The C Terminus of SNAP25 Is Essential for Ca2+-dependent Binding of Synaptotagmin to SNARE Complexes

Abstract

The plasma membrane solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca2+-dependent step in regulated exocytosis, but their precise roles and regulation by Ca2+ are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg180-Ile181, completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln197-Arg198, is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca2+ concentrations. BoNT A treatment essentially increased the Ca2+ concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca2+ regulation of exocytosis. Synaptotagmin, a proposed Ca2+ sensor for exocytosis, was found to bind SNAP25 in a Ca2+-stimulated manner. Ca2+-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca2+concentration required for binding. The C terminus of SNAP25 was also essential for Ca2+-dependent synaptotagmin binding to SNAP25·syntaxin and SNAP25·syntaxin·VAMP SNARE complexes. These results clarify classical observations on the Ca2+ reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca2+-dependent interactions between synaptotagmin and SNARE protein complexes. The plasma membrane solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca2+-dependent step in regulated exocytosis, but their precise roles and regulation by Ca2+ are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg180-Ile181, completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln197-Arg198, is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca2+ concentrations. BoNT A treatment essentially increased the Ca2+ concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca2+ regulation of exocytosis. Synaptotagmin, a proposed Ca2+ sensor for exocytosis, was found to bind SNAP25 in a Ca2+-stimulated manner. Ca2+-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca2+concentration required for binding. The C terminus of SNAP25 was also essential for Ca2+-dependent synaptotagmin binding to SNAP25·syntaxin and SNAP25·syntaxin·VAMP SNARE complexes. These results clarify classical observations on the Ca2+ reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca2+-dependent interactions between synaptotagmin and SNARE protein complexes. vesicle-associated membrane protein solubleN-ethylmaleimide-sensitive factor attachment protein receptor synaptosome-associated protein botulinum neurotoxin glutathione S-transferase 3-(cyclohexylamino)propanesulfonic acid Regulated neurotransmitter secretion is a specialized version of a general membrane fusion mechanism in which exocytotic fusion is strictly Ca2+-regulated. Studies of this process have yielded insights into universal mechanisms for intracellular membrane fusion and the identity of core components of the fusion machinery (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, 2.Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (312) Google Scholar, 3.Martin T.F.J. Trends Cell Biol. 1997; 7: 271-276Abstract Full Text PDF PubMed Scopus (123) Google Scholar). VAMP,1 syntaxin, and SNAP25 are the neural protein substrates for clostridial neurotoxins, a family of highly selective proteases that potently inhibits neurosecretion (4.Montecucco C. Schiavo G. Trends Biochem. Sci. 1993; 18: 324-327Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 5.Niemann H. Blasi J. Jahn R. Trends Cell Biol. 1994; 4: 179-183Abstract Full Text PDF PubMed Scopus (293) Google Scholar). These proteins are solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that mediate the membrane association ofN-ethylmaleimide-sensitive factor, a protein required for membrane fusion (6.Sollner 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). The SNARE proteins are capable of assembling into extremely stable heterotrimeric complexes (7.Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, 8.Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar, 9.Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) that consist of a four-helix bundle in parallel alignment (10.Hanson P.I. Roth R. Morisaki H. Jahn R. Heuser J.R. Cell. 1997; 90: 523-535Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 11.Lin R.C. Scheller R.H. Neuron. 1997; 19: 1087-1094Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12.Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.-K. Bennett M.K. Nature Struct. Biol. 1998; 5: 765-769Crossref PubMed Scopus (419) Google Scholar, 13.Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar). A current hypothesis suggests that the formation of SNARE complexes in transacross apposing membranes promotes intimate bilayer interactions and provides the energy to drive membrane fusion (10.Hanson P.I. Roth R. Morisaki H. Jahn R. Heuser J.R. Cell. 1997; 90: 523-535Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 13.Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, 14.Jahn R. Hanson P.I. Nature. 1998; 393: 14-15Crossref PubMed Scopus (79) Google Scholar, 15.Fiebig K.M. Rice L.M. Pollock E. Brunger A.T. Nature Struct. Biol. 1999; 6: 117-123Crossref PubMed Scopus (237) Google Scholar, 16.Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). Studies and that complexes mediate bilayer and fusion T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). to the fusion by T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google neurosecretion is and Ca2+-dependent that SNARE formation (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, E. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). have an role for the vesicle protein synaptotagmin in neurosecretion M. Y. C. Sudhof T.C. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, M. M. Sci. S. 1994; PubMed Scopus Google Scholar, Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). Ca2+-dependent interactions syntaxin N. Sudhof T.C. Jahn R. PubMed Scopus Google Scholar, R.B. Sudhof T.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) to suggested role a Ca2+ sensor for exocytosis (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, M. Y. C. Sudhof T.C. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, N. Sudhof T.C. Jahn R. PubMed Scopus Google R.B. Sudhof T.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The precise role of synaptotagmin and Ca2+-dependent interactions syntaxin in regulated neurosecretion to Studies of regulated exocytosis in membrane from that are required for a late Ca2+-dependent step that vesicle and and fusion J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T.F.J. J.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). toxin and botulinum neurotoxin (BoNT) and E completely the Ca2+-dependent of exocytosis, which that syntaxin, and SNAP25 in to at BoNT A, which BoNT E cleaves was only partially for fusion of SNAP25 J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). SNAP25 at for BoNT E and for BoNT G. J. F. C. 1993; PubMed Scopus Google and T. Blasi J. Yamasaki S. E. Sudhof T.C. Jahn R. Niemann H. J. Biol. Chem. 1994; Full Text PDF PubMed Google was that the the C terminus of SNAP25 between the toxin a role in Ca2+-dependent membrane fusion J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the this of SNAP25 is to essential for Ca2+-dependent interactions classical observations that BoNT A inhibition of neurosecretion is partially by Ca2+ H. S. S. J. Sci. Full Text PDF PubMed Scopus Google Scholar). the results suggest that an role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca2+-dependent interactions between synaptotagmin and SNARE protein complexes. PC12 in and for at J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R. T.F.J. J. PubMed Scopus Google Scholar) and by a for of a plasma membrane that T.F.J. J.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). in at in and Ca2+ to was in of are by to the of BoNT A, and E by B. R. and BoNT by S. by treatment of membrane was for at to Ca2+ for secretion is to by BoNT A and E but only at in that to SNAP25 by at P. R. G. C. 1997; PubMed Scopus Google Scholar, M. C. S. S. H. Binz T. J. 1999; PubMed Scopus Google Scholar). is that the protein is the of BoNT inhibition in PC12 cell S-transferase fusion proteins by by synaptotagmin and by R. H. Scheller Scheller R.H. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, J. N. Bennett M.K. Scheller R.H. Neuron. 1994; 13: Full Text PDF PubMed Scopus Google Scholar). The synaptotagmin was by M. and M. T. J. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The syntaxin was by E. Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). fusion proteins by glutathione for for the was by by toxin for at for binding and and SNARE complexes by at of syntaxin, at of of synaptotagmin for at which by and in of to in for of heterotrimeric SNARE complexes was by T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). to for the a the of synaptotagmin a a to a to a SNAP25 by M. from a to a to syntaxin a to by R. A a Ca2+-dependent binding of synaptotagmin to SNAP25 was in binding on by in of a at for in an by at for and in in protease and to for binding SNAP25 by of at SNAP25 on protein Studies of the late Ca2+-dependent step of exocytosis in PC12 that BoNT E completely whereas BoNT A was only at of SNAP25 J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R. T.F.J. J. PubMed Scopus Google Scholar). for this difference between was suggested by that BoNT A inhibition of neurosecretion was in by Ca2+ in H. S. S. J. Sci. Full Text PDF PubMed Scopus Google Scholar). the of Ca2+ on the BoNT inhibition of regulated secretion in a that PC12 cell plasma membranes T.F.J. J.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). BoNT E treatment Ca2+-dependent at Ca2+ BoNT A treatment Ca2+-dependent at Ca2+ but inhibition was at BoNT E and BoNT A at and of SNAP25 not BoNT A essentially increased the for Ca2+ in exocytosis to for was not the result of a of exocytosis at the inhibition by BoNT A Ca2+ that for The Ca2+ reversal of inhibition by toxin was for BoNT A and was not for BoNT which cleaves G. F. B. P. C. Nature. PubMed Scopus Google for BoNT which cleaves syntaxin G. Bennett M.K. Scheller R.H. C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). These Ca2+-dependent at Ca2+ The Ca2+ reversal of BoNT A inhibition of secretion in this PC12 cell membrane is to that for BoNT inhibition in the and H. S. S. J. Sci. Full Text PDF PubMed Scopus Google Scholar, S. Physiol. PubMed Scopus Google Scholar, M. J. 1997; PubMed Google Scholar, T. Binz T. Niemann H. E. Nature 1998; PubMed Scopus Google Scholar). of SNAP25 by BoNT A increased the for of exocytosis, the that SNAP25 was in the Ca2+ regulation of exocytosis. The vesicle protein synaptotagmin proposed to a Ca2+ sensor for exocytosis (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google M. Y. C. Sudhof T.C. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, N. Sudhof T.C. Jahn R. PubMed Scopus Google synaptotagmin the of SNAP25 was found to bind an synaptotagmin A, was G. G. Rothman J.E. Sollner T.H. Sci. S. 1997; PubMed Scopus Google Scholar). the of Ca2+ in the binding SNAP25 binding to synaptotagmin A, the binding of the synaptotagmin to SNAP25 was also and the binding was by of Ca2+ A, of binding in that Ca2+ the of SNAP25 synaptotagmin by binding to SNAP25 in the of of but Ca2+ increased the of the to a The of the binding at in the of Ca2+ required to synaptotagmin binding to SNAP25 at Ca2+ at and not the interactions syntaxin to a Ca2+ and Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). that mediate Ca2+-dependent of the protein T.C. J. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the of synaptotagmin required for the Ca2+-stimulated proteins the a the a the The protein SNAP25 binding in the of Ca2+ the fusion protein Ca2+-stimulated interactions SNAP25 not the of the These results for SNAP25 to for synaptotagmin interactions syntaxin the Ca2+-dependent binding Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The syntaxin binding of synaptotagmin to consist of the a a of the J. Fasshauer D. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of SNAP25 essential for synaptotagmin SNAP25 was BoNT A BoNT E. SNAP25 by toxin to synaptotagmin in the of Ca2+ G. G. Rothman J.E. Sollner T.H. Sci. S. 1997; PubMed Scopus Google Scholar). the Ca2+-dependent of SNAP25 synaptotagmin was completely abolished by treatment BoNT E and partially by treatment BoNT A The of inhibition of synaptotagmin binding BoNT SNAP25 was the Ca2+ concentration in the binding the inhibition at Ca2+ BoNT A treatment essentially increased the for Ca2+ of synaptotagmin binding to on synaptotagmin and SNAP25 in Ca2+-dependent interactions between proteins to proteins The of SNAP25 from in the of synaptotagmin A, which was by of Ca2+ The Ca2+-stimulated of synaptotagmin in the SNAP25 was by treatment BoNT E the of syntaxin in the SNAP25 and the of an a synaptotagmin in Ca2+-dependent on Ca2+ synaptotagmin binding the concentration was in protein binding the proteins of the C terminus of SNAP25 BoNT A E the Ca2+-dependent binding of synaptotagmin to SNAP25 Ca2+ toxin treatment the binding of syntaxin to between the of Ca2+ in BoNT A inhibition of exocytosis and in BoNT A inhibition of synaptotagmin interactions that the synaptotagmin that in Ca2+ have C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, T.C. J. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Ca2+-dependent interactions of synaptotagmin syntaxin at Ca2+ whereas synaptotagmin Ca2+-dependent interactions syntaxin at C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). was found to Ca2+-stimulated interactions SNAP25 at Ca2+ required for synaptotagmin The of binding an for Ca2+ of which is to the for vesicle exocytosis The Ca2+-dependent binding of synaptotagmin to SNAP25 required of SNAP25 by BoNT A and E the binding Ca2+ partially in synaptotagmin binding to BoNT SNAP25 and to a to BoNT SNAP25 not SNAP25 and syntaxin stable complexes of a of syntaxin to SNAP25 D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). complexes the of SNAP25 in membranes, SNAP25 binding to synaptotagmin in the of syntaxin the binding of syntaxin to synaptotagmin was not in the of Ca2+ A, of SNAP25 Ca2+-dependent syntaxin binding to synaptotagmin which increased SNAP25 concentration The results that Ca2+-dependent syntaxin binding is by the formation of and the Ca2+-dependent binding of the to synaptotagmin for Ca2+-dependent interactions synaptotagmin SNAP25 The Ca2+-dependent binding of syntaxin to synaptotagmin was by of the C terminus of SNAP25 BoNT E and to a BoNT A the C terminus of SNAP25 is not required for the formation of S. N. Jahn R. J. Biol. Chem. 1994; Full Text PDF PubMed Google the results that Ca2+-dependent binding of SNARE complexes to synaptotagmin the C terminus of suggested that Ca2+ in exocytosis to the formation of SNARE complexes in to drive bilayer fusion R. Hanson P.I. Nature. 1998; 393: 14-15Crossref PubMed Scopus (79) Google Scholar). synaptotagmin also Ca2+-stimulated interactions SNARE was in the the Ca2+-dependent binding of to synaptotagmin was whereas binding was in the of SNAP25 syntaxin in the of The results that Ca2+-dependent binding is SNARE binding to synaptotagmin which was by that synaptotagmin SNARE proteins in complexes not and SNARE complexes Ca2+-dependent binding to synaptotagmin by SNAP25 of to heterotrimeric complexes syntaxin binding to that of SNAP25 which is the of and SNARE complexes D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The Ca2+-dependent binding of to synaptotagmin was BoNT BoNT SNAP25 was to complexes The inhibition of binding result from of complexes by BoNT treatment T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar) from inhibition of the binding of complexes to synaptotagmin by the of essential of SNAP25 by toxin BoNT E treatment was found to SNARE complexes T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google and this for the inhibition of Ca2+-dependent binding to synaptotagmin BoNT A treatment on the of SNARE complexes T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). the in SNARE binding to synaptotagmin from BoNT A treatment that SNAP25 by BoNT A are essential for the Ca2+-dependent of synaptotagmin SNARE complexes. The in this that synaptotagmin a Ca2+-dependent SNAP25 and an essential for Ca2+-dependent synaptotagmin binding is the C terminus of The results an for the reversal of BoNT A inhibition of neurosecretion at elevated Ca2+ in classical also suggest a for the Ca2+ regulation of exocytosis. and H. S. S. J. Sci. Full Text PDF PubMed Scopus Google Scholar) that treatment a that of Ca2+ partially the of BoNT A in the J. Google Scholar) that BoNT A the Ca2+ of neurotransmitter and suggested that BoNT A to the of Ca2+ in neurotransmitter to a of of inhibition by Ca2+ is to BoNT A and not for BoNT BoNT BoNT S. Physiol. PubMed Scopus Google F. PubMed Scopus Google Scholar, J.A. PubMed Scopus Google Scholar, J. D. M. N. S. J. Physiol. Google Scholar) and only to a for BoNT E J. S. Physiol. PubMed Scopus Google Scholar, PubMed Google Scholar). These classical the of BoNT the of the that the of BoNT A is to the Ca2+ regulation of neurotransmitter Studies of toxin in cell have found BoNT A to in Ca2+-dependent secretion BoNT E in to effectiveness in J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R. T.F.J. J. PubMed Scopus Google J. C. R. S. J. Cell Biol. 1995; PubMed Scopus Google Scholar). The results clarify this of BoNT A by that the Ca2+ to activate exocytosis in results in a reversal of BoNT A are in cell for between cell and cell on the of BoNT A an of regulated secretion P. N. 1997; PubMed Scopus Google Scholar). inhibition by BoNT A also in cell are to exocytosis T. Binz T. Niemann H. E. Nature 1998; PubMed Scopus Google Scholar). BoNT A of SNAP25 the of regulated exocytosis, that SNAP25 was in the Ca2+ of exocytosis. binding of SNAP25 synaptotagmin a proposed Ca2+ sensor for exocytosis (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, M. Y. C. Sudhof T.C. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, N. Sudhof T.C. Jahn R. PubMed Scopus Google Scholar). A between which not the C terminus of was G. G. Rothman J.E. Sollner T.H. Sci. S. 1997; PubMed Scopus Google Scholar). The binding in a Ca2+-dependent between SNAP25 and synaptotagmin that the C terminus of of SNAP25 from to by BoNT E treatment abolished Ca2+-dependent synaptotagmin which the of this toxin on exocytosis. of SNAP25 from to by BoNT A treatment, in the Ca2+ for the binding of synaptotagmin to SNAP25 by to Ca2+ an that was to that of BoNT A on exocytosis. The inhibition by BoNT A and reversal at elevated for Ca2+-dependent binding and for Ca2+-dependent exocytosis provides a is a role for SNAP25 an for synaptotagmin in the of exocytosis. for the Ca2+ reversal of BoNT A inhibition of exocytosis is the in the SNARE the BoNT A R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, D. R.B. Brunger A.T. Jahn R. Sci. S. 1998; PubMed Scopus Google Ca2+ binding to this to a of Ca2+-dependent interactions for synaptotagmin synaptotagmin and syntaxin R.B. Sudhof T.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, R.H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Jahn R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Sollner T.H. Rothman J.E. Sci. S. 1996; PubMed Scopus Google but of the essential role of synaptotagmin in exocytosis. The of the synaptotagmin a to for the synaptotagmin Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, C. B. N. Sudhof T.C. Nature. 1995; 375: PubMed Scopus Google Scholar, C. Sudhof T.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). are at Ca2+ are not by and in the of synaptotagmin T.C. J. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). synaptotagmin for interactions syntaxin and at to required for vesicle exocytosis whereas synaptotagmin the Ca2+ of vesicle exocytosis (1.Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, 2.Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (312) Google Scholar, 3.Martin T.F.J. Trends Cell Biol. 1997; 7: 271-276Abstract Full Text PDF PubMed Scopus (123) Google Scholar, E. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of Ca2+-dependent synaptotagmin binding to suggests the that plasma membrane complexes are the Ca2+-dependent for Ca2+-dependent synaptotagmin binding to SNAP25 to of the binding to The for Ca2+-dependent synaptotagmin interactions is Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google whereas an for Ca2+-dependent synaptotagmin binding. at the Ca2+-dependent SNAP25 binding to synaptotagmin was whereas that of syntaxin was not binding to synaptotagmin in the of SNAP25 complexes and to synaptotagmin in a Ca2+-dependent manner. The C terminus of SNAP25 was required for Ca2+-dependent interactions between synaptotagmin and SNARE complexes. A Ca2+-dependent binding of SNARE complexes to synaptotagmin was which also required the C terminus of These results that Ca2+-dependent interactions between synaptotagmin and SNAP25 SNARE complexes are in the C terminus of was that Ca2+-dependent synaptotagmin binding to SNAP25 to SNARE complexes was SNAP25 a but in and SNARE complexes D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). SNAP25 in complexes The of SNAP25 a for Ca2+-dependent synaptotagmin binding Ca2+-dependent binding to a SNAP25 and a SNAP25 inhibits binding. C. The the core of this binding BoNT E Ca2+-dependent interactions The binding by BoNT A synaptotagmin binding in a that is partially by the The of a core of the SNARE of a four-helix bundle in parallel R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar) a for Ca2+-dependent interactions of The of syntaxin to mediate Ca2+-dependent interactions synaptotagmin Hanson P.I. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. J. 1996; PubMed Google Scholar, and J. Fasshauer D. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google but also C. Sudhof T.C. J. Neuron. 1997; 18: Full Text Full Text PDF PubMed Scopus Google is to the C terminus of SNAP25 syntaxin SNAP25 of 13.Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar). from by SNAP25 and syntaxin in SNARE complexes the essential Ca2+-dependent binding for The Ca2+ of membrane fusion by the Ca2+-dependent binding of synaptotagmin on the vesicle to syntaxin and SNAP25 on the plasma Ca2+ synaptotagmin binding to which on the vesicle into to SNARE complexes that fusion T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). Ca2+ the binding of synaptotagmin to partially SNARE complexes the vesicle and plasma membrane to and the of fusion (10.Hanson P.I. Roth R. Morisaki H. Jahn R. Heuser J.R. Cell. 1997; 90: 523-535Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 14.Jahn R. Hanson P.I. Nature. 1998; 393: 14-15Crossref PubMed Scopus (79) Google Scholar, 15.Fiebig K.M. Rice L.M. Pollock E. Brunger A.T. Nature Struct. Biol. 1999; 6: 117-123Crossref PubMed Scopus (237) Google Scholar, Scheller R.H. Nature. 1998; 395: PubMed Scopus Google Scholar). interactions drive the of complexes between synaptotagmin and SNARE proteins (7.Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar). The Ca2+-dependent between synaptotagmin and SNAP25 mediate interactions the that a The C terminus of SNAP25 is for by of the clostridial E, A, and but precise role in neurotransmitter secretion not Studies have that the of SNAP25 by BoNT A are required for interactions S. N. Jahn R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and that of the by BoNT E SNARE formation T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). results that the of SNAP25 the Ca2+ of exocytosis and the of synaptotagmin binding to The of SNAP25 between BoNT A and E is essential for membrane fusion J.A. T.F.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and for Ca2+-dependent synaptotagmin binding. The that a SNAP25 is capable of Ca2+-dependent of BoNT exocytosis the that the C terminus of SNAP25 in the Ca2+ of exocytosis Scheller R.H. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Ca2+-dependent interactions between synaptotagmin and SNARE the C terminus of SNAP25 a of a Ca2+ for regulated exocytosis.