Syntaxin 7 Complexes with Mouse Vps10p Tail Interactor 1b, Syntaxin 6, Vesicle-associated Membrane Protein (VAMP)8, and VAMP7 in B16 Melanoma Cells
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m010838200
ISSN1083-351X
AutoresNicholas M. Wade, Nia J. Bryant, Lisa Connolly, Richard J. Simpson, J. Paul Luzio, Robert C. Piper, David E. James,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoSyntaxin 7 is a mammalian target solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), α-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis. Syntaxin 7 is a mammalian target solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), α-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis. solubleN-ethylmaleimide-sensitive factor attachment protein receptor(s) target vesicle vesicle-associated membrane protein soluble N-ethylmaleimide-sensitive factor attachment protein synaptosomal-associated protein of 25 kDa suppressor of the erd2 deletion mutant Bet one suppressor secretion blocked early in transport vacuolar morphology Vps10p tail interactor new yeast v-SNARE glutathione S-transferase Syntaxin early endosomal antigen 1 polyacrylamide gel electrophoresis green fluorescent protein enhanced green fluorescent protein high performance liquid chromatography mass spectrometry tandem mass spectrometry t-SNARE of the late Golgi peptidase-deficient gene mouse-Vti1 In eukaryotic cells, proteins are transported between intracellular organelles by a series of membrane transport steps. The ability of discrete organelles to fuse in a highly specific way is central to all membrane-trafficking events and relies on a series of molecular events. One event is the formation of a protein complex between sets of molecules found within the transport vesicle (v-SNAREs)1 and the target membrane (t-SNAREs). Much of the work that has led to the formulation of this hypothesis has been performed in the mammalian synapse (1Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar). Here the R- or v-SNARE, VAMP2, forms a complex with two Q- or t-SNARE proteins, Syntaxin 1a and SNAP25. This ternary complex consists of a four-α-helical bundle containing one helix from both Syntaxin 1a and VAMP2 with the remaining two helices being contributed by SNAP25 (2Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar). SNARE complexes that regulate traffic to the cell surface in both mammalian and yeast cells contain three distinct proteins, whereas most intracellular SNARE complexes appear to be comprised of four separate proteins: one v-SNARE and three t-SNAREs (3McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (534) Google Scholar, 4Fukuda R. McNew J.A. Weber T. Parlati F. Engel T. Nickel W. Rothman J.E. Sollner T.H. Nature. 2000; 407: 198-202Crossref PubMed Scopus (193) Google Scholar). For example, inSaccharomyces cerevisiae endoplasmic reticulum to Golgi transport is regulated by the Sed5p·Bos1p·Sec22p·Bet1p complex, whereas vacuolar transport is regulated by a complex comprising Vam3p·Vam7p·Vti1p·Nyv1p (3McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (534) Google Scholar). The Syntaxin isoform, or t-SNARE heavy chain (3McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (534) Google Scholar), associated with each complex appears to be highly specific to a particular vesicle transport step, whereas the light chain t-SNAREs associate with multiple complexes. Vti1p, for example, interacts with Syntaxin homologs involved in Golgi/endosome, intra-Golgi, vacuolar, and prevacuolar transport steps (5Fischer von Mollard G. Stevens T.H. Mol. Biol. Cell. 1999; 10: 1719-1732Crossref PubMed Scopus (135) Google Scholar). Sequencing projects are revealing the existence of an ever increasing number of SNARE family members (6Bock J.B. Scheller R.H. Nature. 1997; 387: 133-135Crossref PubMed Scopus (91) Google Scholar). In the yeast S. cerevisiae there are eight different Syntaxin-like proteins, and in mammalian cells, at least 20 have been identified to date (7Pelham H.R. Exp. Cell Res. 1999; 247: 1-8Crossref PubMed Scopus (133) Google Scholar, 8Weimbs T. Mostov K. Low S.H. Hofmann K. Trends Cell Biol. 1998; 8: 260-262Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). A major challenge is to establish which transport steps each of these proteins regulates. This will require establishing the intracellular location of these proteins as well as their binding partners. Syntaxins 1, 2, 3, and 4 localize to the plasma membrane (9Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (591) Google Scholar); Syntaxin 5 operates between the endoplasmic reticulum and the Golgi apparatus; and Syntaxins 6, 7, 8, 13, and 16 are found in the endosomal system of mammalian cells (10Tang B.L. Low D.Y. Lee S.S. Tan A.E. Hong W. Biochem. Biophys. Res. Commun. 1998; 242: 673-679Crossref PubMed Scopus (51) Google Scholar, 11Subramaniam V.N. Loh E. Horstmann H. Haberman A. Xu Y. Coe J. Griffiths G. Hong W. J. Cell Sci. 2000; 113: 997-1008Crossref PubMed Google Scholar, 12Prekeris R. Klumperman J. Chen Y.A. Scheller R.H. J. Cell Biol. 1998; 143: 957-971Crossref PubMed Scopus (236) Google Scholar, 13Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar, 14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar). The present study focuses on Syntaxin 7. Syntaxin 7 is localized to compartments within the endosomal system where it regulates transport between the late endosome and the lysosome (14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar, 15Nakamura N. Yamamoto A. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Ward D.M. Pevsner J. Scullion M.A. Vaughn M. Kaplan J. Mol. Biol. Cell. 2000; 11: 2327-2333Crossref PubMed Scopus (112) Google Scholar). We have reported previously that Syntaxin 7 interacts with at least one v-SNARE, VAMP8 (14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar). Recent analysis of VAMP8 has demonstrated a requirement for this v-SNARE in the homotypic fusion of both early endosomes and late endosomes (17Antonin W. Holroyd C. Tikkanen R. Honing S. Jahn R. Mol. Biol. Cell. 2000; 11: 3289-3298Crossref PubMed Scopus (127) Google Scholar). Another v-SNARE, VAMP7, has also been implicated in late endosomal transport (16Ward D.M. Pevsner J. Scullion M.A. Vaughn M. Kaplan J. Mol. Biol. Cell. 2000; 11: 2327-2333Crossref PubMed Scopus (112) Google Scholar, 18Advani R.J. Yang B. Prekeris R. Lee K.C. Klumperman J. Scheller R.H. J. Cell Biol. 1999; 146: 765-776Crossref PubMed Scopus (156) Google Scholar), but its relationship to Syntaxin 7 has not yet been explored. This highlights the need for a more thorough characterizaton of Syntaxin 7 binding partners. The aim of the present study was to identify Syntaxin 7-binding proteins using a biochemical approach. To this end, we took advantage of the fact that Syntaxin 7 is expressed at very high levels in the melanoma cell line, B16. Using B16 cells as a source of Syntaxin 7, we used an immunoprecipitation approach combined with mass spectrometry to identify mVti1b, Syntaxin 6, and α-SNAP as Syntaxin 7-binding proteins. In addition, we find that Syntaxin 7 forms a complex with both VAMP7 and VAMP8, suggesting that the t-SNARE Syntaxin 7 may regulate distinct fusion events within the endocytic pathway by associating with distinct subsets of partner SNARE proteins. B16 Melanoma cells were kindly provided by Dr. Peter Parsons (QIMR, Brisbane, Australia) and were cultured in RPMI medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.). For immunoblot analysis, total membranes were prepared as follows. Cells were lysed by passage through a 27-gauge needle in HES buffer (20 mmHepes, 0.5 mm EDTA, 250 mm sucrose) containing protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 250 μm phenylmethylsulfonyl fluoride) before centrifugation at 150,000 × g for 30 min. The membrane pellet was resuspended in HES buffer containing 1% Triton X-100 and incubated on ice for 1 h, after which insoluble material was removed by centrifugation at 17,500 × g for 10 min. Triton X-100-soluble extracts of rat tissues, obtained from male Wistar rats, were prepared by homogenization of the relevant tissues in HES buffer containing protease inhibitors. Homogenates were incubated for 30 min at 4 °C in the presence of 1% Triton X-100, after which insoluble material was removed by centrifugation at 17,500 ×g for 10 min. Polyclonal antiserum against mouse Syntaxin 7 was obtained by immunizing rabbits with a chimeric protein consisting of the cytosolic domain of Syntaxin 7 fused to GST (pGST-Syn7a) (14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar). Antibodies raised against the GST portion of the antigen were removed by passing the antiserum over a column of GST linked to Affi-Gel (Bio-Rad). The flow-through from this column was subsequently passed over a second column made by coupling the GST-Syn7antigen to Affi-Gel. Syntaxin 7-specific antibodies were eluted from this column with 100 mm glycine, 150 mm NaCl (pH 2.8). These affinity-purified antibodies were then titrated to pH 7.5 using Tris-HCl (pH 8). The VAMP8 antibodies have been described previously (14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar). Antibodies to VAMP7 were raised against GST fusion protein containing the entire cytosolic portion of VAMP7, which was expressed from pPL815. Similarly, antibodies against Syntaxin 6 were raised by immunizing rabbits with a GST protein containing the entire cytosolic tail of this protein (pGST-Syn6; 14) or were purchased from Transduction Laboratories. A plasmid encoding the cytosolic tail of human Syntaxin 13 fused to GST and an antibody produced against this fusion protein were kindly provided by Dr. R. Teasdale (Institute for Molecular Bioscience, University of Queensland). The EEA1 antibody was the generous gift of Dr. Ban Hock Toh (Monash Medical School, University of Melbourne, Australia). Antibodies against mVti1b (19Antonin W. Riedel D. von Mollard G.F. J. Neurosci. 2000; 20: 5724-5732Crossref PubMed Google Scholar) were kindly provided by Dr. G. Fischer von Mollard (University of Gottingen, Germany). Antibodies against cytosolic domains of Syntaxin 4 and VAMP3 have been described previously (20Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Membrane samples were subjected to SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. Membranes were blocked in 1% (w/v) non-fat dried milk and then incubated with primary antibodies in phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 1% (w/v) non-fat dried milk for 1 h at room temperature at dilutions that were optimized for each antibody. Visualization of antibody-labeled bands was achieved with the use of horseradish peroxidase-labeled secondary antibodies purchased from Amersham Pharmacia Biotech, and Supersignal Dura chemiluminescent substrate was from Pierce. Immunoblotting competition assays were performed by including the relevant GST fusion protein antigen (100 μg/ml) during incubation with the primary antibody. The GFP-Syn6 plasmid that encodes the full-length murine Syntaxin 6 fused to the C terminus of enhanced GFP (eGFP) was a kind gift from Dr. Jeffrey Pessin (University of Iowa) (21Watson R.T. Pessin J.E. J. Biol. Chem. 2000; 275: 1261-1268Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cells were grown to 70% confluence on glass coverslips and fixed in 2% paraformaldehyde for 30 min. In some cases B16 cells were transiently transfected with the GFP-Syntaxin 6 vector using the LipofectAMINE reagent according to the manufacturer's instructions (Life Technologies, Inc.). This plasmid generated a protein of the expected molecular mass (∼60 kDa) which could be specifically immunoblotted with antibodies specific for either Syntaxin 6 or GFP (data not shown). After fixation, cells were quenched for 5 min in 150 mm glycine, washed in phosphate-buffered saline, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 15 min. After blocking coverslips with 2.5% normal swine serum for 30 min at room temperature primary antibody incubations were performed in 1% normal swine serum in phosphate-buffered saline for 1 h at room temperature, followed by an appropriate fluorescein isothiocyanate- or Texas Red-labeled secondary antibody (Molecular Probes, Eugene, OR) for 30 min at room temperature. Cells were viewed using a 63×/1.4 Ziess oil immersion objective on a Zeiss Axiovert fluorescence microscope, equipped with a Bio-Rad MRC-600 laser confocal imaging system. Images were imported into Adobe Photoshop™ (Adobe Systems Inc.) and assembled as indicated. Affinity matrices were prepared by first binding antibodies (affinity-purified anti-Syn7 antibodies with or without an excess of GST-Syn7 fusion protein and purified rabbit IgG antibodies) to protein A-Sepharose beads (Amersham Pharmacia Biotech). Antibodies were covalently cross-linked to protein A beads using dimethyl pimelimidate (Pierce). Triton X-100-soluble membrane extracts prepared from B16 cells, which had been cultured for 7 days, were prepared as outlined above and incubated with antibody-coated beads for 1 h in the presence of 150 mm NaCl, 20 mm Hepes, and 0.5 mm EDTA. The beads were washed in the same buffer containing 1% Nonidet P-40. Elution of bound proteins was achieved by boiling for 10 min in Laemmli sample buffer containing 4% SDS. Proteins were separated on a 5–15% SDS-polyacrylamide gradient gel and visualized by staining with either silver or Coomassie Brilliant Blue. Individual protein bands were excised and subjected to sequence analysis. In-gel proteolytic digestion (using 0.5 μg of trypsin) of resolved proteins was performed as described previously (22Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). An electrospray ionization ion trap mass spectrometer (LCQ Finnigan MAT, San Jose, CA) coupled on-line with a capillary HPLC (Hewlett-Packard model 1090A) modified for capillary chromatography was used for peptide sequencing. The column used in this study was a 150 × 0.20-mm (inner diameter) capillary column (Brownlee RP-300, 7 μm C8) manufactured using a polyvinylidene difluoride end frit A 60-min linear gradient (flow rate 1.7 μl/min) was used from 0–100% B, where solvent A was 0.1% v/v aqueous trifluoroacetic acid, and solvent B was 0.1% aqueous trifluoroacetic acid in 60% acetonitrile. The electrospray ionization parameters were as follows: spray voltage, 4.5 kV; sheath gas and auxiliary gas flow rates, 5 and 30 (arbitrary value), respectively; capillary temperature, 150 °C; capillary voltage, 20 V; and tube lens offset, 16 V. The sheath liquid used was 2-methoxyethanol (99.9% HPLC grade) delivered at a flow rate of 3 μl/min. The electron multiplier was set to −860 V. In both MS and MS/MS modes, the trap was allowed a maximum injection time of up to 200 ms. The automatic gain control parameter was turned on for all experiments, ensuring that the number of ions in the trap was automatically kept to a constant preset value. The range scanned in MS mode was 350–2,000 kDa, and in MS/MS the range varied according to the mass of the ion selected for MS/MS. After acquiring one scan in MS, the most intense ion in that spectrum above a threshold of 1 × 105 was isolated for subsequent zoom scan (to determine charge state), then collision-induced dissociation or MS/MS in the following scans. The dissociation energy for MS/MS was set to 55%. All spectra were recorded in centroid mode. The sequences of individual peptides were identified using the SEQUEST algorithm (incorporated into the Finnigan Xcalibur-Biomass software) (23Yates J.R.D. Eng J.K. McCormack A.L. Schieltz D. Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1109) Google Scholar). Spectra not identified by SEQUEST were interpreted manually using de novo methods. A Syntaxin 7-specific antibody was raised against a bacterial fusion protein containing the cytosolic tail (residues 1–234) of Syntaxin 7 fused to GST. Antibodies that specifically recognize the Syntaxin 7 portion of this fusion protein were affinity purified as described under "Materials and Methods." This antibody recognizes one band of molecular mass 40 kDa (Fig. 1 A, lane 1). Immunolabeling of this band was completely inhibited in the presence of an excess of the GST-Syn7 antigen (lane 2). In contrast, a fusion protein consisting of the analogous region of Syntaxin 13 fused to GST had no significant effect on the recognition of this band (lane 3). In addition, the mobility of the polypeptide recognized by the Syntaxin 7 antibody was significantly different from the bands labeled with antibodies specific for either Syntaxin 13 or Syntaxin 6 (Fig. 1 A, lanes 4 and 5). To validate further the specificity of the Syntaxin 7 antibody we performed immunofluorescence microscopy using B16 melanoma cells. The antibody labeled a tubulovesicular compartment that was concentrated in the perinuclear region of the cell (Fig. 1 B). This labeling was not significantly different upon inclusion of excess GST-Syn13 fusion protein. However, in the presence of excess GST-Syn7 the immunolabeling was almost completely absent, consistent with the immunoblotting data (Fig. 1 A). Collectively, these data show that our Syntaxin 7 antibody is specific and does not recognize other closely related Syntaxin isoforms. We have shown previously that Syntaxin 7 is widely expressed in rodent tissues and that it regulates a membrane transport step within the late endosome/lysosomal system of mammalian cells (14Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (127) Google Scholar). In view of this function we reasoned that cell types that are specialized for late endosomal/lysosomal biogenesis might up-regulate the machinery that is involved in these transport steps. Melanocytes are a specialized cell type whose primary function is the biogenesis of melanosomes, which represent lysosome-like organelles (24Dell'Angelica E.C. Mullins C. Caplan S. Bonifacino J.S. FASEB J. 2000; 14: 1265-1278Crossref PubMed Google Scholar). Consistent with this notion is our finding that the levels of the Syntaxin 7 protein in the B16 melanosome cell line is ∼10-fold higher than in all other tissues and cell lines tested (Fig. 2). In contrast, the level of Syntaxin 4, which localizes to the plasma membrane, was much more uniform across all tissues and cell types, including B16 cells. A similar uniformity was observed for Syntaxin 6 (Fig. 2) and Syntaxin 13 (data not shown) across these tissues and cell types. Intriguingly, VAMP8, a v-SNARE that has also been implicated in late endosomal transport (17Antonin W. Holroyd C. Tikkanen R. Honing S. Jahn R. Mol. Biol. Cell. 2000; 11: 3289-3298Crossref PubMed Scopus (127) Google Scholar), is also expressed at very high levels in B16 cells compared with other tissues and cell lines (Fig. 2). The high levels of Syntaxin 7 which we observed in B16 cells gave us a good opportunity to find interacting proteins using a coimmunoprecipitation approach. The Syntaxin 7-specific antibody (Fig. 1) was covalently linked to protein A-Sepharose, forming an affinity matrix. This was used to immunoprecipitate Syntaxin 7-containing protein complexes from a B16 cell extract. Using a non-ionic detergent solubilized extract of B16 cells we were able to immunoprecipitate Syntaxin 7 with high efficiency and specificity (Fig. 3). Initially, to identify proteins that interact with Syntaxin 7, immunoprecipitates were subjected to SDS-PAGE followed by silver staining (Fig. 3 B). We were able to detect a number of proteins that coprecipitated with Syntaxin 7 under these experimental conditions (see first and fourth lanes). Most of these protein bands could not be detected when the immunoprecipitation was performed using a control IgG (second lane) or when the incubation with the Syntaxin 7 antibody was performed in the presence of excess recombinant GST-Syn7 (fifth lane). The most prominent bands detected were of average molecular mass 230, 130, 120, 90, 35, and 30 kDa. To identify these proteins we increased the scale of the immunoprecipitation procedure so that bands could be visualized by staining with Coomassie Brilliant Blue (see Fig.3 C). These bands were excised, and tryptic peptide products were sequenced using electrospray mass spectrometry. The sequences obtained are shown in Table I. Several of the bands yielded very clear sequence data and gave positive identities when these sequences were used for BLAST searching of the protein data base. The doublet at 130/120 kDa contained sequences corresponding to the protein phosphatase 110-kDa regulatory subunit (25Chen Y.H. Chen M.X. Alessi D.R. Campbell D.G. Shanahan C. Cohen P. Cohen P.T. FEBS Lett. 1994; 356: 51-55Crossref PubMed Scopus (128) Google Scholar) and a tyrosine kinase associated protein known as BAP-135 (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar). In an effort to verify the presence of BAP-135 in the Syntaxin 7 immunoprecipitate B16 cells were incubated with pervanadate because this causes a marked stimulation of BAP-135 tyrosine phosphorylation in B cells (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar). However, immunoblotting of the Syntaxin 7 immunoprecipitate, obtained from these cells, using a phosphotyrosine antibody failed to reveal any detectable product corresponding to BAP-135 (data not shown). Of particular interest, we were able to detect a number of peptide sequences in the Syntaxin 7 immunoprecipitate corresponding to Syntaxin 7, α-SNAP (27Whiteheart S.W. Griff I.C. Brunner M. Clary D.O. Mayer T. Buhrow S.A. Rothman J.E. Nature. 1993; 362: 353-355Crossref PubMed Scopus (225) Google Scholar), mVti-1b (GenBank NP058080), and VAMP8 (28Wong S.H. Zhang T. Xu Y. Subramaniam V.N. Griffiths G. Hong W. Mol. Biol. Cell. 1998; 9: 1549-1563Crossref PubMed Scopus (99) Google Scholar). The t-SNARE, Syntaxin 6, was also identified in the same band that contained α-SNAP, migrating with an average molecular mass of 32 kDa. The abundant band at 55 kDa corresponded to IgG. None of the other bands that were identified using this approach yielded any detectable peptide sequences.Table IPeptide sequences predicted from mass spectrometer analysisProteinSequenceMolecular masskDaPredictedMeasuredProtein phosphatase 1 M 110-kDa regulatory subunit, Rattus sp. aortaAQLHDTNmELTDLK109.6120YDSSSTSSSDRYDSLLGRRSTQGVTLTDLQEAEKVGQTAFDVADEDILGYLEELQKKBAP-135 homolog, Mus musculusTPTQTNGSNVPFKPR110.3115Syntaxin 7,M. musculusETDKYIKEFGSLPTTPSEQR29.835EKNLVSWESQTQPQVQVQDEEITEDDLRDRLVAEFTTSLTNFQKQLEADIMDINEIFKDLGMMIHEQGDmIDSIEANVESAEVHVQQANQQLSRITQcSVEIQREFGSLPTTPSEQRTLNQLGTPQDSPELRac-SYTPGIGGDSAQLAQRYIKEFGSLPTTPSEQRA-soluble SNAP, M. musculusAIAHYEQSADYYKGEESNSSANK33.933LLEAHEEQNVDSYTEAVKHDAATcFVDAGNAFKKADPQEAINcLMRAIDIYEQVGTSAmDSPLLKLLEAHEEQNVDSYTEAVKEYDSISRNSQSFFSGLFGGSSKLAVQKYEELFPAFSDSRHHISIAEIYETELVDVEKLDQWLTTMLLRSyntaxin 6, M. musculus (low level product)IGGELEEQAVmLDDFSHELESTQSR29.032Putative v-SNARE Vtilb, M. musculusEFGSLPTTPSEQR29.830TLNQLGTPQDSPELRLVAEFTTSLTNFQKQLEADImDINEIFKVAMP8, M. musculusNKTEDLEATSEHFK11.415Immunoprecipitated proteins visualized in Fig. 3 were subjected to MS analysis as described under "Materials and Methods." Sequence matches for the tryptic peptides obtained. Also shown is the predicted molecular mass of the complete matched protein and the apparent molecular mass by SDS-PAGE of the excised band from which those peptide matches were derived. Open table in a new tab Immunoprecipitated proteins visualized in Fig. 3 were subjected to MS analysis as described under "Materials and Methods." Sequence matches for the tryptic peptides obtained. Also shown is the predicted molecular mass of the complete matched protein and the apparent molecular mass by SDS-PAGE of the excised band from which those peptide matches were derived. To verify the identity of these proteins as Syntaxin 7-interacting partners we immunoblotted immunoprecipitates obtained from B16 cells using antibodies specific for a variety of these proteins. As shown in Fig. 4, Syntaxin 6, VAMP8, and mVti1b were all enriched in the Syntaxin 7 immunoprecipitate to almost the same extent as Syntaxin 7. None of these proteins was present when the immunoprecipitation was performed using preimmune serum-coated protein A beads (Fig. 4, second lane). The immunoprecipitation efficiency of Syntaxin 7 was ∼50% (Fig. 4, compare firstand third lanes). In the case of Syntaxin 6, VAMP8, and mVti1b ∼20%, 20, and 10% of each protein was coprecipitated with Syntaxin 7, indicating that each of these proteins was highly enriched in the Syntaxin 7 complex suggesting that they likely form stable SNARE complexes in vivo. It has been reported recently that another v-SNARE, VAMP7, can play a role in the delivery of epidermal growth factor to a degradative compartment, most likely the late endosomal/lysosomal system, where VAMP7 is localized (18Advani R.J. Yang B. Prekeris R. Lee K.C. Klumperman J. Scheller R.H. J. Cell Biol. 1999; 146: 765-776Crossref PubMed Scopus (156) Google Scholar). Although we did not detect this protein in the Syntaxin 7 immunoprecipitate by MS/MS we were able to detect coprecipitation of VAMP7 with Syntaxin 7 by immunoblotting (Fig. 4). VAMP7 was enriched in the Syntaxin 7 immunoprecipitate to the same extent as VAMP8 (Fig. 4). Although a significant proportion of VAMP7 was found in the Syntaxin 7 immunoprecipitate, our inability to detect it by MS/MS raised the possibility that it may be expressed at lower abundance than other proteins. We also immunoblotted these fractions with antibodies specific for Syntaxin 4 and VAMP3 to investigate the specificity of the immunoprecipitatio
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