramine maleate 10 min before injection of ranitidine or histamine (2 mg/kg iv). In the case of histamine, three injections were given at 10-min intervals. After treatment, under ketamine-xylazine anesthesia, the abdominal aorta was isolated and the celiac axis was retrograde perfused under high pressure with PBS. The fundic mucosa was scraped from the serosa and homogenized in a Teflon-on-glass homogenizer in five volumes of buffer A (in mM: 113 mannitol, 37 sucrose, 0.4 EDTA, 5 MES, pH 6.7, 5 benzamidine, 0.1 AEBSF) with four strokes at 300 rpm (Master Servodyne, Cole Parmer). The homogenate was centrifuged consecutively at 50 g for 5 min and at 1,000 g for 10 min. The 1,000-g pellet was resuspended in 18% Ficoll in buffer B (in mM: 300 sucrose, 0.2 EDTA, 5 HEPES, pH 7.4) and overlayed with buffer B. The gradient was then centrifuged at 135,000 g for 2 h in an SW28 rotor. SA vesicles were harvested from the interface of the Ficoll and sucrose buffer layers and, after fourfold dilution with buffer B, were pelleted at 135,000 g in a 50.2 Ti rotor for 60 min. Immunoblotting and quantitation were performed as described above.
Immunofluorescence studies of primary cultures of parietal cells. Parietal cells were isolated from New Zealand White rabbits and maintained on Matrigel-coated coverslips in primary culture, as previously described (8, 41). Parietal cells maintained in culture for 24 h were incubated in the presence of 100 µM ranitidine (resting) or 100 µM histamine-50 µM IBMX for 20 min. After stimulation, cells were rapidly washed in PBS and then fixed in 4% paraformaldehyde for 15 min at 4°C. Cells were permeabilized and blocked with 17% goat serum-0.3% Triton X-100 in PBS for 30 min and then incubated with monoclonal murine anti-H-K-ATPase (MAb 12.18 ascites, 1:2,000), monoclonal murine anti-Rab11a (MAb 8H10 ascites, 1:100), or monoclonal murine anti-dynamin (1:1,000; Transduction Laboratories) for 2 h at room temperature. After they were washed in PBS, all cells were incubated with Bodipy-phallacidin (Molecular Probes) for 60 min at room temperature. Cells were then incubated with Cy5-conjugated donkey anti-mouse IgG or donkey anti-rabbit IgG for 30 min. After a final wash, cells were mounted in Prolong Antifade solution (Molecular Probes) and examined with scanning confocal fluorescence microscopy (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
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Syntaxins on immunoisolated tubulovesicles.
We have utilized immunoisolation of rabbit tubulovesicles to identify
definitively components of H-K-ATPase-containing vesicles (7). Although
syntaxin 1 immunoreactivity was present in gradient-isolated vesicles,
no syntaxin 1 was recovered in the immunoisolated vesicles (7). Thus,
although we did identify a v-SNARE, VAMP-2, on tubulovesicles, no
t-SNARE was definitively identified. Therefore, to obtain further integral membrane markers in tubulovesicles, we investigated the presence of other syntaxins in tubulovesicle fractions
immunoisolated with antibodies against the H-K-ATPase
-subunit (Fig. 1). The gradient-isolated
tubulovesicle fraction contained immunoreactivity for syntaxin 3 and
syntaxin 4. Neither syntaxin isoform was recovered in association with
beads coated with a nonspecific subclass-matched IgG2b monoclonal
antibody. Similarly, although the majority of H-K-ATPase was recovered
in association with anti-H-K-ATPase-coated beads, none of the syntaxin
4 was recovered on immune beads. In contrast, syntaxin 3 immunoreactivity was recovered in the immunoisolated vesicles to an
extent similar to H-K-ATPase (Table 1).
These results in rabbit tubulovesicle membranes are similar to
results obtained recently by Peng and colleagues (36) in rat gastric vesicles.
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Distribution of Rab11a and Rab25 in resting and stimulated glands. Our earlier investigations suggested that stimulation of parietal cells elicited a redistribution of Rab11a into heavy membrane fractions in parallel with the redistribution of H-K-ATPase (21). In these earlier studies we did not observe an increase in soluble Rab11a during stimulation for 45 min. Because the cycling of Rab proteins on and off membranes may be very rapid, we first sought to investigate the possible movement of Rab11a into the cytosolic fraction during stimulation of tubulovesicle fusion with the canaliculus. We therefore incubated isolated gastric glands in the presence of 100 µM ranitidine (resting) or 100 µM histamine-100 µM IBMX (stimulated) for 10 min, which coincides with the exponential phase of the induction of secretion (1). After incubation, glands were rapidly homogenized and the soluble fraction was separated from whole membranes by centrifugation at 150,000 g. Supernates and membrane pellets were then analyzed for Rab11a content on Western blots to determine distribution. In resting glands only 1.06 ± 0.36% of total gland Rab11a was present in the cytosolic fraction. After 10 min of incubation with ranitidine, we observed 0.90 ± 0.50% of Rab11a in the cytosolic fraction compared with 0.95 ± 0.55% in the cytosol of glands stimulated with a combination of histamine and IBMX. No changes were observed with incubations with histamine and IBMX at any time point from 5 to 45 min (data not shown). When blots were probed for Rab25, no immunoreactivity was detectable in the high-speed supernate fractions from resting or stimulated glands. All the Rab25 immunoreactivity was recovered in the membrane fraction. These findings are similar to those previously published for Rab25 (7), and we have been unable to detect any cytosolic Rab25 in parietal cells. The results suggest that there was no redistribution of Rab11a or Rab25 to the cytosol with maximal stimulation of the parietal cells with a combination of histamine and IBMX.
Movement of tubulovesicle contents into SA vesicles after stimulation. Urushidani and Forte (47) characterized a fraction of SA vesicles isolated from rabbits treated with histamine that are enriched in the canalicular membranes of parietal cells. To examine further the possible distribution of Rab11a during stimulation of parietal cells, we studied the redistribution of Rab11a and other putative components of the tubulovesicle into SA vesicle fractions. Results from a representative experiment are shown in Fig. 2 with quantitation presented in Table 2. As noted previously (47), SA vesicle fractions from stimulated rabbits demonstrate a marked increase in H-K-ATPase compared with fractions from rabbits treated with the histamine-receptor antagonist ranitidine. Rab11a showed a similar increase in immunoreactivity in the SA vesicle fractions from stimulated animals. This increase in immunoreactivity was also noted for syntaxin 3 as well as SCAMPs and Rab25, all of which have been localized to immunoisolated tubulovesicle membranes (7). In contrast, although prominent immunoreactivity was observed for dynamin, a large GTPase associated with membrane endocytosis (13, 48), the concentration of this protein was not significantly altered in membranes from stimulated rabbits.
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Immunocytochemical redistribution of Rab11a during parietal cell stimulation. We and others reported that the redistribution of H-K-ATPase to the canalicular membrane of stimulated parietal cells can be demonstrated immunocytochemically in preparations of isolated glands and cultured parietal cells (24, 41). We therefore sought to compare the redistribution of H-K-ATPase to the F-actin-containing canaliculus with that for Rab11a. We utilized primary cultures of rabbit parietal cells, because the canalicular architecture is simplified with internalization of the canalicular membrane as a large intracellular vesicle structure that is clearly separable from the tubulovesicle population (41). Figure 3 demonstrates in cultured parietal cells that stimulation with histamine elicits a redistribution of Rab11a immunoreactivity from an intracellular punctate distribution to a more linear staining pattern colocalizing with F-actin staining. As previously described (41), a similar pattern was observed for H-K-ATPase with redistribution of staining from an intracellular punctate pattern to one that coincided with F-actin.
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DISCUSSION |
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The coordination of vesicle trafficking is critical to the dynamic processes within all cells. Inherent in these pathways are the orderly vectorial fusion and retrieval of membrane vesicles. Although SNARE proteins may mediate the interaction of fusing membranes (25, 38), it appears likely that other factors are necessary to account for the orderly recycling of membrane domains. The results presented here and in previous investigations (7, 9, 36) establish the trafficking of the parietal cell H-K-ATPase as a critical model for regulated apical recycling with discrete localization of SNARE proteins, Rab proteins, and apical endocytotic machinery.
Forte et al. (16) first proposed the membrane recycling hypothesis for parietal cell secretion in 1977. Studies from Peng and colleagues (36) in rat and from our own laboratory in rabbit (7) demonstrated the presence of v- and t-SNARE proteins on tubulovesicle membranes. SNARE proteins have been implicated in the assembly of a critical docking complex necessary for vesicle-membrane fusion (38). We previously demonstrated the presence of VAMP-2, a v-SNARE protein, on immunoisolated tubulovesicles. The present data demonstrate the presence of syntaxin 3, but not syntaxin 4, on immunoisolated tubulovesicles. These results are similar to those observed in rat tubulovesicle membranes (36). Thus, although immunoreactivity for syntaxins 1, 2, 3, and 4 can be documented in parietal cells, only syntaxin 3 is present on tubulovesicles. It is not clear which of the syntaxins is present on the canalicular target membranes. However, we have noted localization of syntaxin 4 immunoreactivity to basolateral membranes in isolated gastric glands and cultured parietal cells, as assessed by immunocytochemistry (unpublished results). Unfortunately, none of the other antibodies presently available have allowed definitive immunocytochemical localization. Still, it is of interest to note that syntaxin 3 has been localized to the basolateral membrane in collecting duct cells (29), whereas it is present in the apical membranes of MDCK cells (28) and on zymogen granules in pancreatic acinar cells (17). Thus syntaxin localization may be more complex than previously understood. Nevertheless, the presence of SNARE proteins on tubulovesicles lends further credence to the notion that vesicle fusion mediates the regulated insertion of the H-K-ATPase into the apical membranes of parietal cells.
Previous investigations have hypothesized that the cycling of Rab proteins on and off their associated vesicle membranes is critical for their function. These studies have relied substantially on the analysis of Rab3 family members in neuronal, endocrine, and exocrine systems (12, 14, 26, 27). Although the predominance of reports supports the dissociation of Rab3 from vesicle membranes during the process of vesicle fusion, Bielinski et al. (5) were unable to observe any movement of Rab3a into a cytosolic fraction with depolarization of synaptosomes. Nevertheless, the vast majority of these studies suggested a model for Rab function in which Rab proteins would cycle off vesicles on fusion with target membranes (33). Because Rab-GDI can remove Rab proteins from membranes (37, 45, 49), by implication it has been hypothesized that Rab-GDI would cycle Rab proteins back to mature secretory vesicles through a cytosolic intermediate.
The findings presented here suggest a model for the function of Rab11a different from that for Rab3. The data presented here show more definitively that Rab11a traffics to the canalicular membrane in a sustained fashion without detectable cycling through the cytosol. Isolation of SA vesicles demonstrated that all the tubulovesicle-associated membrane proteins showed similar increases in immunoreactivity after stimulation of secretion in vivo with histamine. Immunoreactivity for Rab11a and Rab25, as well as for SCAMPs and syntaxin 3, showed increases similar to that for H-K-ATPase. Furthermore, immunocytochemical examination of stimulated parietal cells in primary culture also demonstrated a clear and sustained redistribution of Rab11a immunoreactivity to the canalicular membrane. Unfortunately, antibodies against other tubulovesicle components are not of suitable sensitivity for immunocytochemical analysis, so we have not been able to validate a similar redistribution for other tubulovesicle components. These results indicate that Rab11a does not redistribute to the cytosol during tubulovesicle fusion with the intracellular canaliculus.
Recent investigations have implicated Rab11a as a marker for plasma membrane recycling systems. Ullrich et al. (46) and others (23) demonstrated that Rab11a is associated with the plasma membrane recycling system in nonpolarized cells. In polarized cells the analogous structure, the apical recycling system, is responsible for processing of apical recycling and basolateral-to-apical transcytosis (2). We also recently observed that Rab11a and Rab25 are associated with apical membrane recycling systems in polarized MDCK cells (19). Thus the presence of Rab11a and Rab25 on tubulovesicles indicates that this vesicle population is part of an active recycling system. In the case of parietal cells, however, this recycling appears to be highly regulated along its exocytic and endocytic pathways.
The results presented above also provide the first demonstration of
dynamin in gastric parietal cells. Dynamin is a large GTPase, and its
activity is required for clathrin-dependent endocytosis. Although
dynamin is not necessary for the assembly of coatamer complex, it does
appear to act as a critical regulator of vesicle budding back from the
plasma membrane (13, 48). Thus oligomers of dynamin may form
"rings" that pinch off vesicle buds during reinternalization (42,
43). Although previous investigations have failed to identify classical
coated vesicles in parietal cells (6, 16), clathrin immunoreactivity is
present in parietal cells, and -adaptin immunoreactivity is
associated with a population of tubulovesicles (35). In addition, the
parietal cell coatamer complex associates with the
-subunit of
H-K-ATPase (35). These results, along with the presence of a prominent
concentration of dynamin at the canalicular surface, implicate the
canaliculus as a site for membrane recycling. Indeed, Gottardi and
Caplan (22) noted that the
-subunit of H-K-ATPase contains a
critical endocytotic recognition sequence of FRHY in its cytoplasmic
tail. In transgenic animals expressing a mutation of the critical
tyrosine, the proton pump was present constitutively on the canalicular surface (9). All these results demonstrate the importance of membrane
recycling to the proper functioning of the parietal cell.
In summary, the results presented here indicate that Rab11a does not cycle off tubulovesicle membranes of parietal cells during fusion with the apical target surface. In contrast to the results obtained with Rab3 family members, the Rab proteins associated with apical recycling in polarized epithelial cells may remain with the component vesicle membranes during the process of fusion and likely retrieval. Further studies are required to discern whether similar principles are also at work in other systems of regulated epithelial ion transporter recycling to the apical membrane of polarized epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. David Castle for the gift of antibody and Dr. Mark Knepper for sharing results before publication.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48370 and DK-43405 and a Veterans Administration Merit Award (J. R. Goldenring) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31900 (C. S. Chew)
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: J. R. Goldenring, Institute for Molecular Medicine and Genetics, CB-2803, Medical College of Georgia, 1120 Fifteenth St., Augusta, GA 30912-3175.
Received 21 January 1998; accepted in final form 8 April 1998.
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