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Localization and Function of SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein-25 and Vesicle-associated Membrane Protein-2 in Functioning Gastric Parietal Cells

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m207694200

ISSN

1083-351X

Autores

Serhan Karvar, Xuebiao Yao, James M. Crothers, Yuechueng Liu, John G. Forte,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

The solubleN-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Δ181–206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Δ181–226 inhibited the acid secretory response of parietal cells. Also, SNAP Δ181–226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane. The solubleN-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Δ181–206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Δ181–226 inhibited the acid secretory response of parietal cells. Also, SNAP Δ181–226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane. The secretion of HCl by the gastric parietal cell is driven by a heterodimeric protein known as the H,K-ATPase or proton pump. The regulation of the acid secretory process is thought to be accomplished by secretagogue-dependent trafficking of the proton pump to and from the apical membrane (1Forte J.G. Yao X. Trends Cell Biol. 1996; 6: 45-48Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 2Okamoto C.T. Forte J.G. J. Physiol. (Lond.). 2001; 532: 287-296Crossref Scopus (68) Google Scholar). Upon stimulation, the H,K-ATPase-containing cytoplasmic tubulovesicles fuse with the apical (canalicular) membrane and the process is reversed when the stimulus is removed. Evidence to support this recycling model of acid secretion is extensive, although the regulatory mechanisms underlying the trafficking and fusion events remain elusive. Originally from the study of synaptic transmission, we have come to recognize that membrane fusion is mediated by a set of highly conserved proteins called SNARE proteins (3Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1014) Google Scholar, 4Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 98-106Crossref PubMed Scopus (855) Google Scholar). According to the SNARE 1The abbreviations used are: SNARE, solubleN-ethylmaleimide-sensitive factor attachment protein receptor; SNAP-25, soluble N-ethylmaleimide-sensitive factor attachment protein-25, VAMP-2, vesicle-associated membrane protein-2; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; ER, endoplasmic reticulum; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PM, plasma membrane; DIC, differential interference microscopy; IBMX, isobutylmethylxanthine; MEM, minimum Eagle's medium; AP, aminopyrine; TV, tubulovesicle; rAd, adenoviruses with the incorporated cDNA; GST, glutathione S-transferase1The abbreviations used are: SNARE, solubleN-ethylmaleimide-sensitive factor attachment protein receptor; SNAP-25, soluble N-ethylmaleimide-sensitive factor attachment protein-25, VAMP-2, vesicle-associated membrane protein-2; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; ER, endoplasmic reticulum; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PM, plasma membrane; DIC, differential interference microscopy; IBMX, isobutylmethylxanthine; MEM, minimum Eagle's medium; AP, aminopyrine; TV, tubulovesicle; rAd, adenoviruses with the incorporated cDNA; GST, glutathione S-transferase hypothesis, the plasma membrane maintains a set of closely associated proteins known as target SNAREs such as syntaxin-1A and SNAP-25 (5Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). Complementary proteins located on cytoplasmic vesicle membranes are known as v-SNAREs such as VAMP-2. Docking of the vesicle at the plasma membrane occurs through complementary interaction of v-SNAREs and target SNAREs, ultimately to form a stable SNARE complex that perhaps leads to or catalyzes the fusion of the two membranes (4Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 98-106Crossref PubMed Scopus (855) Google Scholar). Additional factors, both membrane-attached and cytosolic ones, ensure the necessary control of the process. In this study, we used a recombinant adenoviral system to express two of these SNARE proteins, SNAP-25 and VAMP-2 fused to spectrally distinct green fluorescent proteins, in primary cultures of gastric parietal cells. Our goal was to localize the SNARE proteins within the cells and to study their dynamic redistribution when the cells were activated from the resting to stimulated state. We also used a C-terminal deletion mutant of SNAP-25 (SNAP-25 Δ181–206) to examine the role of SNAP-25 in the activation of parietal cells. Gastric mucosal cell fractions were obtained from rabbit and guinea pig stomachs as described previously (6Reenstra W.W. Forte J.G. Methods Enzymol. 1990; 192: 151-165Crossref PubMed Scopus (35) Google Scholar) and in accordance with procedures approved by the local Animal Care and Use Committee. After tranquilization, animals were injected with 20 mg/kg cimetidine 1 h before sacrifice to put parietal cells in a resting state. Gastric mucosa was homogenized in 25 volumes of buffer containing 5 mm PIPES/Tris, pH 6.7, 125 mm mannitol, 40 mm sucrose, 1 mm EDTA, 30 mmphenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. After centrifugation at 80 × g for 10 min to remove whole cells and connective tissue, the resulting supernatant was used to generate three crude particulate fractions and a final supernatant by sequential centrifugation steps: P1 (4300 ×g for 10 min) containing large cellular structures including nuclei and plasma membranes; P2 (13,000 × g for 10 min) containing mainly mitochondria and large granules; and P3 (100,000 × g for 1 h) containing ER and H,K-ATPase-rich tubulovesicles (TV). The final high speed supernatant (S3) was saved as the cytosol. An enriched plasma membrane (PM) fraction was prepared from P1 by centrifugation at 100,000 ×g for 1 h through a density step gradient made of 12 and 18% Ficoll (type 400) with membranes collected from atop each layer (6Reenstra W.W. Forte J.G. Methods Enzymol. 1990; 192: 151-165Crossref PubMed Scopus (35) Google Scholar). A TV fraction highly enriched in H,K-ATPase was prepared from P3 by density gradient centrifugation on a sucrose step gradient as described previously (7Tyagarajan K. Chow D.C. Smolka A. Forte J.G. Biochim. Biophys. Acta. 1995; 1236: 105-113Crossref PubMed Scopus (30) Google Scholar). Isolated gastric glands and parietal cells were prepared from New Zealand White rabbits by a combination of high pressure perfusion and collagenase digestion in HEPES-minimal essential medium (MEM), pH 7.4, as described previously (8Chew C.S. Annu. Rev. Physiol. 1994; 56: 445-461Crossref PubMed Scopus (44) Google Scholar). The resulting suspension containing both glands and cells was strained through a 40-μm mesh to remove connective tissue and large debris. The resulting supernatant was left standing on ice for 5–10 min, time sufficient for large gastric glands to settle leaving individual cells suspended in the medium. Intact cells for primary culture were recovered from the suspension by three repetitions of centrifugation (200 ×g for 5 min) followed by resuspension in fresh HEPES-MEM. This low speed centrifugation favors the harvesting of the larger parietal cells, although few other epithelial cells and fibroblasts were recovered. Cells were incubated for 30 min at 37 °C in medium A consisting of Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 20 mm HEPES, 0.2% bovine serum albumin, 10 mm glucose, 8 nm epidermal growth factor, 1× SITE medium (Sigma), 1 mm glutamine, 100 units/ml penicillin/streptomycin, 400 μg/ml gentamicin sulfate, and 15 μg/liter Geneticin or 20 μg/ml novobiocin, pH 7.4, containing 25 μg/ml amphotericin B to destroy contaminating yeast and bacteria. Cells were then plated onto Matrigel (Collaborative Biomedical) coated coverslips in 12-well plates and incubated at 37 °C in culture medium A. For binding studies where we wished to harvest relatively large amounts of SNAP-25 and its C-terminal deletion mutant, settled glands were collected, washed twice in HEPES-MEM, and plated onto Matrigel in medium A as described for cell culture. Membrane fractions were solubilized in SDS-PAGE sample buffer (1% SDS, 0.4 m urea, 5% 0.7m 2-mercaptoethanol, 0.25 mm EDTA, 10% glycerol, 0.0025% bromphenol blue, and 30 mm Tris-HCl, pH 6.8). Samples were run on 12% gels and transferred to nitrocellulose membranes and then probed with a rabbit polyclonal antibody directed against SNAP-25 (provided by H. P. Moore, University of California, Berkeley, CA) followed by secondary probing with horseradish peroxidase-tagged goat anti-rabbit IgG (Jackson Laboratories). For the detection of VAMP, we used a polyclonal antibody made in rabbit using GST-VAMP-2 fusion protein as antigen (provided by Dr. A. W. Lowe, Stanford University, Palo Alto, CA). This was followed by the goat anti-rabbit IgG tagged with horseradish peroxidase. H,K-ATPase was detected by monoclonal antibody 2G11, a mouse monoclonal antibody against its β-subunit (Affinity Bioreagents) followed by secondary antibody with horseradish peroxidase-tagged goat anti-mouse IgG. Bands were visualized by chemiluminescence with the Renaissance kit (PerkinElmer Life Sciences). Stimulation of parietal cells was quantified using the AP uptake assay as described for cultured parietal cells (9Duman J.G. Tyagarajan K. Kolsi M.S. Moore H.P. Forte J.G. Am. J. Physiol. 1999; 277: C361-C372Crossref PubMed Google Scholar). Culture medium was removed from the wells and replaced with 0.5 ml of HEPES-MEM containing 11 nCi/ml [14C]AP. Cells were either held in a resting state by the H2-receptor blocker cimetidine (100 μm) or stimulated by the addition of histamine and IBMX (100 and 30 μm, respectively). Cultures were gently shaken for 30 min at 37 °C. The coverslips were removed from the medium, quickly dipped in phosphate-buffered saline to remove external radioactivity, and incubated in solubilization buffer (125 mm Tris-Cl, 2% SDS, and 10% 2-mercaptoethanol, pH 6.8) for 1 h at room temperature. Cells were then scraped from the coverslip, and the protein content of the cell scrapings was assayed using the filter paper blot method (10Minamide L.S. Bamburg J.R. Anal. Biochem. 1990; 190: 66-70Crossref PubMed Scopus (237) Google Scholar). Aliquots of both the reaction medium and cell scrapings were assayed for [14C]AP by liquid scintillation counting. These data were used to calculate the AP accumulation ratio (ratio of [AP]i:[AP]e). AP uptake values were normalized among the various preparations by expressing data as a fraction of the stimulated control. Adenoviruses were obtained as previously described with the following mouse cDNA constructs (11Xia Z. Zhou Q. Lin J. Liu Y. J. Biol. Chem. 2001; 276: 1766-1771Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Yang Y. Xia Z. Liu Y. J. Biol. Chem. 2000; 275: 29482-29487Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar): SNAP-25 fused to cyan fluorescent protein (CFP); VAMP-2 fused to yellow fluorescent protein (YFP); SNAP-25 wild type and GFP unfused; and C-terminal deletion mutant SNAP-25 Δ181–206 and GFP unfused. Adenoviruses with the incorporated cDNAs (rAd) were used to infect cells and glands with the respective cDNAs. Infections with rAd were executed by the addition of ∼2 × 106 viral particles/ml to the culture medium immediately after isolation of gastric glands or 6-h post-plating for cultured parietal cells. Various concentrations of viruses were tested with our culture system, and we chose our experimental conditions based on the level of fluorescent protein expression. After 36-h infection, AP uptake experiments were performed on cultured cells. Wild type and mutant SNAP-25 were expressed by rAd infection for 18 h in gastric glands, which were then solubilized with Triton X-100 and used for binding assay. After rAd infection, cultured parietal cells were either held in a resting state or stimulated with histamine plus IBMX. In some cases, cells were treated with a proton pump inhibitor (5 μm SCH-28080) as described previously (13Agnew B.J. Duman J.G. Watson C.L. Coling D.E. Forte J.G. J. Cell Sci. 1999; 112: 2639-2646Crossref PubMed Google Scholar). Infected cells were observed in the presence of Dulbecco's modified Eagle's medium (Invitrogen) for the duration of live cell imaging. Images were collected using conventional epifluorescence microscopy (excitation/emission: GFP, 488/509 nm; YFP, 500/20 nm; CFP, 436/10 nm) with a Nikon Microphot FX-2 using Isee Imaging software. In the case of cells double-labeled with CFP-SNAP-25 and YFP-VAMP-2, images are presented for each of the fluorescent proteins along with images recorded by differential interference microscopy (DIC). To compare and distinguish the distribution of two fluorophores, we also performed a subtraction of the YFP image from the CFP image. This was done using Isee Imaging software by first adjusting the total intensity of the two images to approximately the same level and then performing a pixel by pixel subtraction. Recombinant rat GST-syntaxin-1 was expressed in bacteria as fusion protein as described previously (12Yang Y. Xia Z. Liu Y. J. Biol. Chem. 2000; 275: 29482-29487Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Wild type and mutant SNAP-25 were expressed in gastric glands for 18 h by rAd infection, which were then solubilized with Triton X-100 and used for binding assay. For binding assays, aliquots of total soluble protein were added to 20 μl of glutathione-agarose beads pre-adsorbed with GST-syntaxin-1 and incubated overnight at 4 °C in 300 μl of binding buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm EGTA, and 1% Triton X-100). Beads were washed three times with 0.5 ml of binding buffer at 4 °C. Samples were separated into supernatant and pellet, solubilized in sample buffer, and analyzed by Western blot. Blots were first probed with antibody against SNAP-25, stripped, and then probed with antibody against syntaxin-1 (HPC-1, Sigma). The relative abundance of SNAP-25 in crude subcellular fractions and partially purified membrane fractions from rabbit and guinea pig gastric mucosa was assayed by SDS-PAGE and immunoblotting using a SNAP-25 antibody. The same blots were stripped and reprobed for relative abundance of H,K-ATPase using an antibody against the 60–80-kDa β-subunit. As shown in Fig.1 A for crude cell fractions from rabbit stomach, a visible immunoreactive band was detected in the 25-kDa SNAP-25 region of the low speed fraction (P1) and in the microsomal fraction (P3). There was little or no detectable SNAP-25 reactivity in either the mitochondrial-rich fraction (P2) or the cytosolic supernatant (S3). To better characterize the localization of SNAP-25 in parietal cells, P1 and P3 were further fractionated by density gradient centrifugation. SNAP-25 was highly enriched in the PM fraction purified from P3. In contrast, there was virtually no enrichment of SNAP-25 in the H,K-ATPase-enriched TV fraction purified from P3. The cross-reactivity of the anti-SNAP-25 antibody prepared from immunized rabbit serum resulted in the appearance of several minor immunoreactive bands in the rabbit cell fractions, some more prominent than others (e.g. P1). To circumvent this problem, comparable cell fractions were prepared from guinea pig gastric mucosa. The results shown in Fig. 1 B were similar to those for the rabbit; that is SNAP-25 was most highly enriched in the plasma membrane-rich fractions (P1 and PM), whereas H,K-ATPase was most enriched in the membrane fraction derived from TV P3. Earlier experiments from our laboratory (14Peng X.R. Yao X. Chow D.C. Forte J.G. Bennett M.K. Mol. Biol. Cell. 1997; 8: 399-407Crossref PubMed Scopus (79) Google Scholar) and that of Calhoun and Goldenring (15Calhoun B.C. Goldenring J.R. Biochem. J. 1997; 325: 559-564Crossref PubMed Scopus (82) Google Scholar) demonstrated that another SNARE protein, VAMP-2 (also known as synaptobrevin), was associated with the H,K-ATPase-rich tubulovesicle fraction from parietal cells. To distinguish the distribution of SNAP-25 and VAMP-2, another set of cell fractions was prepared from rabbit gastric mucosal homogenate and Western blots were probed for these two proteins along with H,K-ATPase as a control. The results shown in Fig. 2 demonstrate that VAMP-2 was most highly enriched along with tubulovesicles and H,K-ATPase-rich membranes, whereas SNAP-25 was more highly enriched in the fractions containing plasma membranes. To further evaluate the cellular localization of SNAP-25, we used fluorescence microscopy in cultured parietal cells infected with adenovirus containing the CFP-SNAP-25 cDNA construct. By 12 h post-infection, parietal cells clearly expressed CFP, and high levels of expression were observed by 36 h post-infection. Cyan fluorescence was detected in ∼90% of the parietal cells. Fig.3 shows the intracellular localization of CFP-SNAP-25 in resting (non-stimulated) rabbit parietal cells 24 h post-infection. At this low magnification, some diffuse fluorescence was found in the cytoplasm of many parietal cells, but a prominence of CFP fluorescence is clearly seen outlining the apical membrane vacuoles. Because these membrane vacuoles in cultured parietal cells are derived from the apical plasma membrane of parietal cells in situ (13Agnew B.J. Duman J.G. Watson C.L. Coling D.E. Forte J.G. J. Cell Sci. 1999; 112: 2639-2646Crossref PubMed Google Scholar), we call them apical membrane vacuoles. No fluorescence was detected on the "basolateral" membrane surrounding the cells. Thus, the results indicate that SNAP-25 is clearly targeted to the apical plasma membrane. The C terminus of SNAP-25 has been shown to be important in forming a SNARE complex; therefore, we used a C-terminal deletion mutant of SNAP-25 Δ181–206 to probe for a direct functional role of SNAP-25 in parietal cell activation. After 36 h, cultures infected with adenovirus including either wild type or mutant SNAP-25 or a control adenovirus containing the β-galactosidase gene were compared with uninfected cultures for their acid secretory response to stimulants. Parietal cell cultures from each condition were either maintained in a resting state (cimetidine) or stimulated to maximum secretion with histamine plus IBMX. The [14C]AP uptake was measured as an index of acid secretion (Fig.4). To account for variations among four separate preparations, the [14C]AP uptake ratio was normalized to the control non-infected, stimulated parietal cells that was set at 100% for each experiment. Compared with uninfected controls, [14C]AP uptakes were not significantly lower in cells infected with wild type SNAP-25 adenovirus (85.7 ± 19.2% of stimulated control; p = 0.9) or control adenovirus containing β-galactosidase gene but no SNAP-25 (70.4 ± 14.9% of stimulated control; p = 0.24). However, we detected almost 70% inhibition of acid secretion by cells infected with the C-terminal mutant SNAP-25 (AP ratio was 31.0 ± 5.9% of the stimulated control, p < 0.003). To further explore the molecular involvement of SNAP-25 in the gastric acid secretion process, an in vitro binding assay was performed. SNAP-25 wild type and C-terminal deletion mutant were expressed in cultured rabbit gastric glands by adenovirus infection similar to that used for cell cultures. Membrane proteins were solubilized with Triton X-100 and incubated overnight with GST-syntaxin-1A immobilized on glutathione beads. After separating pellet and supernatant by centrifugation, the fractions were separated by SDS-PAGE and successively probed for SNAP-25 and syntaxin-1 by immunoblotting. As shown in Fig. 5, wild-type SNAP-25 was effective in facilitating the assembly of a complex with syntaxin-1, whereas the C-terminal deletion mutant was much less effective. Wild type SNAP-25 was detected entirely in the binding fraction, but the majority of the C-terminal deletion mutant of SNAP-25 was detected in the non-binding fraction. The mutant, which runs at a lower molecular weight, allows native SNAP-25 to be seen recovered only in the binding fraction (also seen in the non-infected gland lysates). In previous studies, we have shown that expressed GFP-VAMP-2 was localized to cytoplasmic membranes along with H,K-ATPase in resting parietal cell cultures and that the two proteins were translocated to apical membrane commensurate with stimulation of acid secretion (16Karvar S. Yao X. Duman J.G. Hybiske K. Liu Y. Forte J.G. Gastroenterology. 2002; 123: 281-290Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Here we sought to monitor the dynamic changes in response to stimulation of live parietal cells that were co-infected with adenovirus containing cDNA constructs for CFP-SNAP-25 and YFP-VAMP-2. Cyan fluorescence was detected in ∼90% of the parietal cells. Yellow fluorescence was detected in ∼75% of the parietal cells, most of which also displayed cyan. The distribution of CFP-SNAP-25 and YFP-VAMP-2 in parietal cells during different functional states is shown in Figs. 6 and7 along with a comparable image using DIC optics. In addition, for each cell there is a "coincident fused image" that is a mathematical reconstruction showing only pixels that were positive for both probes. In a series of resting cells shown in Fig. 6, CFP-SNAP-25 was clearly localized to the apical membrane vacuoles, although there was usually some diffuse cytoplasmic labeling. In these same resting cells, YFP-VAMP-2 was observed almost exclusively throughout the cytoplasm surrounding the apical membrane vacuoles but with very limited yellow fluorescence directly on the apical membrane vacuoles. In no case was either CFP-SNAP-25 or YFP-VAMP-2 associated with the basolateral plasma membrane or the visible lamellipodial extensions. Because there was considerable overlap in the CFP and YFP signals, especially in the cytoplasmic region surrounding the apical vacuoles, we performed a mathematical subtraction of YFP signal from that of CFP. The resulting difference is shown in Fig. 6 as the last column of images. In every case, there is a ring of remaining CFP fluorescence denoting that CFP-SNAP-25 is more preferentially localized to the apical membrane vacuoles in the resting parietal cells. Note that the nuclear membrane, which is denoted on the DIC image by an asterisk, is approximately the same size as the apical vacuoles, but it is not labeled by CFP fluorescence and does not appear in the subtractive image. Thus, separate compartments can be identified for the SNAP-25 and VAMP-2 in resting cells, although there is a subset of overlapping coincidence in the cytoplasm.Figure 7Dynamic changes in the intracellular distribution of CFP-SNAP-25 and YFP-VAMP-2 in live parietal cells and following stimulation. Primary cultures of gastric parietal cells were co-infected with recombinant CFP-SNAP-25 and YFP-VAMP-2 adenoviruses and maintained in culture in the presence of 100 μm cimetidine as described in Fig. 6. After 36 h, cimetidine was removed and cells were stimulated with 100 μm histamine plus 30 μm IBMX, either in the absence (A) or presence (B) of the proton pump inhibitor SCH-28080 (5 μm) as indicated. After 25 min of stimulation, live cells were examined for general morphology (DIC) and for CFP-SNAP-25 fluorescence (CFP .SN25) and YFP-VAMP-2 fluorescence (YFP .VMP2). Apical membrane vacuoles are much enlarged in the stimulated state because of the accumulation of HCl and water, whereas the vacuoles are not enlarged when the H+/K+ pump is inhibited by SCH-28080. For both conditions of stimulation, there is a high degree of co-localization of the CFP-SNAP-25 and YFP-VAMP-2 fluorescence on the apical membrane vacuoles and a relative diminution of signal in the cytoplasm. The co-localization is substantiated by the subtractive image (CFP-YFP) in which the vacuolar signal all but disappears. The asterisk in DIC image indicates nucleus. Bar marker is 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the case of cells stimulated by histamine and IBMX, the apical membrane vacuoles become highly enlarged filling with HCl and water because of the action of newly incorporated H,K-ATPase pump. The images of maximally stimulated cells shown in Fig. 7 A demonstrate an almost coincident distribution of CFP-SNAP-25 and YFP-VAMP-2 on the enlarged apical vacuoles, consistent with the notion that YFP-VAMP-2 translocated to the apical membrane after stimulation. However, the relative diminution of free cytoplasmic space in these maximally stimulated cells makes it difficult to exclude the possibility that some spatial artifact might contribute to apparent co-localization of the two signals. We previously showed that treatment of the cells with a proton pump inhibitor eliminates the swelling artifact by inhibiting the delivery of HCl and water while permitting other events in the signaling cascade including the recruitment of H,K-ATPase to the apical membrane vacuoles (13Agnew B.J. Duman J.G. Watson C.L. Coling D.E. Forte J.G. J. Cell Sci. 1999; 112: 2639-2646Crossref PubMed Google Scholar). Thus, to avoid the swelling artifact parietal cells were stimulated and treated with the proton pump inhibitor SCH-28080. The corresponding images in Fig. 7 B show that CFP-SNAP-25 and YFP-VAMP-2 are primarily associated with the vacuolar membranes, even while the cytoplasmic area was preserved by the treatment of stimulated cells with SCH-28080. Thus, in live parietal cells, there is a dynamic stimulation-dependent co-localization of VAMP-2 and SNAP-25 in the apical plasma membrane vacuoles. In this study, we examined the distribution dynamics and functional importance of SNAP-25 in gastric parietal cells. In the cell fractionation studies, SNAP-25 immunoreactivity was mostly associated with the enriched plasma membrane fraction. SNAP-25 was also detected in the P3 (microsomal) fraction, but because it did not purify with H,K-ATPase-rich tubulovesicles, we suspect that this was may be a contamination of the rough ER. On the other hand, VAMP-2 clearly associates with the P3 fraction and is further enriched when H,K-ATPase is purified by density gradient purification. These data are consistent with earlier work suggesting that VAMP-2 is associated with H,K-ATPase-rich membranes typical of a v-SNARE, whereas SNAP-25 may be associated with the apical plasma membrane more typical of a target SNAREs (14Peng X.R. Yao X. Chow D.C. Forte J.G. Bennett M.K. Mol. Biol. Cell. 1997; 8: 399-407Crossref PubMed Scopus (79) Google Scholar, 17Jons T. Lehnardt S. Bigalke H. Heim H.K. Ahnert-Hilger G. Eur J. Cell Biol. 1999; 78: 779-786Crossref PubMed Scopus (4) Google Scholar). However, the present SNAP-25 data are in disagreement with the findings of Jöns et al. (17Jons T. Lehnardt S. Bigalke H. Heim H.K. Ahnert-Hilger G. Eur J. Cell Biol. 1999; 78: 779-786Crossref PubMed Scopus (4) Google Scholar) who report that SNAP-25, syntaxin-1, and synaptobrevin (VAMP-2) all co-localize with H,K-ATPase in tubulovesicles of resting parietal cells and redistribute to the apical membrane after stimulation. In their study, Jöns et al. (17Jons T. Lehnardt S. Bigalke H. Heim H.K. Ahnert-Hilger G. Eur J. Cell Biol. 1999; 78: 779-786Crossref PubMed Scopus (4) Google Scholar) did not establish co-localization by double labeling but rather used comparative staining patterns observed in sections of resting and secreting gastric mucosa and in isolated parietal cells. Therefore, it is not possible to conclude that none of the three proteins was associated with the apical membrane in resting cells. Furthermore, the isolated parietal cell preparations used by these authors were highly rounded and appeared to lack apical membrane vacuoles, indicating that they may have been functionally and morphologically compromised. To localize SNAP-25 more specifically and because we found that existing commercial antibodies against SNAP-25 were not suitable for immunocytochemistry, we used the recombinant adenovirus technique as an ideal vector for introducing specific gene expression into primary cultures of parietal cells. This allowed us to localize targeted protein expression, observe the dynamic changes associated with functional activity, and introduce mutations to alter that activity. Gastric acid secretion by the parietal cell involves secretagogue-regulated recycling of the H,K-ATPase to and from the apical membrane (1Forte J.G. Yao X. Trends Cell Biol. 1996; 6: 45-48Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 2Okamoto C.T. Forte J.G. J. Physiol. (Lond.). 2001; 532: 287-296Crossref Scopus (68) Google Scholar). These regulated trafficking events, which deliver the pump to the apical cell surface, result in massive morphological transformations of the parietal cell. The trafficking of the H,K-ATPase and the remodeling of the apical membrane during this process probably involve the coordinated function of vesicular trafficking machinery (1Forte J.G. Yao X. Trends Cell Biol. 1996; 6: 45-48Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 13Agnew B.J. Duman J.G. Watson C.L. Coling D.E. Forte J.G. J. Cell Sci. 1999; 112: 2639-2646Crossref PubMed Google Scholar). The dramatic nature of the morphological transformation facilitates superior visualization of changes in the distribution of proteins such as SNAP-25 and VAMP-2, affecting fusion. By introducing both CFP-SNAP-25 and YFP-VAMP-2 adenoviral constructs into the same gastric parietal cells, we have successfully studied the subcellular localization and dynamic distribution of these proteins. In resting cells, YFP-VAMP-2 was distributed throughout the cytoplasm in a pattern similar to that of H,K-ATPase. YFP-VAMP-2 was largely translocated to the apical membrane vacuoles upon stimulation. These data are consistent with previous work where GFP-VAMP-2 was expressed in parietal cells (16Karvar S. Yao X. Duman J.G. Hybiske K. Liu Y. Forte J.G. Gastroenterology. 2002; 123: 281-290Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In the case of parietal cells expressing CFP-SNAP-25, the majority of signal was localized to the apical membrane vacuoles in both resting and stimulated cells; however, there was always a component of CFP-SNAP-25 detected in cytoplasm, especially in the resting state. An artifactual localization is always a possibility when one is inducing overexpression, and the specific location of the expressed protein is frequently related to the integrity of the product such as correct folding, glycosylation, and so forth (18Okamoto C.T. Chow D.C. Forte A.J. Am. J. Physiol. 2000; 278: C727-C738Crossref Google Scholar, 19Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar). In cases of overexpression, we would expect to find abundant signal associated with the ER or recycling endosomal compartment as well as in the final targeted cytolocation. The formation and stability of the SNARE complex including VAMP-2, syntaxin, and SNAP-25 are believed to play a central role in the molecular mechanism underlying exocytosis (3Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1014) Google Scholar, 4Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell. 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More specifically, botulinum neurotoxins A and E cleave the C-terminal domain of SNAP-25, causing complete or partial inhibition of secretion in neuronal and endocrine systems as well as pancreatic acinar cells (22Lawrence G.W. Foran P. Mohammed N. DasGupta B.R. Dolly J.O. Biochemistry. 1997; 36: 3061-3067Crossref PubMed Scopus (48) Google Scholar, 28Hansen N.J. Antonin W. Edwardson J.M. J. Biol. Chem. 1999; 274: 22871-22876Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover, mutation studies have shown important details regarding the participation of SNAP-25 in the exocytic process (12Yang Y. Xia Z. Liu Y. J. Biol. Chem. 2000; 275: 29482-29487Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 29Huang X. Sheu L. Tamori Y. Trimble W.S. Gaisano H.Y. Pancreas. 2001; 23: 125-133Crossref PubMed Scopus (40) Google Scholar, 30Graham M.E. Washbourne P. Wilson M.C. Burgoyne R.D. J. Cell Sci. 2001; 114: 4397-4405Crossref PubMed Google Scholar, 31Wei S. 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In gastric parietal cells, we observed that cells expressing C-terminal deletion mutant SNAP-25 Δ180–206 showed consistent and significant diminution of stimulated acid secretion as compared with wild type expression. In addition, our binding data demonstrate that the C-terminal deletion mutant unlike wild type SNAP-25 was relatively ineffective in associating with syntaxin-1. These secretion and binding data agree with a previous study involving cerebellar neurons, which showed that overexpressed SNAP-25 Δ180–206 mutant altered the vesicle fusion kinetics, reaffirming the importance of SNAP-25 and specifically this C-terminal segment in exocytosis (12Yang Y. Xia Z. Liu Y. J. Biol. Chem. 2000; 275: 29482-29487Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The results also add further evidence for the importance of SNARE proteins for the activation of parietal cells. A major function of the gastric parietal cell is to produce HCl secretion. Because of the massive membrane transformations required for the regulation of its functional activity, the parietal cell may be a model system to characterize the protein-protein interactions that regulate membrane trafficking in other cell types (2Okamoto C.T. Forte J.G. J. Physiol. (Lond.). 2001; 532: 287-296Crossref Scopus (68) Google Scholar, 33Okamoto C.T. Li R. Zhang Z. Jeng Y.Y. Chew C.S. J. Controlled Release. 2002; 78: 35-41Crossref PubMed Scopus (25) Google Scholar). Previous studies demonstrate the presence of v-SNARE and target SNARE proteins on tubulovesicle membranes including the association of VAMP-2 and syntaxin-3 with H,K-ATPase-rich tubulovesicles (14Peng X.R. Yao X. Chow D.C. Forte J.G. Bennett M.K. Mol. Biol. Cell. 1997; 8: 399-407Crossref PubMed Scopus (79) Google Scholar, 15Calhoun B.C. Goldenring J.R. Biochem. J. 1997; 325: 559-564Crossref PubMed Scopus (82) Google Scholar, 34Calhoun B.C. Lapierre L.A. Chew C.S. Goldenring J.R. Am. J. Physiol. 1998; 275: C163-C170Crossref PubMed Google Scholar). SNAP-25 and syntaxin isoforms 1, 2, and 4 were also identified in parietal cell membrane fractions (14Peng X.R. Yao X. Chow D.C. Forte J.G. Bennett M.K. Mol. Biol. Cell. 1997; 8: 399-407Crossref PubMed Scopus (79) Google Scholar, 17Jons T. Lehnardt S. Bigalke H. Heim H.K. Ahnert-Hilger G. Eur J. Cell Biol. 1999; 78: 779-786Crossref PubMed Scopus (4) Google Scholar). Earlier functional studies (16Karvar S. Yao X. Duman J.G. Hybiske K. Liu Y. Forte J.G. Gastroenterology. 2002; 123: 281-290Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 35Ammar D.A. Zhou R. Forte J.G. Yao X. Am. J. Physiol. 2002; 282: G23-G33Crossref PubMed Scopus (53) Google Scholar) demonstrate that VAMP-2 (16Karvar S. Yao X. Duman J.G. Hybiske K. Liu Y. Forte J.G. Gastroenterology. 2002; 123: 281-290Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and syntaxin-3 (35Ammar D.A. Zhou R. Forte J.G. Yao X. Am. J. Physiol. 2002; 282: G23-G33Crossref PubMed Scopus (53) Google Scholar) are essential for the parietal cell activation. The present results now implicate a functional role for SNAP-25 in the regulated trafficking and recycling of H,K-ATPase and add further evidence for the general importance of SNARE proteins for the secretory activation of parietal cells. We thank J. G. Duman, A. Sawaguchi, R. Zhou, R. Jain, and M. Turkoz for technical assistance, C. Chan and Dr. H.-P. Moore (University of California, Berkeley, CA) for SNAP-25 antibody, and Dr. A. W. Lowe (Stanford University, Palo Alto, CA) for VAMP-2 antibody.

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