Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis.

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.

Intercalated and inner medullary collecting duct (IMCD) 1 cells of the kidney mediate the secretion of H ϩ by an apical membrane H ϩ -ATPase (1,2). The rate of H ϩ transport in these cells is regulated in part by exocytic insertion and endocytic retrieval of H ϩ -ATPase-coated vesicles (1,3). We have shown that the exocytic insertion of proton pumps (H ϩ -ATPase) into the apical membrane of rat IMCD cells in culture utilizes a mechanism similar to that described in neurosecretory cells involving SNARE proteins (syntaxin, SNAP-23, and VAMP-2) (4,5). This conclusion is based on the following observations. 1) IMCD cells express all of the SNARES (syntaxins, VAMP-1, and -2, and SNAP 23) along with synaptotagmin, N-ethylmaleimide-sensitive factor, and ␣ and ␥ SNAP that are required for vesicle targeting and fusion (4,5). 2) Clostridial toxins that act as highly specific SNARE proteases inhibit H ϩ -ATPase exocytosis and H ϩ secretion (6,7). 3) Activation of regulated exocytosis by acute acidification of IMCD cells induces the formation of a SNARE complex that includes syntaxin, VAMP-2, SNAP-23, N-ethylmaleimide-sensitive factor, SNAPs, and a subunit of the H ϩ -ATPase (4). However, the targeting signal for these acid secretory vesicles may differ from that of neurosecretory vesicles in that a subunit of the H ϩ -ATPase itself may be required for delivery of these vesicles to the apical membrane.
The role of SNAREs in targeting vesicles is controversial (8,9), and it has been suggested that these proteins only serve as the machinery for fusion (10,11). There is evidence that v-and t-SNAREs are promiscuous, and in in vitro assays a given v-SNARE can form complexes with many different t-SNAREs (12). On the other hand, in several cell types, the distribution of t-SNAREs is restricted to specific membrane domains (10). Such a restriction is consistent with a role of these SNAREs in the targeting process.
In the current study we have examined the hypothesis that H ϩ -ATPase in renal acid-secretory cells interacts directly with an apical membrane receptor, a t-SNARE, and that this t-SNARE is a specific isoform of syntaxin (synt). The specificity of this interaction between H ϩ -ATPase and syntaxin provides for the targeting of the H ϩ -ATPase to the apical membrane of renal epithelial cells. To evaluate this hypothesis, we first demonstrated that, in a pull-down assay, GST fusion syntaxin (1A, 1B, 2, 4, and 5) isoforms, immobilized on glutathione-Sepharose beads and identified in both intact renal medulla and cultured IMCD cells, could all complex with VAMP-1 and SNAP-23 in kidney homogenate. However, only syntaxin-1 could complex with H ϩ -ATPase. Deletion of the SNARE binding (H3) domain of syntaxin-1 interfered with the ability of this t-SNARE to bind other SNAREs and also H ϩ -ATPase. Transfected IMCD cells that express a cytoplasmic (soluble) form of syntaxin-1A (deletion of membrane binding domain, synt-1A⌬C) had a marked reduction in regulated H ϩ -ATPase exocytosis compared with wild type cells or cells that expressed a soluble form of syntaxin-4 (synt-4⌬C). Finally, we have shown in these cells by fluorescent microscopy that syntaxin-1 is localized to the apical membrane. Thus, it can be demonstrated that there is isoform-specific interaction between H ϩ -ATPase and syntaxin-1 and that the specificity of the interaction and localization of syntaxin-1 can account for both targeting and fusion of H ϩ -ATPase vesicles with the apical plasma membrane.

Kidney Inner Medullary Homogenate
Rat kidney inner medulla was homogenized using Teflon-coated Dounce homogenizer in a buffer consisting of HB, 20 mM Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl, 320 mM sucrose. Just before use, 1 mM DTT, 2 g/ml pepstatin, 10 g of leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride was added. The homogenate was centrifuged at 1000 ϫ g at 4°C for 20 min to obtain a post-nuclear supernatant. Triton X-100 was added to the supernatant to a final concentration of 1% and homogenized a second time in a Dounce. This 1% Triton X-100-treated homogenate was centrifuged at 13,000 ϫ g for 45 min at 4°C. The final supernatant was collected and stored at Ϫ80°C for binding assays.

Rt-PCR and Expression Vectors Construction and RNA Purification
Messenger RNA was purified from IMCD cells, rat renal medulla, and brain tissue using a commercial poly(A) pure kit (Ambion Inc.), and this RNA was analyzed by Rt-PCR to identify mRNA expression of syntaxin-1A, -1B, -2, -4, and -5.

PCR Primer Design
Primers for Syntaxin Isoforms to be Cloned into the PGEX Vectors to Express GST-Syntaxin Fusion Proteins-To facilitate the subsequent expression vector constructs, some restriction sites were added to the primers so that the PCR products from these primers can be inserted into the corresponding restriction sites of appropriate vectors. The primers used are as follows: 1) for Synt-1A: upstream, 5Ј-cac gaa ttc aa AAG GAC CGA ACC CAG GAG-3Ј; downstream, 5Ј-taa ctc gag ATC CAA AGA TGC CCC CGA TG-3.
An EcoRI and XhoI restriction site was added to the 5Ј-terminal end of the upstream and downstream primers, respectively. When spliced into the EcoRI and XhoI cloning site of the vector PGEX-4T2, it enables the synt-1A PCR template to fuse with the GST codon beginning at the second codon, including amino acids 2-288 of syntaxin-1A. 2) For Synt 1B: upstream, 5Ј-cac gaa ttc gg ATG AAG GAT CGG ACT CAG-3Ј; downstream, 5Ј-cat ctc gag TTT TGG GAG TGA GCT TGG AG-3Ј.
Again, EcoRI and XhoI sites were introduced into the 5Ј-terminal end of the primers. The PCR product was spliced into the EcoRI and XhoI sites of PGE X-4T2 where it fuses with GST in-frame starting at the first codon of S1B, including amino acids 1-289 of syntaxin-1B. 3) For Synt-2: upstream, 5Ј-aat aga att CGG CTG CCG GAC CTC ACG-3Ј; downstream, 5Ј-gac ctc gag CAT TTG CCA ACC GTC AAG-3Ј.
EcoRI and XhoI sites were also attached to the 5Ј-terminal end of the primers. The PCR product of these primers was spliced into the cloning sites of EcoRI and XhoI of PGEX 4T2 enabling the fourth codon of the synt-2 template to be fused in-frame with GST, including amino acids 4 -291 of syntaxin-2. 4) For Synt-4: upstream, 5Ј-aat gga tc CGC GAC AGG ACC CAT GAG TTG-3Ј; downstream, 5Ј-acg gaa ttc TCC CAG TGC CAA GAA CAT GTG-3Ј.
A BamHI and EcoRI restriction site was added to the 5Ј-terminal end of upstream and downstream primers, respectively, to facilitate the subsequent cloning step. When the PCR product of these primers was spliced into the BamHI and EcoRI cloning site of the vector PGEX 3X, it made the synt-4 template fuse in-frame with GST, starting at the 2nd codon including amino acids 2-299 of syntaxin-4. 5) For synt-5: upstream, 5Ј-caa gga tcc aa ATG TCC TGC CGG GAT C-3Ј; downstream, 5Ј-cac gaa ttc TCA GGC AAG GAA GAC CAC-3Ј.
These primers were designed to splice the synt-1A sequence 2-162 (amino acids) into the cloning site of EcoRI and XhoI of PGEX 4T2 to generate a truncation of the H3 binding and transmembrane domain of synt-1A.

Rt-PCR
Reverse transcription was accomplished by using 0.1 g of mRNA as template and oligo(dT) 16 as reverse transcriptase primer in a total volume of 20 l; after incubation at room temperature for 10 min, the reaction temperature was raised to 42°C for 15 min, denatured at 99°C for 5 min, and then cooled to 5°C for 5 min. Then 1 nmol of each upstream and downstream PCR primer and other components for PCR were added to a total volume of 100 l (PerkinElmer Life Sciences, GeneAmp RNA PCR kit). The PCR was performed on a MiniCycler TM with the time and temperature as follows: melting point, 95°C, 60 s; annealing, 60°C, 30 s; extending, 72°C, 120 s for 30 cycles followed by a 7-min extension at 72°C. PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide.

Construction of Expression Vectors
GST-Syntaxin Isoform Fusion Protein Expression Vectors-Corresponding bands of the PCR products separated on the agarose gel were purified using a commercial kit (QIAEX II, Gel Extraction kit, Qiagen Inc.) and subjected to enzyme restriction cut as indicated by the attached enzyme restriction site in the primers. Vectors PGEX 4T2 and PGEX 3X were also cut with the same enzyme as the PCR product to be inserted. All the cut DNA were electrophoresed on 1% agarose gels and extracted as indicated above.
PCR products encoding synt-1A, -1B, -2, and -1A ⌬H3⌬C sequence were spliced into the EcoRI and XhoI restriction site of PGEX 4T2 for expression of GST fusion protein. PCR products encoding S4 and S5 sequence were cloned into the BamHI and EcoRI cloning site of PGEX 3X for expression of GST fusion protein.
GFP-Syntaxin Isoform Fusion Protein Expression Vectors-Rt-PCR products from primers for cloning into PcDNA 3.1/NT-GFP-TOPO were ligated directly with PcDNA3.1/NT-GFP-TOPO vector, and the clones with the insertion in the right orientation were picked up for transfection of IMCD cells. All the final clones of recombinant vector were DNA sequenced to confirm the correct insertion.

Expression and Purification of GST-Syntaxin Fusion Protein
Proteins were expressed in the Escherichia coli BL 21 strains. Three hours after induction of the E. coli with IPTG, the bacteria were homogenized in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3), and the expressed protein was purified from this homogenate by binding the fusion protein to glutathione-Sepharose 4B beads in accordance with the manufacturer's instruction for batch pu- . After extensive washing of the beads with HB ϩ 0.5% Triton X-100, the proteins on the beads were solubilized in SDS sample buffer and subjected to immunoblot analysis for subunits of the H ϩ -ATPase from the V 1 domain (31-kDa subunit E) and from the V 0 domain (116-kDa subunit a and 16-kDa subunit C) (A) or for VAMP and SNAP-23. These immunoblots are representative of four separate experiments (C). rification (Amersham Biosciences). The purity of the isolated fusion protein was assessed by gel electrophoresis.

In Vitro ''Pull-down'' Binding Assay
GST-syntaxin isoform fusion protein (25 g) fixed on beads and 800 g of kidney homogenate protein were incubated at 4°C overnight in HB buffer (20 mM Hepes, pH 7.2, 100 mM KCl, 2 mM MgCl 2 ) containing 1 mM DTT, 2 g/ml pepstatin, 10 g/ml leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride added just before use) with gentle agitation. Proteins bound to beads were recovered by centrifuging at 500 ϫ g for 5 min and washed three times with buffer (HB containing 0.5% Triton X-100). The protein attached to the washed beads was solubilized in an equal volume of 2ϫ SDS sampler buffer (final concentration: 50 mM Tris, pH 6.8, 100 mM DTT, 10% glycerin, 2% SDS, and 0.1 bromphenol blue) and heated for 5 min at 95°C and then subjected to SDS-PAGE and immunoblot analysis.

Immunoblots
After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes. The membranes were blocked using 5% nonfat dry milk in TBST and incubated overnight at 4°C with primary antibody. After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase, the blots were developed using the enhanced chemiluminescence method. The film were scanned and then subjected to band densitometry and quantification (NIH Image).
Antibodies used in this study are as follows: for H ϩ -ATPase antibodies: rabbit antibody to the 31-kDa subunit of the H ϩ -ATPase (gift of D. Brown); rabbit polyclonal antibody to the 116-kDa subunit (Synaptic Systems GmbH); and a rabbit polyclonal antibody to the 16-kDa subunit (Ductin) (Oncogene Research Products). For Snap-23 antibodies: rabbit antibody to Snap-23, from Synaptic Systems GmbH. For synaptobrevin/VAMP: mouse monoclonal antibody to synaptobrevin/VAMP, from Synaptic System GmbH. For syntaxin 1A: mouse monoclonal antibody to syntaxin-1A, clone HPC-1 (Sigma). For syntaxin-4: rabbit antibody to syntaxin-4 Synaptic Systems GmbH.
The secondary antibodies for the immunoblot were peroxidase-conjugated goat anti-rabbit and -mouse IgG and were purchased from Jackson ImmunoResearch. . Cloning with the right ligation was confirmed with PCR using the GFP forward and syntaxin isoformspecific reverse primers. Purified recombinant plasmids were used to transfect the IMCD cell using calcium phosphate precipitation and a selection medium containing 800 g/ml G418. Survived cell were further selected to yield monoclonal cell lines using the method of limited dilution. Stable cell lines that express GFP-syntaxin mutant protein were confirmed by directly viewing the GFP fluorescence, immunostaining, and immunoblotting with GFP antibody and syntaxin isoformspecific antibodies. Positive clones were used for experiments.

Effect of Expression of Syntaxin Mutant Proteins on H ϩ -ATPase Translocation to the Apical Membrane of IMCD Cells
Wild type IMCD cells and IMCD cells transfected to express various GFP-syntaxin constructs were used to assess the effect of acute cellular acidification on the exocytic insertion of proton pumps into the apical membrane. Apical membranes were isolated from monolayers of wild type IMCD and IMCD cells transfected with GFP-syntaxin constructs by a vesiculation method modified in our laboratory for polarized epithelial cells (7). Control (pH i ϳ7.2) or acid-loaded cells, in which the pH i falls to 6.5, were incubated for 90 min at 37°C in a vesiculation medium (CHB or NHB (7)) that contained 50 mM paraformaldehyde, 2 mM DTT, and protease inhibitors. Paraformaldehyde and DTT induced the formation of apical membrane vesicles that are released into the incubation medium. At the end of 90 min, the medium containing the released apical membrane vesicles were collected, filtered through 37-m nylon mesh to remove whole cells, and centrifuged at 20,000 rpm at 4°C in a Sorval RC5B centrifuged for 1 h to pellet the vesicles. The pellet was aliquoted for protein determination and dissolved in SDS sample buffer for subjecting to SDS-PAGE and immunoblotting.

Syntaxin Isoforms Expressed in Rat Renal
Medulla-Although there are a large number of isoforms of syntaxin, we have restricted our studies to the isoforms of syntaxin that are localized to the apical plasma membrane of renal collecting duct cells and one isoform (syntaxin-5) that is largely restricted to Golgi membranes (10). We were able to demonstrate that rat renal medulla expresses syntaxin-1A, -1B, -2, -4, and -5 utilizing appropriate primers and Rt-PCR of messenger RNA isolated from rat renal medulla (Fig. 1). The products obtained were the appropriate size for syntaxin-1A (882 bp, 2nd and 3rd lanes), -1B (984 bp, 4th and 5th lanes), -2 (878 bp, 6th and 7th lanes), 4 (924 bp, 8th and 9th lanes) and 5 (925 bp, 10th and 11th lanes). No PCR products were identified if the Rt step was omitted. Molecular identification of these products was confirmed by sequence analysis, and all products were Ͼ97% identical to the published sequences for these rat syntaxins. All of these isoforms were expressed in both kidney and brain with the exception of syntaxin-2, which was only expressed in kidney. Rt-PCR analysis of mRNA derived from cultured IMCD cells also documented that these epithelial cells expressed these isoforms of syntaxin (data not shown). Thus, renal tubular cells in the distal nephron express multiple isoforms of syntaxin including syntaxin-1A that has been thought to be restricted to neuroendocrine tissue (13).
Binding of VAMP, SNAP-23, and H ϩ -ATPase to Syntaxin Fusion Proteins-To test the hypothesis that syntaxin-1A specifically binds H ϩ -ATPase in addition to the other v-and t-SNAREs necessary to form a fusion complex, we compared the capacity of immobilized fusion GST syntaxins to bind SNAP-23, VAMP, and H ϩ -ATPase. The fusion proteins were produced by transformed, IPTG-induced E. coli purified and bound to glutathione-Sepharose G4 beads (Fig. 2). Rat kidney homogenate was incubated with beads coated with either GST alone or equivalent amounts of GST-syntaxin isoforms 1A, 1B, 2, 4, 5, and 1A⌬C⌬H3, a truncated syntaxin-1A missing the C-terminal and H3 domain (Fig. 3). After extensive washing of the beads with HB ϩ 0.5% Triton X-100, the proteins on the beads were solubilized in SDS sample buffer and subjected to immunoblot analysis as follows: (a) subunits of the H ϩ -ATPase from the V 1 domain (31-kDa subunit E) and from the V 0 domain (116-kDa subunit a, and 16-kDa subunit c) (Fig. 3A) (14 -16); (b) VAMP; and (c) SNAP-23 (Fig. 3B). Compared with control beads coated only with GST, beads coated with syntaxin-1A or -B bound appreciable amounts of H ϩ -ATPase. Syntaxin-1Acoated beads bound 8 times (p Ͻ 0.05, n ϭ 6) and syntaxin-1B coated beads bound 3 times (p Ͻ 0.05, n ϭ 5) more 31-kDa subunit of the H ϩ -ATPase than did control beads. The other isoforms, syntaxin-2, -4, -5, and -1A⌬H3 bound no more 31-kDa subunit of the H ϩ -ATPase than background. The binding pattern of the 16-and 116-kDa (V o ) subunits of the H ϩ -ATPase to syntaxin isoforms was similar to that of the 31-kDa subunit (Fig. 3A). The binding of both of V 1 (31 kD) and V o (16 and 116 kDa) subunits to syntaxin-1 indicates that intact proton pump units interact with this t-SNARE. In contrast, all of the fulllength syntaxin isoforms bound VAMP and SNAP-23 (Fig. 3B). Deletion of the H3 domain of syntaxin-1A (17) resulted in a mutant that, as expected, did not bind VAMP or SNAP-23, but surprisingly, this construct also did not bind H ϩ -ATPase.

Expression of Syntaxin Fusion Proteins by IMCD Cells and Effects on Exocytic Insertion of H ϩ -ATPase into Apical Plasma
Membrane-To demonstrate that the specificity of binding documented in our in vitro studies occurs also within intact cells, we generated several transfected IMCD cell lines that expressed various GFP-syntaxin constructs. Our working assumption was that the expression of syntaxin-1A that lacked a membrane binding domain but retained the v-SNARE and H ϩ -ATPase binding domain could act as an inhibitor of regulated H ϩ -ATPase exocytosis. For these studies we developed 4 cell lines: one cell line expressed full-lengthGFP-syntaxin-1A (GFP-synt-1A), a second expressed a truncation of syntaxin-1A in which there was a deletion of the C-terminal membrane binding domain (GFP-synt-1A⌬C) (18), the third, GFP-synt-1A⌬C⌬H3, was a truncation of both the membrane binding domain and the H3, v-SNARE binding domain, and the last cell line expressed a truncation of syntaxin-4 that also had a deletion of the C-terminal membrane binding domain (GFP-synt-4⌬C).
All cell lines expressed a similar amount of fusion protein as judged by immunoblot analysis of whole cell homogenate. The fusion protein was detected utilizing an antibody to the GFP portion of the fusion protein (Fig. 4A). However, the distribution of full-length GFP-synt-1A was distinct from the truncated form, GFP-synt-1A⌬C. The full-length form was found to be primarily localized to the membrane fraction (Fig. 4B), whereas the truncated form was primarily in the cytosolic fraction (Fig.  4B). Immunohistochemical studies also confirmed this biochemical observation (Fig. 4C). GFP-synt-1A was localized in discrete vesicles and plasma membranes, and GFP-synt1A⌬C was diffusely distributed throughout the cytosol but not the plasma membrane (Fig. 4C). The distribution of endogenous synt-1A in non-transfected cells is similar to the distribution of GFP-synt-1A in transfected cells in that both are restricted to membranes. In non-transfected cells, immunohistochemical studies indicate that endogenous synt-1A is primarily localized to the apical membrane (Fig. 5, left).
When wild rat IMCD cells in culture are subjected to cellular acidification, exocytic insertion of H ϩ -ATPase vesicles into the apical membrane is activated (7). This induced exocytic response to cellular acidification results in an enhancement in the amount of H ϩ -ATPase present in the apical membrane of wild type IMCD cells without change in a resident apical membrane protein such as GP-135 (Fig. 6). Transfected cells expressing GFP-synt-1A, GFP-synt-1A⌬C⌬H3, or GFP-synt-4⌬C, when subjected to cellular acidification, displayed a similar degree of apical membrane H ϩ -ATPase amplification (Fig. 6) to that of non-transfected cells. However, when cells that express GFP-synt-1A⌬C, a form of syntaxin that retains the binding sites for v-SNAREs and H ϩ -ATPase but lacks the membrane binding domain, are subjected to cellular acidification, apical membrane H ϩ -ATPase amplification was abolished (Fig. 6). Thus, this soluble, truncated construct of syntaxin-1A appears to act as a specific inhibitor of regulated exocytosis. To evaluate this hypothesis further, we determined whether any of these constructs of syntaxin could be co-immunoprecipitated with H ϩ -ATPase (Fig. 7). Both GFP-synt-1A and GFP-synt-1A⌬C could be co-immunoprecipitated with H ϩ -ATPase, but GFPsynt-4⌬C could not. These observations indicate that soluble GFP-synt-1A⌬C binds to H ϩ -ATPase and by so doing interferes with its targeting to the apical membrane.

DISCUSSION
Regulation of H ϩ secretion in the collecting duct of the mammalian kidney involves amplification of the number of apical membrane proton pumps (H ϩ -ATPase) by regulated exocytic insertion and reduction by endocytic retrieval of these pumps into a subapical population of recycled vesicles (1,3). The recycled proton pump containing vesicles is unusual in that their cytoplasmic surface is almost entirely coated with the V 1 portion of the H ϩ -ATPase (19). In cultured rat IMCD cells we have demonstrated that the process of regulated exocytic insertion of H ϩ -ATPase containing vesicles into the apical membrane involves a process analogous to neurosecretion. The vesicle fusion process in these cells involves SNAREs with the formation of a large, clostridial, toxin-sensitive complex that includes syntaxin, SNAP-23, VAMP, N-ethylmaleimide-sensitive factor, and ␣ and ␥ SNAP (4). The complex differs in that it also contains the H ϩ -ATPase (4,5).
In addition to mediating fusion, the SNARE proteins may be involved in the targeting of the H ϩ -ATPase vesicles to the apical membrane. This latter role of SNAREs in targeting is controversial because SNARE pairing can be promiscuous (12). Thus, factors other than the specificity of SNARE pairing may allow these proteins to participate in the targeting process. Important factors that could confer a specific role of a SNARE protein in targeting may include selective affinity of this t-SNARE for a specific non-SNARE in the vesicle and segregation of the target SNARE to a single membrane domain (apical). Consistent with this kind of targeting scheme is the hypothesis that the renal acid secretory vesicles are targeted, in part, by an interaction between an apical membrane syntaxin and the vesicular coating protein, the H ϩ -ATPase. To test this hypothesis we initially examined the ability of fusion GSTsyntaxin isoforms to form complexes with v-SNAREs and H ϩ -ATPase derived from detergent (Triton-X-100)-solubilized extracts from the renal medulla of rats. The isoforms of syntaxin evaluated (syntaxin-1A, -1B, -2, -4, and -5) included only the syntaxins that are expressed by rat kidney and present in the apical membrane of intact tubular epithelial cells with the exception of syntaxin-5, a non-plasma membrane syntaxin that served as a control for these assays (10). Syntaxin 3 which is also expressed in the plasma membrane of renal epithelial cells was not evaluated because it has been shown to be localized to only the basolateral membrane of renal tubular epithelial cells of the collecting duct (20). In the pull-down assay, utilizing these GST syntaxins bound to Sepharose beads, we demonstrated isoform specificity in that only syntaxin-1 (1A Ͼ Ͼ 1B) bound both V 1 and V o H ϩ -ATPase subunits, whereas all the other isoforms (syntaxin-2, -4, and -5) did not. All syntaxin isoforms examined bound VAMP and SNAP-23. Thus, the interaction between the proton pump and syntaxin appears not to be mediated by the binding of the proton pump to the other SNAREs that form the fusion complex. Paring of syntaxin-1 with H ϩ -ATPase is direct and a likely process for targeting these vesicles to the apical membrane.
To identify the region of syntaxin involved in binding to H ϩ -ATPase, we determined whether the proton pump could be bound by truncated forms of syntaxin-1A. Truncation of the C-terminal membrane-binding region did not alter the binding characteristics for H ϩ -ATPase or other SNAREs. On the other hand, deletion of the H3-SNARE binding domain abolished not only VAMP and SNAP-23 binding but also H ϩ -ATPase binding. It would appear that the proton pump interacts with the same general region of syntaxin-1A as do other SNAREs but does not attach indirectly to syntaxin through VAMP or SNAP-23.
These in vitro studies also are consistent with the view that there is syntaxin isoform specificity in the process of exocytic insertion of proton pumps into the apical membrane. To evaluate this proposal in an in vivo system, we examined the effect of expressing constructs of syntaxin in cultured IMCD cells on apical membrane H ϩ -ATPase amplification. We developed cell lines that express truncated forms of syntaxin-1A and syntaxin-4 that had deletions of the membrane binding domain but not the SNARE binding domain. These cells expressed a soluble form of syntaxin (Fig. 4). We also generated a line that overexpressed full-length syntaxin-1A, a form that was primarily membrane-bound. Finally, we generated a cell line that expressed a form of syntaxin-1A that had truncations for both the membrane binding and SNARE binding domains. Based on the results of the pull-down assay, we predicted that the only truncation form of syntaxin that could act as an inhibitor of exocytosis of the proton pump would be the form that could bind to H ϩ -ATPase but would not be localized to the plasma membrane (synt-1A⌬C). Our results confirmed this prediction because only the expression of synt-1A⌬C but not other forms (synt-1A⌬C⌬H3 or synt-4⌬C) resulted in an appreciable inhibition of H ϩ -ATPase exocytosis. We also have demonstrated that synt-1A⌬C but not synt-4⌬C co-immunoprecipitates with H ϩ -ATPase. This observation indicates that these two molecules also interact in vivo. Finally, we have shown that syntaxin-1A is primarily distributed to the apical membrane of cultured IMCD cells. This series of observations is consistent with the hypothesis that interaction between the isoform of syntaxin, syntaxin-1A, and the H ϩ -ATPase is specifically re-quired for the targeting and fusion of proton pump laden vesicles with the apical membrane Although there is compelling evidence in our cultured IMCD cell line (4) and in non-renal tissue (21) for a role for SNAREs in fusion targeting of H ϩ -ATPases to the plasma membrane, to date there has been little evidence that indicates that SNAREs regulate exocytic insertion of the proton pump in the intact nephron. In the present study we demonstrate that the rat renal medulla expresses all of the constituents necessary to form fusion complexes. In addition the proteins derived from renal medullary homogenates interact with GST-syntaxin-1A and form a complex that has the same constituents that are observed in cultured IMCD cells when H ϩ -ATPase exocytosis is stimulated. Therefore, we predict that as in the cultured cell model system, in the intact nephron, targeting of proton pumps involves the same set of SNAREs and has the same isoform specificity for syntaxin-1A as in the IMCD cell in culture.