Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

Guangmu Li,1,2 Qiongqiong Yang,1,2,5 Edward A. Alexander,1,2,3 and John H. Schwartz1,2,4

1Renal Section, Boston University Medical Center, and Departments of 2Medicine, 3Physiology, and 4Pathology, Boston University School of Medicine, Boston, Massachusetts; and 5Department of Nephrology, First Affiliated Hospital, Zhongshan University, Guangzhou, China

Submitted 2 February 2005 ; accepted in final form 27 April 2005


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 ABSTRACT
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H+ transport in the collecting duct is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H+-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H+-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H+-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1A{Delta}C [amino acids (aa) 1–264] formed complexes with H+-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H+-ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H+-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H+-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H+-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H+-ATPase exocytosis.

soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H++ transport


THE INTERRELATIONSHIP and response of the SNARE proteins [soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors] have provided a universal model for vesicle targeting, trafficking and fusion in eukaryotic cells (7, 22, 24). It is believed that when the target membrane resident Snares (t-SNARE, syntaxin, and SNAP or SNAP23) pair with the Snares from the vesicle [v-SNARE, vesicle-associated membrane polypeptide (VAMP)], a SNARE core complex will form and membrane fusion (exocytosis) will occur.

In the kidney, inner medullary collecting duct (IMCD) cells are in part responsible for acid-base homeostasis. H+ secretion by these cells is primarily mediated by an apical membrane H+-ATPase (9). These multisubunit enzymes reside in high density on vesicles that undergo regulated exocytotic insertion into the apical membrane when the cells are acidified and endocytotic retrieval to vesicles when cell pH recovers (1). Previous work from our laboratory (4) showed that SNAREs are involved in the regulation of exocytotic insertion of H+-ATPase-laden vesicles into the apical membrane, affecting the rate of H+ transport by these cells. H+-ATPase forms complexes with SNARE proteins (syntaxin 1A, SNAP23, and VAMP2) both in vitro and in vivo. In addition, the rate of H+ secretion is inhibited either by a cytosolic form of syntaxin 1A or by clostridial toxins that specifically cleave the SNARE proteins syntaxin 1A and SNAP23 (4). These observations confirm that syntaxin 1A and SNAP23 are the t-SNAREs that regulate exocytotic insertion of H+-ATPase containing vesicles. In an in vitro binding assay, solid-phase glutathione S-transferase (GST)-syntaxin fusion proteins were used to pull down syntaxin binding candidates from the kidney homogenate. Several syntaxin isoforms (syntaxin 1A, 1B, 2, 4, 5) were able to form complexes with other SNARE proteins (SNAP23 and VAMP 2) through a conserved H3 domain. However, only syntaxin 1A, also mediated by the H3 domain, bound H+-ATPase (10). On the basis of this latter observation we hypothesize that a specific H+-ATPase binding site is present in the H3 domain of the syntaxin 1A molecule but not in the H3 domain of other syntaxin isoforms.

Syntaxin 1A, like other syntaxin isoforms, contains three domains: an amino-terminal SNARE N, which is believed to have inhibitory regulatory function in SNARE-mediated fusion (17), an H3 domain, which provides one {alpha}-helix to form the synaptic fusion core complex with the cytoplasmic domain of synaptobrevin II (the second helix of the core) and the amino- and carboxy-terminal domains of SNAP25 or SNAP23 (the third and fourth helices) (6, 18, 23), and a carboxy-terminal transmembrane domain (TMD), which enables syntaxin to anchor to the plasma membrane. The H3 domain is the domain responsible for both SNARE and H+-ATPase binding and consists of amino acid residues 161–264 (10).

The purpose of the current study was to identify the regions of the H3 domain of syntaxin 1A responsible for H+-ATPase and SNARE (SNAP23 and VAMP) binding. To this end, a series of pull-down assays were performed with truncated constructs of this H3 domain. In addition, we constructed chimeric forms of syntaxin 1A in which the SNARE N was ligated to fragments of the H3 domain. Our results indicate that the amino-terminal half of the H3 domain of syntaxin 1A has elements that bind SNAP23 and VAMP, whereas the carboxy-terminal half has elements that bind H+-ATPase.


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All experiments were approved by the Institutional Animal Use and Care Committee of Boston University School of Medicine, and the animals were euthanized by pentobarbital overdose after termination of the study.

Kidney Inner Medullary Homogenate

Rat kidney inner medulla was homogenized with a Teflon-coated Dounce homogenizer in HEPES buffer (HB) consisting of (in mM) 20 HEPES, pH 7.5, 100 KCl, 2 MgCl, and 320 sucrose. Just before use, 1 mM DTT, 2 µg/ml pepstatin, 10 µg/l leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride were added. The homogenate was centrifuged at 1,000 g and 4°C for 20 min to obtain a postnuclear supernatant. Triton X-100 was added to the supernatant to a final concentration of 1% and homogenized a second time with a Dounce homogenizer. This 1% Triton X-100-treated homogenate was centrifuged at 13,000 g for 45 min at 4°C. The final supernatant was collected and used for binding assays.

Sample Preparation of Postnuclear, Cytoplasm, and Membrane Fractions

Cultured IMCD cells were homogenized in buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) with a Teflon-coated homogenizer, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4oC to remove whole cells, nuclei, and mitochondria. This postnuclear supernatant was centrifuged at 200,000 g for 1 h to produce a pellet (membrane fractions enriched for both plasma membranes and intracellular membrane) and a supernatant (cytoplasm fraction).

RNA Purification

Messenger RNA was purified from IMCD cells and rat renal medulla with a commercial poly(A) pure kit (Ambion), and this RNA was analyzed by RT-PCR to identify mRNA expression of syntaxins in the IMCD cells.

RT-PCR

Reverse transcription was accomplished by using 0.1 µg of mRNA as template and oligo(dT)16 as reverse transcriptase primer in a total volume of 20 µl. 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. Upstream and downstream PCR primer (0.1 nmol each) and other components for PCR were then added to a total volume of 100 µl (GeneAmp RNA PCR kit, PerkinElmer Life Sciences). PCR was performed on a MiniCycler 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 420-s extension at 72°C. PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide.

Expression and Purification of GST-Syntaxin Fusion Protein

GST-syntaxin fusion proteins were expressed in Escherichia coli BL 21 strains with the recombinant pGEX 4T2 and purified as reported previously (10).

Preparation for Syntaxin 1A Truncated and Chimeric Proteins

For truncated syntaxin 1A and syntaxin 1A fragments, rat syntaxin 1A cDNA [Gene Bank Identifier (GI):6665796] fragments were cloned into the plasmid pGEX-5x-1 EcoRI and XhoI cloning site. The primer pairs used in PCR amplification of the cDNA fragments were Syn F1 combined with Syn1–264R ({Delta}1), Syn 1–250R ({Delta}2), Syn 1–235R ({Delta}3), Syn 1–220R ({Delta}4), Syn 1–205R ({Delta}5), Syn 1–190R ({Delta}6), and Syn 1–161R ({Delta}7), Syn F190/F230 combined with Syn1–264R (F8/F9), or Syn F190 with Syn 1–220R (F10). In making the chimeric molecule, XhoI-cut PCR products of primer pairs Syn F1/Syn1–161R, F190N/R264N (C190), or F220N/R264N (C220) were ligated by T4 DNA ligase. Ligated DNA fragments were subjected to EcoRI/NotI digestion and cloned into the corresponding sites of plasmid pGEX-5X-1. Recombinant vectors were transformed into E. coli BL 21 for expression and purification of GST-syntaxin 1A fragment fusion proteins as shown above.

The primer sequences were as follows: Syn F1: cac gaa ttc atg aag gac cga acc cag gag ctc; Syn F190: cac gaa ttc gcc ctc agt gag atc gag acc agg c; Syn F230: cac gaa ttc gac agg atc gag tac aat gtg gaa c; Syn1–264R: taa ctc gag ctt cct gcg tgc ctt gct ctg gta c; Syn1–250R: taa ctc gag gtc aga cac ggc cct ctc cac gta; Syn 1–235R: taa ctc gag gta ctc gat cct gtc aat cat ctc; Syn 1–220R: taa ctc gag ggc cat gtc cat gaa cat atc gtg t; Syn 1–205R: taa ctc gag caa ctt gat gat ctc act gtg cct; Syn1–190R: taa ctc gag ctg ctt cga gat gct gga gtc cat; Syn1–161R: taa ctc gag ggt cgt ggt ccg gcc agt gat ctc; F190N: aga ctc gag gcc ctc agt gag atc gag acc ag; F220N: aga ctc gag atg ctg gtg gag agc cag ggg ga; R264N: taa gcg gcc gcc ttc ctg cgt gcc ttg ctc tg.

All the primer DNA-oligo samples were synthesized by Integrated DNA Technologies. Constructs were sequenced to ensure that each construct was correct.

Cloning of Syntaxin 1A Chimeric Construct C220 into Pc DNA3.1/NT-GFP-TOPO for Expression of Green Fluorescent Protein-Syntaxin 1A Chimeric Protein

The primer pair 5'-atg aag gac cga acc cag gag-3' (upstream) and 5'-cta tgc ctt gct ctg gta ctt g-3' (downstream) was used in the PCR reaction to amplify the template of the previous recombinant construct C220. DNA fragments from PCR coding the chimerical SNARE N (aa 1–161) and residues 220–264 of syntaxin 1A were ligated into the pc DNA3.1/NT-GFP-TOPO vector (Invitrogen). The final clone of the recombinant chimeric syntaxin 1A vector was sequenced to ensure that the construct was correct.

Transfection and Selection of Stable Transfected Cell Lines

Purified recombinant plasmid pc DNA3.1/NT-GFP-syntaxin 1A C220 was used to transfect IMCD cells by calcium phosphate precipitation. Transfected cells were selected in medium containing 800 µg/ml G418. Surviving cells were further selected to yield monoclonal cell lines, using the method of limited dilution. Stable cell lines that expressed GFP-syntaxin 1A C220 protein were confirmed by directly viewing the green fluorescent protein (GFP) fluorescence, immunostaining, and immunoblotting with GFP antibody. Positive clones were used for immunoprecipitation experiments and functional assay.

In Vitro Pull-Down Binding Assay

GST-syntaxin isoform fusion proteins (25 µg) fixed on beads and 800 µg of kidney homogenate protein were incubated at 4°C overnight in HB buffer (20 mM HEPES, pH 7.2, 100 mM KCl, and 2 mM MgCl2, containing 1 mM DTT, 2 µg/ml pepstatin, 10 µg/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 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 2x SDS sampler buffer (final concentration: 50 mM Tris, pH 6.8, 100 mM DTT, 10% glycerin, 2% SDS, and 0.1 bromphenol blue), heated for 5 min at 95°C, and then subjected to SDS-PAGE and immunoblot analysis.

Immunoprecipitation

Cell homogenates [800 µg in 1 ml immunoprecipitation (IP) buffer] were precleared with 30 µl of suspended protein A beads plus 5 µl of normal rabbit serum for 1 h at 4°C. Precleared supernatants were incubated with primary rabbit antibody (1:200) for 2 h at 4°C, followed by addition of 30 µl of protein A beads, and incubated overnight at 4°C with gentle agitation. Bound proteins were washed three times with IP buffer, dissolved in 30 µl of 2x SDS sample buffer, and subjected to immunoblot analysis.

Immunoblots

After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween 20 and incubated overnight at 4°C with primary antibody. After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase, the blots were developed by the enhanced chemiluminescence method. The film was scanned and then subjected to band densitometry and quantification (NIH Image).

Antibodies used in this study are as follows: rabbit antibody to the 31-kDa subunit of the H+-ATPase (gift of Dr. Dennis Brown, Harvard University) and rabbit polyclonal antibody to the 116-kDa subunit (Synaptic Systems), rabbit antibody to SNAP23 (Synaptic Systems), mouse monoclonal antibody to synaptobrevin/VAMP (Synaptic Systems), and mouse monoclonal antibody to syntaxin-1A, clone HPC-1 (Sigma). The secondary antibodies for the immunoblot were peroxidase-conjugated goat anti-rabbit and anti-mouse IgG and were purchased from Jackson ImmunoResearch.

H+-ATPase-Mediated Proton Transport

The rate of Na+-independent intracellular pH (pHi) recovery (H+-ATPase-mediated H+ transport) was determined in monolayers of wild-type IMCD cells and transfected IMCD cells that express GFP-Syntaxin 1A C220 chimeric protein. Quiescent IMCD cells grown on glass coverslips were incubated for 1 h at 37°C in Na+-HEPES buffer (NHB) [in mM: 110 NaCl, 50 HEPES acid, 5 KCl, 1 MgCl2, 5 KH2PO4, 1 CaCl2, and 5 glucose (pH 7.2)] containing 10 µM BCECF-AM. The monolayer was washed three times with NHB and then suspended in 1 ml of NHB. Fluorescence intensity was measured in a PerkinElmer model LS 650-10 fluorospectrophotometer equipped with a thermostatically controlled (37°C) cuvette holder at excitation wavelengths of 505 and 455 nm with a slit width of 5 nm and an emission wavelength of 560 nm with a slit width of 10 nm. At the end of each experiment, the fluorescence intensity ratio was calibrated to pHi with potassium HEPES buffer containing 10 mg/ml nigericin. Na+-independent pHi recovery after a 20 mM NH4Cl-induced acid load when incubated in choline-HEPES buffer [in mM: 110 choline chloride, 50 HEPES acid, 5 KCl, 1 MgCl2, 5 KH2PO4, 1 CaCl2, 5 glucose, and 10 potassium acetate (pH 7.2)] was determined as previously described (2, 21).


    RESULTS
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 MATERIALS AND METHODS
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Expression of Syntaxin 1A in IMCD Cells

To demonstrate that the proton-secreting IMCD cells express the t-SNARE syntaxin 1, RT-PCR and Western blot analysis were performed. Positive PCR products with the expected size for syntaxin 1A (882 bp) and syntaxin 1B(984 bp) were obtained by using the IMCD cell mRNA as a template for RT and the appropriate primers for PCR amplification (Fig. 1). These data demonstrate that syntaxin 1 (1A and 1B) is expressed in the IMCD cell at the mRNA level and confirm our earlier observation that syntaxin 1A is expressed by this cell line (11).



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Fig. 1. RT-PCR amplification of syntaxin 1A and 1B mRNA from inner medullary collecting duct (IMCD) cells. Lane 1, DNA ladder; lane 2, template positive, syntaxin 1A; lane 3, template negative, syntaxin 1A; lane 4, template positive, syntaxin 1B; lane 5, template negative, syntaxin 1B.

 
Further evidence that syntaxin 1 is expressed by IMCD cells was documented by Western blot analysis (Fig. 2). Syntaxin 1A is primarily confined to a membrane fraction, as assessed by cell fractionation and Western blot analysis. It is readily detected in the membrane preparation as a 35-kDa band but is found in minimal amounts in the cytoplasmic fraction. This result is consistent with the physiological function of syntaxin 1A as a target t-SNARE on the plasma membrane. Similar results were obtained by RT-PCR and Western blot for the expression and localization of syntaxin in rat kidney (data not shown).



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Fig. 2. Western blot analysis of syntaxin 1A expression in IMCD cell preparation. Equal amounts of protein (80 µg/lane) from cell cytoplasm (Cyto), membrane (Mem), and postnuclear (Pnuc) preparation were subjected to Western blot analysis for syntaxin 1. Syntaxin 1A is expressed in the IMCD cell as a 35-kDa protein, predominantly in the membrane and absent in the cytoplasm preparation.

 
Specific Binding Site for H+-ATPase in Syntaxin 1A H3 Domain

Syntaxin 1A is the main t-SNARE on the presynaptic membrane of neurons. It mediates the targeting and fusion of synaptic vesicles and therefore transmitter release. The plasma membrane localization of syntaxin 1A in the IMCD cell suggests a role for this t-SNARE in mediating the fusion of the cellular vesicles such as H+-ATPase-coated vesicles (10). We showed previously that protein-protein interactions between syntaxin 1A and H+-ATPase occur in both in vitro and in vivo systems (10). It also appears that the H3 domain in syntaxin 1A is critical to this interaction (10).

The SNARE theory for synaptic fusion during exocytosis is based mainly on the fact that the SNARE core complex formation occurs when a t-SNARE pairs with a v-SNARE. Our previous in vitro studies (10) showed that although GST-syntaxin isoforms 1A, 1B, 2, 4, and 5 all form SNARE complexes with SNAP23 and VAMP, only syntaxin 1A, through its H3 domain, not only binds to SNAP23 and VAMP but also binds to H+-ATPase. From this observation we infer that syntaxin 1A encodes a specific binding site in its H3 domain for H+-ATPase. To explore this possibility, a series of truncation and chimeric experiments were performed.

Effect of truncation of H3 domain on binding. Syntaxins consist of three domains: SNARE N, SNARE binding (H3), and carboxy-terminal TMD. The TMD does not contribute to the binding of syntaxin 1A to either H+-ATPase or SNARE proteins (SNAP23 and VAMP) (Figs. 3 and 4). GST-syntaxin 1A 1–264 ({Delta}1, TMD-deleted construct) bound H+-ATPase and SNARE proteins to the same extent as GST-full-length syntaxin 1A (1A). Deleting the first 15 amino acids from the carboxy-terminal end of the H3 domain (construct {Delta}2: syntaxin 1A 1–250) did not impair the ability of syntaxin 1A to bind either H+-ATPase or SNARE proteins, but when residues 220–250, especially 235–250, were deleted ({Delta}3, {Delta}4), syntaxin 1A lost its ability to complex with H+-ATPase while it retained its ability to complex with SNAP23 and VAMP. Therefore, it is likely that residues 220–250 are the subdomain for H+-ATPase binding. Further deletion of the H3 domain ({Delta}5, {Delta}6, and {Delta}7) severely impaired the remaining ability to bind SNAP23 and VAMP, suggesting that residues 161–235, especially residues 190–220, are the segment responsible for SNAP23 and VAMP binding.



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Fig. 3. Specific binding of syntaxin 1A to H+-ATPase: effect of truncation of the H3 domain. Rat kidney homogenate was incubated with beads coated with glutathione S-transferase (GST), GST fusion syntaxin 1A, or GST fusion truncated syntaxin 1A missing the carboxy-terminal transmembrane and/or an indicated fragment of H3 soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) binding domain ({Delta}1–{Delta}7). After extensive washing of the beads with HB + 0.5% Triton X-100, the proteins bound on the beads were solubilized in SDS sample buffer and subjected to immunoblot analysis for H+-ATPase (116-kDa and 31-kDa subunits) and SNARE proteins [SNAP23 and vesicle-associated membrane polypeptide (VAMP)]. To document that equivalent amounts of GST or GST fusion proteins were added in the pull-down assay, gels were stained with Ponceau S red (top). Blots are representative of 4 consecutive experiments. GST, GST beads; 1A, GST-syntaxin 1A full length; {Delta}1, GST-syntaxin 1A 1–264; {Delta}2, GST-syntaxin 1A 1–250; {Delta}3, GST-syntaxin 1A 1–235; {Delta}4, GST-syntaxin 1A 1–220; {Delta}5, GST-syntaxin 1A 1–205; {Delta}6, GST-syntaxin 1A 1–190; {Delta}7, GST-syntaxin 1A 1–161.

 


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Fig. 4. Schematic illustration of syntaxin 1A constructs used in the pull-down assay and their relative binding capacities to H+-ATPase and SNARE proteins. aa1, aa161, aa264, and aa 288 indicate the amino acid (aa) positions in the syntaxin 1A molecule. SNARE N represents the amino-terminal part of the syntaxin 1A and includes aa 1–161; H3 represents the SNARE binding domain and includes aa 162–264; TMD represents the transmembrane domain and includes aa 264–288. Dashed lines in C190 and C220 constructs are the missing parts (162–190 in C190 and 162–220 in 220N) in the H3 domain of syntaxin. All of these constructs are expressed with a GST at the amino-terminal end of the fusion protein and were coupled to beads when used in the pull-down assay. Relative binding is expressed as the amount bound relative to that bound by the full-length construct (set to 100), determined by densitometric analysis (NIH Image) of the bands of 4 separate studies.

 
Binding of H+-ATPase, SNAP23, or VAMP by H3 fragments. If residues 220–250 in syntaxin 1A encode the site for H+-ATPase binding and residues 190–220 the site for SNAP23, then these isolated fragments fused directly to GST (F8, F9, and F10) should be able to bind H+-ATPase, SNAP23, or both. Unexpectedly, all of the three fragments used in the experiments did not pull down H+-ATPase, whether it included the putative encoded binding site for H+-ATPase (F8 and F9) or not (F10) (Figs. 4 and 5), but when the constructs contained the putative SNARE protein binding segments (F8 and F10), they complexed with SNAP23 and VAMP (Fig. 5; see Fig. 7). However, another construct, F9, lacking the 190–220 SNARE protein binding site, did not pull down SNAP23 and VAMP, whereas the F10 construct, which does have this site, did, further confirming the results in the truncation experiments that residues 190–220 are the site for SNAP23 binding to syntaxin 1A.



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Fig. 5. Protein binding by H3 fragments. Rat kidney homogenate was incubated with beads coated with GST-fused syntaxin 1A H3 domain fragments (F8-AA190–264, F9-AA230–264, and F10-AA190–220). After extensive washing of the beads with HB + 0.5% Triton X-100, the proteins bound on the beads were solubilized in SDS sample buffer and subjected to immunoblot analysis for H+-ATPase (116-kDa subunit a and 31-kDa subunit E) and SNARE proteins (SNAP23 and VAMP). Blot is representative of 4 individual studies.

 


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Fig. 7. Expression of GFP-syntaxin 1A C220 in transfected IMCD cells. A: immunoblot analysis: homogenates of the GFP-syntaxin 1A C220 chimera-transfected IMCD cells were separated on SDS-PAGE and transferred onto nitrocellulose membrane for immunoblotting with anti-green fluorescent protein (GFP) antibody. The expressed GFP-syntaxin 1A C220 was detected as a 53-kDa band by the anti-GFP antibody. B: immunohistochemistry: GFP-syntaxin 1A C220 chimera-transfected IMCD cells grown on glass coverslips were fixed and incubated with rabbit anti-GFP IgG followed by a Cy3-conjugated goat anti-rabbit secondary antibody. The chimeric construct of syntaxin 1A C220 showed a cytosolic distribution in transfected cells.

 
Binding of H+-ATPase, SNAP23, or VAMP by H3 chimera fragments. To perform a specific function, proteins need to have not only the correct sequence of amino acids (primary structure) but also the right conformation (secondary and tertiary structure). Syntaxin 1A may need its SNARE N domain, which is lacking in the F8 and F9 constructs, to provide the appropriate conformation or space for pairing with H+-ATPase. To test this hypothesis, we ligated the SNARE N domain to the F8 and F9 fragments to make chimera SNARE N-F8 or -F9 proteins. Ligation of SNARE N to F8 and F9 restored the ability of these two H3 fragments (C190 and C220) to bind H+-ATPase in the pull-down assay (Figs. 4 and 6).



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Fig. 6. Protein binding by H3 chimera fragments. Rat kidney homogenate was incubated with beads coated with GST-fused syntaxin 1A H3 domain fragments and SNARE N chimera (C190-SNARE N-190–264 and C220-SNARE N-220–264). After extensive washing of the beads with HB + 0.5% Triton X-100, the proteins bound on the beads were solubilized in SDS sample buffer and subjected to immunoblot analysis for H+-ATPase (116-kDa subunit a and 31-kDa subunit E) and SNARE proteins (SNAP23 and VAMP). Blot is representative of 4 individual studies.

 
Expression of H+-ATPase binding H3 chimera fragment in IMCD cells. To demonstrate that the carboxy-terminal half of the H3 domain of syntaxin 1A is critical for the binding and regulation of exocytosis of H+-ATPase, IMCD cells were transfected with a construct that consisted of syntaxin 1A C220 chimera in which the SNARE N domain (residues 1–161) and the carboxy-terminal half of the H3 domain (residues 220–264) were ligated and then fused with GFP to express a GFP fusion protein. DNA sequencing and BLAST evaluation of the recombinant plasmid showed it was a correct, in-frame construct. Transfection of the recombinant vector into the IMCD cells and clone selection yielded a stable cell line with a high level of GFP fusion protein expression as shown by immunoblotting (Fig. 7A). The GFP-syntaxin 1A C220 chimera (mass of 52–53 kDa) was identified with a GFP antibody. Immunohistochemistry revealed that the expressed GFP-syntaxin 1A chimera had a cytosolic distribution in the IMCD cells (Fig. 7B).

To document in vivo that the syntaxin 1A H3 subdomain binds the H+-ATPase we evaluated our ability to coimmunoprecipitate the H+-ATPase along with the H3 chimera from homogenate derived from cells that were transfected to express this fusion protein. With GFP antibody to immunoprecipitate the GFP-syntaxin 1A C220 chimera, the 31-kDa subunit of H+-ATPase was detected by immunoblot in the immunoprecipitate. This observation indicates that protein-protein interaction takes place between GFP-syntaxin 1A C220 and H+-ATPase in cells (Fig. 8A).



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Fig. 8. Interaction of syntaxin 1A C220 with H+-ATPase in vivo. A: immunoprecipitation: homogenates of GFP-syntaxin 1A C220-transfected IMCD cells were immunoprecipitated by a rabbit anti-GFP antibody (GFP Ab) and normal rabbit serum (NRS) as negative control. Immunoprecipitates were analyzed by immunoblotting for the 31-kDa subunit of H+-ATPase. A positive band of 31 kDa was detected in the precipitate of the GFP antibody. B: comparison of Na+-independent and -dependent intracellular pH (pHi) recovery in GFP-syntaxin 1A C220-transfected and -nontransfected normal IMCD cell lines. Values are mean ± SE load (n = 4). {Delta}pHi/min, pHi recovery rate after a NH4Cl acid load.

 
Presumably the H+-ATPase binding site within the H3 domain of intact syntaxin 1A functions to target the proton pump to the apical membrane. If this proposal is correct. then the expression of this non-membrane-bound chimera, which contains the putative H+-ATPase binding domain, should interfere with the normal trafficking of the H+-ATPase to the apical plasma membrane and the ability of these cells to transport H+. To evaluate the functional effect of expression of this chimera we determined the rate of Na+-independent and -dependent pHi recoveries after an acute acid load in transfected and nontransfected IMCD cells. Although the rate of Na+-dependent pHi recovery remained unchanged in both cell lines, the Na+-independent (H+-ATPase mediated) rate was inhibited by 75% in GFP-syntaxin 1A C220 chimera-transfected cells to a level of 0.011 ± 0.006 {Delta}pHi/min, compared with 0.040 ± 0.013 {Delta}pHi/min in normal IMCD cells (Fig. 8B).


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
Crystallography of the synaptic fusion complex has revealed that the four twisted and parallel helix bundles are made from a conserved polar interaction between an arginine residue (Arg56) from synaptobrevin (VAMP) and three glutamine residues, one from syntaxin (Gln226) and two from SNAP25 (Gln53 and Gln174) (6, 23). Competitive inhibition experiments using the peptides that span the syntaxin H3 domain also identified a minimal binding domain (residues 189–220) for syntaxin binding to SNAP25 (26). Our results demonstrating that the cassette in syntaxin 1A for binding SNAP23 covers residues 190–235 are consistent with reports that the SNAP23 and VAMP binding domain in syntaxin 1A is located in the amino-terminal half of the H3 domain (26). We have also uncovered another domain in the H3 segment of syntaxin 1A based on the observation that deletion of amino acids 235–250 from syntaxin 1A diminished its ability to complex with H+-ATPase and the chimeric constructs of H3 missing only residues 161–199 coupled with H+-ATPase in in vitro pull-down assays. When this construct is expressed in IMCD cells it not only interacts with the H+-ATPase but inhibits activation of H+ secretion. This latter observation is in concurrence with a prior observation that the expression of a similar fusion protein that contained the entire H3 domain of syntaxin 1A inhibited trafficking of the H+-ATPase to the apical membrane but the expression of the H3 domain of syntaxin 4 does not (10).This additional domain within the H3 segment of syntaxin 1A is an H+-ATPase binding domain. Thus the syntaxin 1A H3 domain can be divided into at least two parts: the carboxyl-terminal TMD adjacent half (residues 235–250), an H+-ATPase binding domain, and the amino-terminal half (next to SNARE N), a SNARE binding domain.

Our previous observation that many syntaxin isoforms can form a SNARE core complex but only syntaxin 1 interacts with H+-ATPase suggests that the SNARE binding domain is conserved throughout the syntaxin family whereas the H+-ATPase binding domain is specific for a single isoform, syntaxin 1. This assumption was examined by performing a BLAST sequence analysis of the NCBI protein data base with syntaxin 1A residues 190–220, the SNARE binding domain, and with residues 235–250, the H+-ATPase binding domain. The peptide sequence of the SNARE binding domain of syntaxin 1A had homology with H3 domain sequences in syntaxin 1B (96%), 2 (93%), 3 (93%), 4 (80%), and 11 (86%). On the other hand, the peptide sequence of the H+-ATPase binding domain of syntaxin 1A had homology with only syntaxin 1B. Thus the H+-ATPase binding domain is a unique sequence in syntaxin 1.

It was reported that the amino-terminal domain of syntaxin (SNARE N) has a negative regulatory function in the fusion of the phospholipid vesicles (17). Removal of the SNARE N domain of syntaxin accelerated the rate of vesicle fusion but did not affect the rate of v-/t-SNARE assembly (17). In our study, the fragment of syntaxin H3 SNARE binding domain (F8) bound SNAP23 and VAMP to an equivalent degree whether or not it was fused with the SNARE N domain (C190). This demonstrates that SNARE N has no effect on the rate of SNARE core complex formation. However, the H3 fragment encoding the H+-ATPase binding domain (F8 and F9) formed a complex with its partner (H+-ATPase) only when this fragment was ligated to the SNARE N (C220 or C190). These results suggest that SNARE N might play a critical role in facilitating the syntaxin 1A-H+-ATPase interaction. The exact mechanism requires further study.

SNARE proteins may confer the specificity of vesicle targeting, docking, and tethering as well as fusion. However, more recent functional data favor a role only in fusion (7). The interaction among SNARE proteins to form a SNARE complex appears to be critical for synaptic fusion. However, a novel binding partner of the syntaxin family, taxilin, has been recently implicated in intracellular vesicle traffic. Although its exact binding property for the syntaxin family is uncertain, it has been shown that this protein interacts with syntaxin family members without forming the SNARE complex (16). This binding pattern may provide a new way for syntaxin to play a role in the specificity of vesicle targeting and docking. The fact that syntaxin 1A, through a domain not implicated in SNARE binding, interacts with H+-ATPase may be one of the examples in which interactions between cargo protein of the vesicle (H+-ATPase) and the target SNARE protein provide the information for specificity of targeting and/or docking. We propose that the binding of syntaxin 1A to H+-ATPase pilots the H+-ATPase-laden vesicles to the apical membrane in IMCD cells. After targeting of the vesicles, subsequent cascades may take place to form a SNARE complex, leading to the fusion between the apical membrane and the vesicle membrane resulting in the insertion of H+-ATPase into the apical membrane.

Syntaxin 1A was initially believed to be involved only in neurotransmitter release at synaptic clefts in the central nervous system (5). In recent years, evidence has accumulated documenting that syntaxin 1A also plays various roles in a variety of cells in other organs. It interacts with the cystic fibrosis transmembrane conductance regulator Cl channel, inhibiting its activity by means of hydrophilic interactions that are distinct from the hydrophobic interactions that mediate SNARE complex formation (8, 1214). Syntaxin 1A was also reported to increase the net externalization of the epithelial Na+ channel (ENaC) complex through the SNARE machinery (19, 20) and to interact with the LD subtype of voltage-gated Ca2+ channels in pancreatic {beta}-cells (25). These data, along with our previous results that indicate that the exocytic insertion of H+-ATPase into the plasma membrane involves SNARE fusion machinery (3, 4, 10, 15) and the finding in this study that syntaxin 1A interacts with H+-ATPase in an isoform-specific manner, indicate that syntaxin 1A has wide diversity in both tissue distribution and function.

In IMCD cells, the interaction between syntaxin 1A and H+-ATPase involves both non-SNARE and SNARE proteins to mediate and regulate the insertion of the proton pump into the apical plasma membrane of these cells. The initial coupling between syntaxin 1A and H+-ATPase through the specific H+-ATPase binding domain takes place by a non-SNARE mechanism, whereas subsequent events (fusion) may involve the formation of SNARE core complex between t-SNARE, syntaxin 1A, and SNAP23 in the plasma membrane and v-SNARE and VAMP from the H+-ATPase-containing vesicles. Thus syntaxin 1A subserves a dual role in the process of H+-ATPase apical membrane amplification: targeting of the H+-ATPase to the apical membrane via a specific cassette in the H3 domain and fusion via a second H3 cassette that mediates SNARE interaction.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59529.


    ACKNOWLEDGMENTS
 
We appreciate the gift of rabbit antibody to the 31-kDa subunit of H+-ATPase from Dr. Dennis Brown.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Schwartz, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118-2908 (e-mail: jhsch{at}bu.edu)

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. Section 1734 solely to indicate this fact.


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