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SNAP-29 Is a Promiscuous Syntaxin-Binding SNARE
Biochemical and Biophysical Research Communications 285, 167–171 (2001) doi:10.1006/bbrc.2001.5141, available online at http://www.idealibrary.com on
SNAP-29 Is a Promiscuous Syntaxin-Binding SNARE Anita C. Hohenstein and Paul A. Roche 1 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received May 29, 2001
SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways. Key Words: SNARE; SNAP-29; SNAP-23; protein transport.
The movement of membrane and protein in all eukaryotic cells is a highly regulated process. With very few exceptions, proteins traffic from compartment-tocompartment in cells by a process involving vesicle budding from a donor organelle, vesicle docking with an appropriate target membrane, and the vesicle fusion with the target membrane (1). The strict regulation of this process ensures that cargo is transferred from one compartment to another in an orderly fashion. While the players that regulate the specificity with which a specific vesicle docks and fuses with a particular target membrane are under intense investigation, it is clear that one class of proteins, termed SNAREs, play an important part in the process of membrane1 To whom correspondence should be addressed at NIH, Bldg. 10, Room 4B36, Bethesda, MD 20892. Fax: (301) 496-0887. E-mail: [email protected].
membrane fusion (2, 3). The SNARE complex that regulates synaptic vesicle fusion with presynaptic membranes exists as a four helical bundle of coiled-coils (4). In this macromolecular complex, one coil is derived from the vesicle-associated SNARE protein VAMP and three coils are derived from the target SNARE (tSNARE) complex. In neurons, the t-SNARE complex consists of the presynaptic plasma membrane proteins syntaxin (possessing one coiled-coil) and SNAP-25 (possessing two coiled-coils). It is likely that each intracellular organelle can serve as a target membrane for transport vesicles or secretory granules. In agreement with the idea that specific SNARE combinations regulate specific transport steps, up to 35 v-SNARE and t-SNARE isoform are thought to exist in H. sapiens (5), and in many cases these proteins reside on distinct intracellular organelles (2). Unlike the wide variety of VAMPs and syntaxins present in mammalian cells, only two ubiquitously expressed homologs of SNAP-25 have been identified thus far. One of these, SNAP-23, is the product of duplication of an ancestral SNAP-25 gene (6), has similar SNAREbinding domains as SNAP-25 (7), and functions in regulated exocytosis from non-neuronal secretory cells (7– 9). In agreement with the proposed role of SNAP-23 as a plasma membrane target SNARE for secretory vesicles, in vitro binding studies have shown that SNAP-23 binds well to plasma membrane syntaxins but not the syntaxins present on internal membranes (10, 11). In contrast with SNAP-23, there is conflicting data on the binding properties and function of SNAP-29 in protein transport. Unlike SNAP-23, SNAP-29 has been reported to be present on many different internal membranes and binds to a wide variety of syntaxin isoforms (11). These data suggest that SNAP-29 could participate in SNARE complex assembly in a variety of intracellular membrane fusion events. On the other hand, another report has shown that SNAP-29 is present in the Golgi apparatus and binds to Golgi syntaxins almost exclusively (12), suggesting a very specific role for SNAP-29 in the regulation of intra-Golgi traffic. In this study, we have examined the ability of SNAP-29 to bind to different syntaxin isoforms using a variety of binding assays. We find that unlike SNAP-
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23, SNAP-29 is a relatively promiscuous syntaxin binding protein. Although binary SNAP-29/syntaxin t-SNARE complexes are formed in vitro and in vivo, the presence of a vesicle SNARE (i.e., VAMP) dramatically augments the binding of SNAP-29 to both Golgi and non-Golgi syntaxins. Furthermore, SNAP-29 augments the ability of syntaxin to bind to VAMP, a process required for the formation of a ternary SNARE fusion complex. We conclude that SNAP-29 functions by participating in SNARE-dependent trafficking events between vesicles and the numerous intracellular target organelles in eukaryotic cells. MATERIALS AND METHODS Plasmids and antibodies. The pcDNA3-myc-SNAP-29, pRCCMV-FLAG-VAMP 2, and pCMV4-myc-VAMP 8 expression vectors were the kind gift of Richard Scheller (Stanford, University, CA). The cDNAs for full-length human syntaxin 4, human syntaxin 6, and human SNAP-23 were subcloned into pcDNA3 and the sequence was confirmed by automated sequence analysis. GST fusion protein vectors for syntaxin 4, syntaxin 6, syntaxin 7, and VAMP 2 have been described previously (7, 13, 14). A SNAP-29 antiserum was obtained by immunizing rabbits with a carboxyl-terminal human SNAP-29 peptide (amino acids 242–258) coupled to maleimide-activated KLH (Pierce, Rockford, IL) using standard protocols. The anti-SNAP-23 amino-terminus rabbit antisera have been described previously (15). Syntaxin 6 and syntaxin 4 monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). In vitro binding assays. Proteins were translated in the presence of [ 35S]methionine using the TNT Quick in vitro transcription and translation kit (Promega). GST-fusion proteins were obtained using standard purification protocols and were immobilized onto glutathione-Sepharose beads (Pharmacia) prior to use. In vitro translated material was incubated with 5 g of GST alone, GST-syntaxin 4, GST-syntaxin 7, and GST-syntaxin 6 fusion proteins on ice in a binding buffer of 150 mM NaCl, 20 mM Hepes (pH 7.0), 1 mM MgCl 2, 0.1% Triton X-100, 0.5 mg/ml BSA and protease inhibitors (16). After incubation for 1 h on ice, the beads were washed extensively and the bound material was analyzed by SDS–PAGE. Cell culture, immunoprecipitation, and immunoblotting. Subconfluent HeLa cells were maintained and transfected using the LipofectAmine Plus method as described previously (16). Immunoprecipitations from cell lysates were performed using antibodies immobilized on protein A-Sepharose beads as described previously (17). Immunoblot analysis of cell extracts and immunoprecipitates was performed using ECL detection (NEN/Dupont) as described previously (17).
RESULTS AND DISCUSSION SNAP-29 binding to syntaxin in vitro. There is conflicting data regarding the ability of SNAP-29 to bind to different syntaxins (11, 12). In an attempt to determine if SNAP-29 is able to bind syntaxins present on distinct internal membranes, we examined the ability of in vitro translated SNAP-29 to bind to a variety of GST-syntaxin fusion proteins that are representative of the diverse family of syntaxins present on cellular organelles. SNAP-29 bound equally well to syntaxins that reside on late endosomes (syntaxin 7), the plasma membrane (syntaxin 4), and in the Golgi apparatus/
FIG. 1. SNAP-29 binds to multiple syntaxins in vitro. Equivalent amounts of in vitro translated [ 35S]methionine-SNAP-29 and [ 35S]methionine-SNAP-23 were incubated with the indicated immobilized GST fusion proteins on ice. The amount of SNAP-29 and SNAP-23 bound to each fusion protein was analyzed by SDS–PAGE and fluorography. Gels were stained with Coomassie brilliant blue R-250 to confirm that equal amounts of GST fusion proteins were present in each incubation. An aliquot (1/20th) of the reaction mixture was also analyzed to confirm that equal amounts of radiolabeled SNAP-29 and SNAP-23 were present in the incubations.
TGN (syntaxin 6), although under these conditions less than 5% of the input SNAP-29 bound to each syntaxin (Fig. 1). The ability to bind to syntaxins present on many different internal compartments is unique to SNAP-29, as the ubiquitously expressed SNAP-25 homolog SNAP-23 bound very well to syntaxin 4 but did not bind to either syntaxin 7 or syntaxin 6. Additional binding studies revealed that SNAP-23 also bound well to GST fusion proteins of the plasma membrane syntaxins syntaxin 1A, syntaxin 2, and syntaxin 3 (Ref. 11 and ACH, unpublished observation). As these syntaxins are present primarily on the plasma membrane of mammalian cells (18), these data reveal that SNAP-23 forms binary t-SNARE complexes with plasma membrane syntaxins while SNAP-29 is able to form t-SNARE complexes with syntaxins present on many different internal membranes. SNAP-29 binding to syntaxins in vivo. To confirm that the results obtained in the in vitro binding assay agreed with t-SNARE interactions in vivo, we examined the interaction of SNAP-29 and syntaxin 4 or syntaxin 6 in HeLa cells. The endogenous expression of these proteins is very low in these cells, and for this reason we overexpressed the proteins by transient transfection. Analysis of SNAP-29 immunoprecipitates revealed that SNAP-29 bound to syntaxin 4 quite well in vivo, whereas its binding to syntaxin 6 was low but clearly detectable (Fig. 2A). In contrast, SNAP-23 bound syntaxin 4 efficiently but did not bind to syntaxin 6 in vivo (Fig. 2B). The failure of SNAP-23 to bind to syntaxin 6 was not a consequence of the differential intracellular location of these proteins in vivo, as even cytosolically-expressed mutants of SNAP-23 and syn-
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with VAMPs (19 –21). To determine if SNAP-29 also augments syntaxin associations with VAMP, we examined the ability of immobilized GST-VAMP 2 to bind to syntaxin from HeLa cell extracts expressing syntaxin alone or syntaxin together with SNAP-29. Coexpression with SNAP-29 dramatically augments the ability of both syntaxin 4 and syntaxin 6 to bind to VAMP 2 in vitro (Fig. 4). In fact, syntaxin 6 binding to VAMP 2 was undetectable unless SNAP-29 was coexpressed with syntaxin 6. These data, together with our results showing that SNAP-29 association with syntaxin occurs in vivo, demonstrates that syntaxins present in binary t-SNARE complexes with SNAP-29 has increased affinity for VAMP. VAMP binding augments syntaxin 6 binding to SNAP-29. Given our data showing that association with SNAP-29 augments syntaxin binding to VAMP in vitro, we set out to examine whether coexpression with VAMP augments the formation of SNAP-29/syntaxin 6 complexes. For these studies we chose to transfect either VAMP 2 or VAMP 8, two distinct v-SNAREs, in FIG. 2. SNAP-29 binds to syntaxin 4 and syntaxin 6 in vivo. HeLa cells were mock-transfected or transfected with expression vectors of either syntaxin 4 or syntaxin 6 together with SNAP-29 (A) or together with SNAP-23 (B). Equivalent aliquots of anti-SNAP-29 or anti-SNAP-23 immunoprecipitates were analyzed by SDS–PAGE and immunoblotted with a syntaxin 4 mAb (for cells expressing syntaxin 4) or a syntaxin 6 mAb (for cells expressing syntaxin 6). Each immunoprecipitate was also blotted with antisera recognizing either SNAP-29 or SNAP-23. Immunoblotting of equivalent fractions of the cell lysates confirmed the expression of the various constructs in the transfected cells.
taxin 6 (generated by truncation of their membraneanchoring domains) were unable to bind in vivo (data not shown). These data demonstrate that unlike SNAP-23, SNAP-29 is able to form binary t-SNARE complexes with both plasma membrane and Golgi syntaxins. We next performed experiments to confirm that the binary t-SNARE complexes observed in our coimmunoprecipitation studies represented true in vivo associations of SNAP-29 with syntaxin and were not an artifact of post-lysis SNARE associations. Coimmunoprecipitation of syntaxin 4 with SNAP-29 was only observed in lysates of cells coexpressing both proteins and was not observed when lysates containing either SNAP-29 alone or syntaxin 4 alone were simply mixed during the immunoprecipitation procedure (Fig. 3). These data clearly demonstrate that our in vivo association assay reflects true intracellular protein associations and, together with data shown above, demonstrate that SNAP-29 is able to form binary t-SNARE complexes with both plasma membrane- and Golgiassociated syntaxins in vivo. SNAP-29 augments syntaxin binding to VAMPs. It is likely that the function of both SNAP-25 and SNAP-23 is to facilitate the interaction of syntaxins
FIG. 3. SNAP-29 does not bind to syntaxin in cell extracts. HeLa cells were mock-transfected or transfected with expression vectors encoding SNAP-29 alone, syntaxin 4 alone, or SNAP-29 together with syntaxin 4. The cells were lysed and equal portions of cell lysates were mixed in the following combinations: SNAP-29 lysate and mock-lysate (lanes 1 and 5), syntaxin lysate and mock-lysate (lanes 2 and 6), SNAP-29/syntaxin 4 lysate and mock-lysate (lanes 3 and 7), and SNAP-29 lysate together with syntaxin lysate (lanes 4 and 8). Each lysate combination was incubated on ice for 1 h and subjected to immunoprecipitation with preimmune rabbit serum (control IP) or with a SNAP-29 antiserum (SNAP-29 IP). The immunoprecipitates were analyzed by immunoblotting with anti-syntaxin 4 mAb (upper panel). Equivalent portions of each cell lysate were also analyzed for expression of syntaxin 4 or SNAP-29 by immunoblot analysis (lower panel).
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HeLa cells expressing SNAP-29 and syntaxin 6. Whereas only a small amount of syntaxin 6 was associated with SNAP-29 in cells not overexpressing either VAMP, co-expression of either VAMP 2 or VAMP 8 dramatically increased syntaxin 6 binding to SNAP-29 (Fig. 5). These data demonstrate that the association of syntaxin 6 with SNAP-29 is enhanced by the binding of a VAMP to the complex. The results presented here and by Steegmaier et al. (11) showing that SNAP-29 has the capacity to bind to many different syntaxins is at odds with data showing that SNAP-29 bound almost exclusively to the Golgi SNARE syntaxin 6 (12). While we and Steegmaier et al. examined syntaxin binding to in vitro translated SNAP-29, Wong et al. used a Golgi membrane preparation as the source of SNAP-29. Since this extract contained not only SNAP-29 but also Golgi syntaxins and Golgi VAMPs, we have now addressed the possibility that the binding of SNAP-29 to syntaxin 6 could be augmented by overexpression of VAMPs in vivo. Although we did not specifically address whether Golgi VAMPs could mediate this effect, we did observe a remarkable increase in SNAP-29 binding to syntaxin 6 in cells co-expressing either VAMP 2 or VAMP 8, thereby providing a possible mechanism to resolve this discrepancy in binding data. Analysis of SNARE complexes which regulate synaptic vesicle fusion with the presynaptic plasma membrane of neurons (4), secretory vesicle fusion with the plasma membrane of yeast (22), and ER-derived transport vesicle fusion with the Golgi apparatus (23) strongly suggest that a four helical bundle of coiledcoils is required to facilitate SNARE-dependent membrane fusion. Since SNAP-25 and SNAP-23 are ex-
FIG. 5. Coexpression with VAMP augments SNAP-29 binding to syntaxin. HeLa cells were transfected with expression vectors encoding SNAP-29 and syntaxin 6 alone, together with VAMP 8, or together with VAMP 2. The cells were lysed and the amount of syntaxin 6 present in anti-SNAP-29 immunoprecipitates was analyzed by immunoblot analysis using a syntaxin 6 mAb. The SNAP-29 immunoprecipitates were also analyzed for expression of SNAP-29 and equal portions of each cell lysate were analyzed for expression of syntaxin 6 to confirm that their expression was not altered by cotransfection with VAMP. Additional blots confirmed the expression of the indicated VAMP in the cell lysates.
pressed predominantly on the plasma membrane of mammalian cells, SNAP-29 seemed to be a reasonable candidate to replace the two coiled-coils of SNAP-25 in the formation of various intra-organelle SNARE-pairs. We now show that SNAP-29 does indeed have the capacity to interact with a representative group of syntaxins that reside on a variety of intracellular organelles. Given the widespread distribution of SNAP-29 we and others have observed (11), we suggest that SNAP-29 does indeed play a role as a syntaxinbinding t-SNARE involved in the formation of SNAREpairs for a variety of intracellular transport steps. It is interesting to note that like SNAP-23, SNAP-29 bound to plasma membrane syntaxins in our in vitro and in vivo binding assays. Given the demonstrated role for SNAP-23 in regulated exocytosis, it is possible that complexes of SNAP-29 with plasma membrane syntaxins regulate constitutive but not regulated protein transport. Although speculative, the wide range of syntaxin binding activities of SNAP-29 is in very good agreement with this hypothesis. ACKNOWLEDGMENTS
FIG. 4. Coexpression with SNAP-29 augments syntaxin binding to VAMP. HeLa cells were mock-transfected or transfected with expression vectors encoding syntaxin alone, syntaxin together with SNAP-29, or SNAP-29 alone. The syntaxin expression vectors encoded either syntaxin 4 (upper panel) or syntaxin 6 (lower panel). Equivalent portions of each cell lysate were incubated with immobilized GST alone or GST-VAMP 2 fusion proteins. The amount of syntaxin 4 bound (upper panel) or syntaxin 6 bound (lower panel) to the fusion proteins was analyzed by immunoblotting. Equivalent portions of each cell lysate were also examined for expression of syntaxin 4 or syntaxin 6 by immunoblot analysis.
We than Richard Scheller and Jonathan Pevsner for generously providing SNARE expression vectors and David Winkler for peptide synthesis, oligonucleotide synthesis, and automated sequence analysis.
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3. Lin, R. C., and Scheller, R. H. (2000) Annu. Rev. Cell Dev. Biol. 16, 19 – 49. 4. Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347–353. 5. Bock, J. B., Matern, H. T., Peden, A. A., and Scheller, R. H. (2001) Nature 409(6822), 839 – 841. 6. Vaidyanathan, V. V., and Roche, P. A. (2000) Gene 247(1–2), 181–189. 7. Vaidyanathan, V. V., Puri, N., and Roche, P. A. (2001) J. Biol. Chem., in press. 8. Rea, S., Martin, L. B., McIntosh, S., Macaulay, S. L., Ramsdale, T., Baldini, G., and James, D. E. (1998) J. Biol. Chem. 273, 18784 –18792. 9. Guo, Z., Turner, C., and Castle, D. (1998) Cell 94, 537–548. 10. Araki, S., Tamori, Y., Kawanishi, M., Shinoda, H., Masugi, J., Mori, H., Niki, T., Okazawa, H., Kubota, T., and Kasuga, M. (1997) Biochem. Biophys. Res. Commun. 234, 257–262. 11. Steegmaier, M., Yang, B., Yoo, J. S., Huang, B., Shen, M., Yu, S., Luo, Y., and Scheller, R. H. (1998) J. Biol. Chem. 273, 34171– 34179. 12. Wong, S. H., Xu, Y., Zhang, T., Griffiths, G., Lowe, S. L., Subramaniam, V. N., Seow, K. T., and Hong, W. (1999) Mol. Biol. Cell 10, 119 –134.
13. Bock, J. B., Lin, R. C., and Scheller, R. H. (1996) J. Biol. Chem. 271, 17961–17965. 14. Wang, H., Felin, L., and Pevsner, J. (1997) Gene 199, 39 – 48. 15. Low, S. H., Roche, P. A., Anderson, H. A., van Ijzendoorn, S. C. D., Zhang, M., Mostov, K. E., and Weimbs, T. (1998) J. Biol. Chem. 273, 3422–3430. 16. Valdez, A. C., Cabaniols, J. P., Brown, M. J., and Roche, P. A. (1999) J. Cell Sci. 112, 845– 854. 17. Anderson, H. A., and Roche, P. A. (1998) J. Immunol. 160, 4850 – 4858. 18. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863– 873. 19. Pevsner, J., Hsu, S. C., Braun, J. E., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron 13, 353–361. 20. Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Su¨dhof, T. C., and Niemann, H. (1994) EMBO J. 13, 5051–5061. 21. Kawanishi, M., Tamori, Y., Okazawa, H., Araki, S., Shinoda, H., and Kasuga, M. (2000) J. Biol. Chem. 275(11), 8240 – 8247. 22. Rossi, G., Salminen, A., Rice, L. M., Brunger, A. T., and Brennwald, P. (1997) J. Biol. Chem. 272, 16610 –16617. 23. Xu, D., Joglekar, A. P., Williams, A. L., and Hay, J. C. (2000) J. Biol. Chem. 275(50), 39631–39639.
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SNAP-29 Is a Promiscuous Syntaxin-Binding SNARE Anita C. Hohenstein and Paul A. Roche 1 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received May 29, 2001
SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways. Key Words: SNARE; SNAP-29; SNAP-23; protein transport.
The movement of membrane and protein in all eukaryotic cells is a highly regulated process. With very few exceptions, proteins traffic from compartment-tocompartment in cells by a process involving vesicle budding from a donor organelle, vesicle docking with an appropriate target membrane, and the vesicle fusion with the target membrane (1). The strict regulation of this process ensures that cargo is transferred from one compartment to another in an orderly fashion. While the players that regulate the specificity with which a specific vesicle docks and fuses with a particular target membrane are under intense investigation, it is clear that one class of proteins, termed SNAREs, play an important part in the process of membrane1 To whom correspondence should be addressed at NIH, Bldg. 10, Room 4B36, Bethesda, MD 20892. Fax: (301) 496-0887. E-mail: [email protected].
membrane fusion (2, 3). The SNARE complex that regulates synaptic vesicle fusion with presynaptic membranes exists as a four helical bundle of coiled-coils (4). In this macromolecular complex, one coil is derived from the vesicle-associated SNARE protein VAMP and three coils are derived from the target SNARE (tSNARE) complex. In neurons, the t-SNARE complex consists of the presynaptic plasma membrane proteins syntaxin (possessing one coiled-coil) and SNAP-25 (possessing two coiled-coils). It is likely that each intracellular organelle can serve as a target membrane for transport vesicles or secretory granules. In agreement with the idea that specific SNARE combinations regulate specific transport steps, up to 35 v-SNARE and t-SNARE isoform are thought to exist in H. sapiens (5), and in many cases these proteins reside on distinct intracellular organelles (2). Unlike the wide variety of VAMPs and syntaxins present in mammalian cells, only two ubiquitously expressed homologs of SNAP-25 have been identified thus far. One of these, SNAP-23, is the product of duplication of an ancestral SNAP-25 gene (6), has similar SNAREbinding domains as SNAP-25 (7), and functions in regulated exocytosis from non-neuronal secretory cells (7– 9). In agreement with the proposed role of SNAP-23 as a plasma membrane target SNARE for secretory vesicles, in vitro binding studies have shown that SNAP-23 binds well to plasma membrane syntaxins but not the syntaxins present on internal membranes (10, 11). In contrast with SNAP-23, there is conflicting data on the binding properties and function of SNAP-29 in protein transport. Unlike SNAP-23, SNAP-29 has been reported to be present on many different internal membranes and binds to a wide variety of syntaxin isoforms (11). These data suggest that SNAP-29 could participate in SNARE complex assembly in a variety of intracellular membrane fusion events. On the other hand, another report has shown that SNAP-29 is present in the Golgi apparatus and binds to Golgi syntaxins almost exclusively (12), suggesting a very specific role for SNAP-29 in the regulation of intra-Golgi traffic. In this study, we have examined the ability of SNAP-29 to bind to different syntaxin isoforms using a variety of binding assays. We find that unlike SNAP-
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23, SNAP-29 is a relatively promiscuous syntaxin binding protein. Although binary SNAP-29/syntaxin t-SNARE complexes are formed in vitro and in vivo, the presence of a vesicle SNARE (i.e., VAMP) dramatically augments the binding of SNAP-29 to both Golgi and non-Golgi syntaxins. Furthermore, SNAP-29 augments the ability of syntaxin to bind to VAMP, a process required for the formation of a ternary SNARE fusion complex. We conclude that SNAP-29 functions by participating in SNARE-dependent trafficking events between vesicles and the numerous intracellular target organelles in eukaryotic cells. MATERIALS AND METHODS Plasmids and antibodies. The pcDNA3-myc-SNAP-29, pRCCMV-FLAG-VAMP 2, and pCMV4-myc-VAMP 8 expression vectors were the kind gift of Richard Scheller (Stanford, University, CA). The cDNAs for full-length human syntaxin 4, human syntaxin 6, and human SNAP-23 were subcloned into pcDNA3 and the sequence was confirmed by automated sequence analysis. GST fusion protein vectors for syntaxin 4, syntaxin 6, syntaxin 7, and VAMP 2 have been described previously (7, 13, 14). A SNAP-29 antiserum was obtained by immunizing rabbits with a carboxyl-terminal human SNAP-29 peptide (amino acids 242–258) coupled to maleimide-activated KLH (Pierce, Rockford, IL) using standard protocols. The anti-SNAP-23 amino-terminus rabbit antisera have been described previously (15). Syntaxin 6 and syntaxin 4 monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). In vitro binding assays. Proteins were translated in the presence of [ 35S]methionine using the TNT Quick in vitro transcription and translation kit (Promega). GST-fusion proteins were obtained using standard purification protocols and were immobilized onto glutathione-Sepharose beads (Pharmacia) prior to use. In vitro translated material was incubated with 5 g of GST alone, GST-syntaxin 4, GST-syntaxin 7, and GST-syntaxin 6 fusion proteins on ice in a binding buffer of 150 mM NaCl, 20 mM Hepes (pH 7.0), 1 mM MgCl 2, 0.1% Triton X-100, 0.5 mg/ml BSA and protease inhibitors (16). After incubation for 1 h on ice, the beads were washed extensively and the bound material was analyzed by SDS–PAGE. Cell culture, immunoprecipitation, and immunoblotting. Subconfluent HeLa cells were maintained and transfected using the LipofectAmine Plus method as described previously (16). Immunoprecipitations from cell lysates were performed using antibodies immobilized on protein A-Sepharose beads as described previously (17). Immunoblot analysis of cell extracts and immunoprecipitates was performed using ECL detection (NEN/Dupont) as described previously (17).
RESULTS AND DISCUSSION SNAP-29 binding to syntaxin in vitro. There is conflicting data regarding the ability of SNAP-29 to bind to different syntaxins (11, 12). In an attempt to determine if SNAP-29 is able to bind syntaxins present on distinct internal membranes, we examined the ability of in vitro translated SNAP-29 to bind to a variety of GST-syntaxin fusion proteins that are representative of the diverse family of syntaxins present on cellular organelles. SNAP-29 bound equally well to syntaxins that reside on late endosomes (syntaxin 7), the plasma membrane (syntaxin 4), and in the Golgi apparatus/
FIG. 1. SNAP-29 binds to multiple syntaxins in vitro. Equivalent amounts of in vitro translated [ 35S]methionine-SNAP-29 and [ 35S]methionine-SNAP-23 were incubated with the indicated immobilized GST fusion proteins on ice. The amount of SNAP-29 and SNAP-23 bound to each fusion protein was analyzed by SDS–PAGE and fluorography. Gels were stained with Coomassie brilliant blue R-250 to confirm that equal amounts of GST fusion proteins were present in each incubation. An aliquot (1/20th) of the reaction mixture was also analyzed to confirm that equal amounts of radiolabeled SNAP-29 and SNAP-23 were present in the incubations.
TGN (syntaxin 6), although under these conditions less than 5% of the input SNAP-29 bound to each syntaxin (Fig. 1). The ability to bind to syntaxins present on many different internal compartments is unique to SNAP-29, as the ubiquitously expressed SNAP-25 homolog SNAP-23 bound very well to syntaxin 4 but did not bind to either syntaxin 7 or syntaxin 6. Additional binding studies revealed that SNAP-23 also bound well to GST fusion proteins of the plasma membrane syntaxins syntaxin 1A, syntaxin 2, and syntaxin 3 (Ref. 11 and ACH, unpublished observation). As these syntaxins are present primarily on the plasma membrane of mammalian cells (18), these data reveal that SNAP-23 forms binary t-SNARE complexes with plasma membrane syntaxins while SNAP-29 is able to form t-SNARE complexes with syntaxins present on many different internal membranes. SNAP-29 binding to syntaxins in vivo. To confirm that the results obtained in the in vitro binding assay agreed with t-SNARE interactions in vivo, we examined the interaction of SNAP-29 and syntaxin 4 or syntaxin 6 in HeLa cells. The endogenous expression of these proteins is very low in these cells, and for this reason we overexpressed the proteins by transient transfection. Analysis of SNAP-29 immunoprecipitates revealed that SNAP-29 bound to syntaxin 4 quite well in vivo, whereas its binding to syntaxin 6 was low but clearly detectable (Fig. 2A). In contrast, SNAP-23 bound syntaxin 4 efficiently but did not bind to syntaxin 6 in vivo (Fig. 2B). The failure of SNAP-23 to bind to syntaxin 6 was not a consequence of the differential intracellular location of these proteins in vivo, as even cytosolically-expressed mutants of SNAP-23 and syn-
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with VAMPs (19 –21). To determine if SNAP-29 also augments syntaxin associations with VAMP, we examined the ability of immobilized GST-VAMP 2 to bind to syntaxin from HeLa cell extracts expressing syntaxin alone or syntaxin together with SNAP-29. Coexpression with SNAP-29 dramatically augments the ability of both syntaxin 4 and syntaxin 6 to bind to VAMP 2 in vitro (Fig. 4). In fact, syntaxin 6 binding to VAMP 2 was undetectable unless SNAP-29 was coexpressed with syntaxin 6. These data, together with our results showing that SNAP-29 association with syntaxin occurs in vivo, demonstrates that syntaxins present in binary t-SNARE complexes with SNAP-29 has increased affinity for VAMP. VAMP binding augments syntaxin 6 binding to SNAP-29. Given our data showing that association with SNAP-29 augments syntaxin binding to VAMP in vitro, we set out to examine whether coexpression with VAMP augments the formation of SNAP-29/syntaxin 6 complexes. For these studies we chose to transfect either VAMP 2 or VAMP 8, two distinct v-SNAREs, in FIG. 2. SNAP-29 binds to syntaxin 4 and syntaxin 6 in vivo. HeLa cells were mock-transfected or transfected with expression vectors of either syntaxin 4 or syntaxin 6 together with SNAP-29 (A) or together with SNAP-23 (B). Equivalent aliquots of anti-SNAP-29 or anti-SNAP-23 immunoprecipitates were analyzed by SDS–PAGE and immunoblotted with a syntaxin 4 mAb (for cells expressing syntaxin 4) or a syntaxin 6 mAb (for cells expressing syntaxin 6). Each immunoprecipitate was also blotted with antisera recognizing either SNAP-29 or SNAP-23. Immunoblotting of equivalent fractions of the cell lysates confirmed the expression of the various constructs in the transfected cells.
taxin 6 (generated by truncation of their membraneanchoring domains) were unable to bind in vivo (data not shown). These data demonstrate that unlike SNAP-23, SNAP-29 is able to form binary t-SNARE complexes with both plasma membrane and Golgi syntaxins. We next performed experiments to confirm that the binary t-SNARE complexes observed in our coimmunoprecipitation studies represented true in vivo associations of SNAP-29 with syntaxin and were not an artifact of post-lysis SNARE associations. Coimmunoprecipitation of syntaxin 4 with SNAP-29 was only observed in lysates of cells coexpressing both proteins and was not observed when lysates containing either SNAP-29 alone or syntaxin 4 alone were simply mixed during the immunoprecipitation procedure (Fig. 3). These data clearly demonstrate that our in vivo association assay reflects true intracellular protein associations and, together with data shown above, demonstrate that SNAP-29 is able to form binary t-SNARE complexes with both plasma membrane- and Golgiassociated syntaxins in vivo. SNAP-29 augments syntaxin binding to VAMPs. It is likely that the function of both SNAP-25 and SNAP-23 is to facilitate the interaction of syntaxins
FIG. 3. SNAP-29 does not bind to syntaxin in cell extracts. HeLa cells were mock-transfected or transfected with expression vectors encoding SNAP-29 alone, syntaxin 4 alone, or SNAP-29 together with syntaxin 4. The cells were lysed and equal portions of cell lysates were mixed in the following combinations: SNAP-29 lysate and mock-lysate (lanes 1 and 5), syntaxin lysate and mock-lysate (lanes 2 and 6), SNAP-29/syntaxin 4 lysate and mock-lysate (lanes 3 and 7), and SNAP-29 lysate together with syntaxin lysate (lanes 4 and 8). Each lysate combination was incubated on ice for 1 h and subjected to immunoprecipitation with preimmune rabbit serum (control IP) or with a SNAP-29 antiserum (SNAP-29 IP). The immunoprecipitates were analyzed by immunoblotting with anti-syntaxin 4 mAb (upper panel). Equivalent portions of each cell lysate were also analyzed for expression of syntaxin 4 or SNAP-29 by immunoblot analysis (lower panel).
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HeLa cells expressing SNAP-29 and syntaxin 6. Whereas only a small amount of syntaxin 6 was associated with SNAP-29 in cells not overexpressing either VAMP, co-expression of either VAMP 2 or VAMP 8 dramatically increased syntaxin 6 binding to SNAP-29 (Fig. 5). These data demonstrate that the association of syntaxin 6 with SNAP-29 is enhanced by the binding of a VAMP to the complex. The results presented here and by Steegmaier et al. (11) showing that SNAP-29 has the capacity to bind to many different syntaxins is at odds with data showing that SNAP-29 bound almost exclusively to the Golgi SNARE syntaxin 6 (12). While we and Steegmaier et al. examined syntaxin binding to in vitro translated SNAP-29, Wong et al. used a Golgi membrane preparation as the source of SNAP-29. Since this extract contained not only SNAP-29 but also Golgi syntaxins and Golgi VAMPs, we have now addressed the possibility that the binding of SNAP-29 to syntaxin 6 could be augmented by overexpression of VAMPs in vivo. Although we did not specifically address whether Golgi VAMPs could mediate this effect, we did observe a remarkable increase in SNAP-29 binding to syntaxin 6 in cells co-expressing either VAMP 2 or VAMP 8, thereby providing a possible mechanism to resolve this discrepancy in binding data. Analysis of SNARE complexes which regulate synaptic vesicle fusion with the presynaptic plasma membrane of neurons (4), secretory vesicle fusion with the plasma membrane of yeast (22), and ER-derived transport vesicle fusion with the Golgi apparatus (23) strongly suggest that a four helical bundle of coiledcoils is required to facilitate SNARE-dependent membrane fusion. Since SNAP-25 and SNAP-23 are ex-
FIG. 5. Coexpression with VAMP augments SNAP-29 binding to syntaxin. HeLa cells were transfected with expression vectors encoding SNAP-29 and syntaxin 6 alone, together with VAMP 8, or together with VAMP 2. The cells were lysed and the amount of syntaxin 6 present in anti-SNAP-29 immunoprecipitates was analyzed by immunoblot analysis using a syntaxin 6 mAb. The SNAP-29 immunoprecipitates were also analyzed for expression of SNAP-29 and equal portions of each cell lysate were analyzed for expression of syntaxin 6 to confirm that their expression was not altered by cotransfection with VAMP. Additional blots confirmed the expression of the indicated VAMP in the cell lysates.
pressed predominantly on the plasma membrane of mammalian cells, SNAP-29 seemed to be a reasonable candidate to replace the two coiled-coils of SNAP-25 in the formation of various intra-organelle SNARE-pairs. We now show that SNAP-29 does indeed have the capacity to interact with a representative group of syntaxins that reside on a variety of intracellular organelles. Given the widespread distribution of SNAP-29 we and others have observed (11), we suggest that SNAP-29 does indeed play a role as a syntaxinbinding t-SNARE involved in the formation of SNAREpairs for a variety of intracellular transport steps. It is interesting to note that like SNAP-23, SNAP-29 bound to plasma membrane syntaxins in our in vitro and in vivo binding assays. Given the demonstrated role for SNAP-23 in regulated exocytosis, it is possible that complexes of SNAP-29 with plasma membrane syntaxins regulate constitutive but not regulated protein transport. Although speculative, the wide range of syntaxin binding activities of SNAP-29 is in very good agreement with this hypothesis. ACKNOWLEDGMENTS
FIG. 4. Coexpression with SNAP-29 augments syntaxin binding to VAMP. HeLa cells were mock-transfected or transfected with expression vectors encoding syntaxin alone, syntaxin together with SNAP-29, or SNAP-29 alone. The syntaxin expression vectors encoded either syntaxin 4 (upper panel) or syntaxin 6 (lower panel). Equivalent portions of each cell lysate were incubated with immobilized GST alone or GST-VAMP 2 fusion proteins. The amount of syntaxin 4 bound (upper panel) or syntaxin 6 bound (lower panel) to the fusion proteins was analyzed by immunoblotting. Equivalent portions of each cell lysate were also examined for expression of syntaxin 4 or syntaxin 6 by immunoblot analysis.
We than Richard Scheller and Jonathan Pevsner for generously providing SNARE expression vectors and David Winkler for peptide synthesis, oligonucleotide synthesis, and automated sequence analysis.
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