Abrogating Munc18-1-SNARE Complex Interaction Has Limited Impact on Exocytosis in PC12 Cells

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins initiate membrane fusion through the formation of the SNARE complex, a process tightly regulated by Sec1/Munc18-1 (SM) proteins. The emerging trend is that SM proteins promote SNARE-mediated membrane fusion by binding to a Syntaxin N-terminal motif. Here we report that mutations in the hydrophobic pocket of Munc18-1 (F115E and E132A), predicted to disrupt the N-terminal Sx1a interaction have a modest effect on binding to Sx1a in its free state, but abolish binding to the SNARE complex. Overexpression of the Munc18-1 mutant in PC12 cells lacking Munc18-1 rescues both neuroexocytosis and the plasma membrane localization of Syntaxin. However, total internal reflection fluorescence microscopy analysis reveals that expression of a Munc18-1 double mutant reduces the rate of vesicle fusion, an effect only detectable at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore critical for SNARE complex binding. However, mutations abrogating this interaction have a limited impact on Ca2+-dependent exocytosis in PC12 cells. Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins initiate membrane fusion through the formation of the SNARE complex, a process tightly regulated by Sec1/Munc18-1 (SM) proteins. The emerging trend is that SM proteins promote SNARE-mediated membrane fusion by binding to a Syntaxin N-terminal motif. Here we report that mutations in the hydrophobic pocket of Munc18-1 (F115E and E132A), predicted to disrupt the N-terminal Sx1a interaction have a modest effect on binding to Sx1a in its free state, but abolish binding to the SNARE complex. Overexpression of the Munc18-1 mutant in PC12 cells lacking Munc18-1 rescues both neuroexocytosis and the plasma membrane localization of Syntaxin. However, total internal reflection fluorescence microscopy analysis reveals that expression of a Munc18-1 double mutant reduces the rate of vesicle fusion, an effect only detectable at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore critical for SNARE complex binding. However, mutations abrogating this interaction have a limited impact on Ca2+-dependent exocytosis in PC12 cells. Following stimulation of neurons, a number of well orchestrated protein/protein (1Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1888) Google Scholar) and protein/lipid (2Söllner T.H. Mol. Membr. Biol. 2003; 20: 209-220Crossref PubMed Scopus (143) Google Scholar) interactions underpin the fusion of secretory vesicles with the presynaptic plasma membrane. In this sequence of interactions, vesicles approach the plasma membrane (tethering and docking), undergo priming and, upon Ca2+ influx, fuse with the plasma membrane, thereby releasing neurotransmitter into the synaptic cleft (1Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1888) Google Scholar). Vesicular exocytosis relies on the function of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) 2The abbreviations used are: SNAREsoluble N-ethylmaleimide-sensitive fusion protein attachment protein receptorSMSec1/Munc18TIRFtotal internal reflection fluorescenceMSDmean square displacementNPYneuropeptide YhPLAPhuman placental alkaline phosphataseSx1aSyntaxin 1aWTwild typeGSTglutathione S-transferasehGHhuman growth hormonePSSphysiological saline solutionPDBProtein Data BankGFPgreen fluorescent protein. proteins as demonstrated by the blockade of neuroexocytosis following SNARE protein cleavage by clostridial neurotoxins (3Schiavo G. Benfenati F. Poulain B. Rossetto O. Polverino de Laureto P. DasGupta B.R. Montecucco C. Nature. 1992; 359: 832-835Crossref PubMed Scopus (1459) Google Scholar). One of the key players in SNARE regulation is the cytosolic regulatory protein, Munc18-1 (Munc18a, nsec-1) (4Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 2003-2007Crossref PubMed Scopus (224) Google Scholar, 5Hata Y. Slaughter C.A. Südhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (594) Google Scholar, 6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 7Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (228) Google Scholar). Although the function of SNARE proteins in mediating exocytosis is well established (2Söllner T.H. Mol. Membr. Biol. 2003; 20: 209-220Crossref PubMed Scopus (143) Google Scholar, 8Jahn R. Lang T. Südhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar), the precise role of Munc18-1 in exocytosis is still a subject of heated debate (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 7Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (228) Google Scholar, 9Peng R. Gallwitz D. EMBO J. 2004; 23: 3939-3949Crossref PubMed Scopus (67) Google Scholar, 10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor Sec1/Munc18 total internal reflection fluorescence mean square displacement neuropeptide Y human placental alkaline phosphatase Syntaxin 1a wild type glutathione S-transferase human growth hormone physiological saline solution Protein Data Bank green fluorescent protein. Munc18-1 belongs to the Sec1/Munc18 (SM) family of proteins that are involved in mediating membrane trafficking events (11Gallwitz D. Jahn R. Trends Biochem. Sci. 2003; 28: 113-116Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12Toonen R.F. Verhage M. Trends Cell Biol. 2003; 13: 177-186Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 13Latham C.F. Meunier F.A. Int. J. Biochem. Cell Biol. 2007; 39: 1576-1581Crossref PubMed Scopus (14) Google Scholar). Mutations in these proteins have recently been associated with infantile epileptic encephalopathy (14Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. Okada I. Yoshimura Y. Hirai S. Kumada T. Hayasaka K. Fukuda A. Ogata K. Matsumoto N. Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (436) Google Scholar). Although the function of Munc18-1 and its interaction with SNAREs have been studied for over 10 years, the molecular mechanism of Munc18-1 regulation of membrane fusion is still not clear. Munc18-1 was originally characterized as a negative regulator of exocytosis as it binds to the target membrane SNARE, Syntaxin 1a (Sx1a) (5Hata Y. Slaughter C.A. Südhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (594) Google Scholar) in a conformation that sequesters the Sx1a SNARE helix and inhibits SNARE complex formation (7Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (228) Google Scholar, 15Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (617) Google Scholar). Other SM proteins have been shown to bind to their cognate syntaxins via an N-terminal motif (16Bracher A. Weissenhorn W. EMBO J. 2002; 21: 6114-6124Crossref PubMed Scopus (131) Google Scholar, 17Hu S.H. Latham C.F. Gee C.L. James D.E. Martin J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8773-8778Crossref PubMed Scopus (112) Google Scholar, 18Latham C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar, 19Yamaguchi T. Dulubova I. Min S.W. Chen X. Rizo J. Südhof T.C. Dev. Cell. 2002; 2: 295-305Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), allowing interactions that are associated with a positive role for SM proteins in SNARE-mediated membrane fusion (20Peng R. Gallwitz D. J. Cell Biol. 2002; 157: 645-655Crossref PubMed Scopus (121) Google Scholar). Despite biochemical evidence supporting a negative regulatory role for Munc18-1, there is strong genetic evidence for a critical positive role for Munc18-1 in exocytosis, as demonstrated by a Munc18-1 knock-out mouse that exhibits a complete blockage of neurotransmission (21Verhage M. Maia A.S. Plomp J.J. Brussaard A.B. Heeroma J.H. Vermeer H. Toonen R.F. Hammer R.E. van den Berg T.K. Missler M. Geuze H.J. Südhof T.C. Science. 2000; 287: 864-869Crossref PubMed Scopus (1008) Google Scholar). Recently, a short N-terminal peptide from Sx1a was also shown to bind to Munc18-1 via a novel interaction that promotes SNARE-mediated fusion of liposomes in vitro (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Moreover, the N-terminal truncation of Sx1a only affects the binding of the open conformation of Sx1a to Munc18-1 occurring near the plasma membrane (10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Oddly, the Munc18-1-Sx1a interaction in solution predominantly involves the closed conformation (10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), which raises the question of the relative contribution of the Sx1a N terminus to the overall Munc18-1-Sx1a interaction. To address the functional significance of the N-terminal interaction, we took a reverse strategy and investigated the molecular nature of the binding of Munc18-1 to the Sx1a N terminus and to the SNARE complex. We mutated Munc18-1:Phe115 and Glu132, which are located in an evolutionarily conserved surface pocket of Munc18-1 (17Hu S.H. Latham C.F. Gee C.L. James D.E. Martin J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8773-8778Crossref PubMed Scopus (112) Google Scholar, 22Burkhardt P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar). We found that whereas these mutations have only a mild effect on the binding of Munc18-1 to Sx1a in its free state, they completely abrogate Munc18-1 binding to the SNARE complex. Expression of these mutants in PC12 cells lacking Munc18-1 rescues exocytosis to a similar extent to that obtained with Munc18-1-WT. Total internal reflection fluorescence (TIRF) microscopy of vesicle docking at the cell surface reveals that these mutations support exocytosis. However, the rate of vesicle fusion is markedly reduced at the onset of stimulation, for a role of Munc18-1 in the of SNARE-mediated vesicle fusion exocytosis. for the complex Data Bank and the obtained from the Protein Data was W. for Sx1a peptide binding to of Munc18-1 was in the following the of Munc18-1 was the of the The peptide binding of the complex was with the in the Munc18-1 in the peptide of the of the complex mutated in to the Sx1a T.J. 1994; PubMed Scopus Google Scholar). Expression of in the been C.F. Meunier F.A. J. 2007; Google Scholar). mutants and from the a and as and and mutant was by a at to the N-terminal Munc18-1 mutant was by an at by and with the to was into also with the mutations the strategy as Y and growth hormone also used to cells as Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). and both in fusion proteins in by in Sx1a was also in the fusion proteins similar to C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar). The was from proteins at in was from glutathione with Sx1a was in with The SNARE complex was by and in a and at in and The complex was on via the on and by to the in the protein with Munc18-1-WT. of on a in a with a of a of a of and of by proteins on with a of protein in the of protein to the was by and a the with and a of was with in and mutant binding to Sx1a was by and for SNARE complex SNARE with a of at in and proteins to by was for of Munc18-1 was as F. Y. Dulubova I. M. X. Südhof T.C. Rizo J. J. Cell Biol. PubMed Scopus Google Scholar). and used for the as C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar). proteins by into and was at a with of in into in of the was to and the binding The free of binding was from the and the binding was from the with protein well the for the of protein in as the of mean of Sx1a Munc18-1 cells with the cells with and with and by and and cells with and and the cells and with by and a as J. Meunier F.A. Mol. Biol. Cell. 2008; PubMed Google Scholar). Munc18-1 PC12 cells at in with of with soluble human placental alkaline and of of the wild type the Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). the cells and in the cells with physiological saline solution and and was with of and was a at by to and at for and the in of The of into the and in the cells by the at for to alkaline and an was for placental alkaline phosphatase with the The total of the was to 10 was by a cells on a with expression proteins as in the in cells with in to the cells with and with and for at was by to and used for the as C. M. H. N. Meunier F.A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). cells on in in The of from cells was as the of an as F.A. J. G. Mol. Biol. Cell. PubMed Scopus Google Scholar). are of in was an with a and with a the by for at cells with human growth hormone and The cells on and to for in and in with Ca2+ following the onset of Following the by The of vesicle was of the of The mean square displacement of vesicle was from the and to vesicle in J.A. W. J. Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The is by the following is an to the of the the the and was obtained by The is by the following in of the of the and the of positive for both and the was obtained with The was therefore from and of and analysis and the as mean Data analysis was at are as mean and are and are key Munc18-1 predicted to the interaction the N-terminal peptide of Sx1a and a predominantly hydrophobic pocket on the surface of Munc18-1 we used a of the Munc18-1-Sx1a N-terminal interaction and interactions with the of the SM protein, Munc18-1-Sx1a was by the for the of the Munc18-1-Sx1a complex recently by and P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar) The key interaction is and (17Hu S.H. Latham C.F. Gee C.L. James D.E. Martin J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8773-8778Crossref PubMed Scopus (112) Google Scholar, 18Latham C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar), which is conserved in the Munc18-1-Sx1a interaction and as well as cognate the N-terminal binding (17Hu S.H. Latham C.F. Gee C.L. James D.E. Martin J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8773-8778Crossref PubMed Scopus (112) Google Scholar). a conserved motif of Sx1a been shown to to interactions with Munc18-1 via the hydrophobic pocket (17Hu S.H. Latham C.F. Gee C.L. James D.E. Martin J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8773-8778Crossref PubMed Scopus (112) Google Scholar, 22Burkhardt P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar, M. Dulubova I. J. H. Rizo J. Südhof T.C. J. Neurosci. 2007; 27: PubMed Scopus Google Scholar). on the an interaction with in this motif. We to the impact of key Munc18-1 and on binding to We mutants of Munc18-1 at and at as well as a double mutant to disrupt the binding pocket and their contribution to the interactions with Sx1a and with the SNARE complex. We the interaction of Sx1a with Munc18-1 and In Munc18-1 for Sx1a which only of Munc18-1 and of and only Sx1a was to and with and the by and In this the was from the N terminus of the Munc18-1 proteins and the Sx1a was to the via its to However, with the Munc18-1 mutants a in Sx1a binding in the of the detectable binding to Sx1a at that the mutants still a as by the strong in is not the peptide binding surface only of the total surface Sx1a is to Munc18-1 of a total of P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar). of the Munc18-1 and to that the Munc18-1 protein is The reduced Sx1a binding for and double mutant was and with Sx1a binding The in in binding to Sx1a with whereas the in in binding to Sx1a The double in a in binding to Sx1a with analysis of the Munc18-1 mutants detectable with the the that reduced binding by a protein To these interactions and the in binding to Sx1a was a of was into the double binds to with a of is the of by P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar) but is in with the obtained by F. Y. Dulubova I. M. X. Südhof T.C. Rizo J. J. Cell Biol. PubMed Scopus Google Scholar), and with obtained by M. Dulubova I. J. H. Rizo J. Südhof T.C. J. Neurosci. 2007; 27: PubMed Scopus Google Scholar, J. J.E. N. Scheller R.H. 1994; 13: Full Text PDF PubMed Scopus Google Scholar). the reduced in the of the Sx1a binding to was not with that of In the binding was by the with obtained for the and for is with that of P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar) also a of in the of binding the Sx1a N-terminal peptide was the binding of is characterized by a contribution for the and to binding is that the overall binding surface is reduced the Sx1a peptide is to but there is a by the of the Sx1a N-terminal upon that the Munc18-1 mutant binding to on the reduced Sx1a binding in which is not an to these binding that of the Munc18-1 hydrophobic predicted to binding to the Sx1a N-terminal affects the and of but not a in the binding for the binding of to Munc18-1 by as mean for at as mean for at as mean for at K. in a In of the that Munc18-1 binds to the SNARE complex, we investigated the role of the Munc18-1 hydrophobic pocket in mediating this interaction (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, C.F. Meunier F.A. J. 2007; Google Scholar). demonstrated (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), we found that bind to the SNARE complex of and and a in the Munc18-1 hydrophobic Munc18-1 interaction with the SNARE complex the double mutant was also to bind the SNARE complex and The Munc18-1 hydrophobic pocket is therefore critical to binding to the SNARE complex, an interaction that been shown to promote membrane fusion (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). is in with that the SNARE Sx1a the (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) an N-terminal truncation of Sx1a I. M. S. I. Südhof T.C. Rizo J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: PubMed Scopus Google Scholar) not with are key of Sx1a and Munc18-1, the N-terminal and SNARE helix of Sx1a P. Hattendorf D.A. Weis W.I. Fasshauer D. EMBO J. 2008; 27: 923-933Crossref PubMed Scopus (197) Google Scholar) the helix to in Munc18-1 binding with cognate it is that the relative of the and N-terminal peptide associated with SNARE In support of by and F. Y. Dulubova I. M. X. Südhof T.C. Rizo J. J. Cell Biol. PubMed Scopus Google Scholar) mutations in the Munc18-1 protein the Sx1a and found that they are for SNARE complex but not binding to free the of the Sx1a N-terminal peptide and support a Munc18-1 at binding a Sx1a conserved and a only Sx1a N-terminal peptide and that is for Munc18-1 with SNARE also that the SNARE complex, with Munc18-1 and that these interactions are involved in a positive regulatory role on SNARE-mediated fusion (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, I. M. S. I. Südhof T.C. Rizo J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: PubMed Scopus Google Scholar, Jahn R. Lang T. Biol. 2006; PubMed Scopus Google Scholar). In that the hydrophobic pocket of Munc18-1 is for interaction with the SNARE complex and to a extent to the free Sx1a binding. demonstrated that mutations in key of the Munc18-1 hydrophobic pocket SNARE complex binding to a extent with free we investigated the functional significance of this interaction. of evidence have recently shown that Munc18-1 Sx1a to the plasma membrane (10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar, P. B. N. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). Moreover, a in the Munc18-1-Sx1a interaction been found to at the of the plasma membrane (10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In the of the plasma membrane, Munc18-1 binding through the N terminus of thereby the interaction of Sx1a with (10Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). raises the question as to the N-terminal interaction is for the of to the plasma membrane. We therefore the Munc18-1 double mutant lacking the to bind to the N terminus of Sx1a was of Sx1a to the plasma membrane. The of Munc18-1 in cells affects the of Sx1a to the plasma membrane as Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). In these expression of plasma membrane localization as shown Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). expression of was also of Sx1a to the plasma membrane not support a role for the N-terminal interaction in the of Sx1a to the plasma membrane. We and mutants of in Munc18-1 PC12 cells. cells with a allowing the expression of with a soluble of which in a Ca2+-dependent and by of alkaline phosphatase of as Y. Y. E. B. S. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). Expression of the of by is also to exocytosis in Munc18-1 knock-out cells A. de H. I. O. Toonen R. J. Verhage M. J. Neurosci. 2007; 27: PubMed Scopus Google Scholar, T. Toonen R.F. de H. T. J. Südhof T.C. E. Verhage M. Full Text Full Text PDF PubMed Scopus Google Scholar). Expression of a of exocytosis, reduced with the by expression of and was also to exocytosis with the functional not from that of and The of in PC12 cells is in We this the of cells with and for and by with that both in the secretory cells with and and of found in the and mutant effect thereby Although the of and was to a extent in cells upon expression of not of the process of exocytosis. To this was cells with and to exocytosis in these cells and a short of following stimulation, the rate of fusion of the vesicle in cells was in cells and However, this effect was and was at stimulation, the vesicle of cells was as with that of cells Although the was not the of vesicles was reduced in cells The effect of on the rate of vesicle fusion and their and was only and the overall impact of these to the of and was However, it is that in the of vesicles in neurotransmitter from neurotransmitter relies on the of synaptic process is on the of fusion and is by cleavage F.A. G. J. J. 2002; PubMed Scopus Google Scholar) through genetic of M. Y. Hammer R.E. C. C.F. Südhof T.C. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). have found that mutations of Munc18-1 that also SNARE complex binding not neurotransmitter in Munc18-1 knock-out and F. Y. Dulubova I. M. X. Südhof T.C. Rizo J. J. Cell Biol. PubMed Scopus Google Scholar, P. A. Biochem. J. PubMed Scopus Google Scholar). However, in these from the Munc18-1 the effect was to a in the of synaptic in the effect of Munc18-1 on the fusion J. Science. PubMed Scopus Google Scholar), an is that the Munc18-1 mutations SNARE complex binding the of of the fusion is to the nature of these Despite the that the Sx1a N-terminal interaction with Munc18-1 is critical for SNARE fusion of vesicles (6Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, M. Dulubova I. J. H. Rizo J. Südhof T.C. J. Neurosci. 2007; 27: PubMed Scopus Google Scholar), that this not to Sx1a to the plasma membrane and only a limited impact on Ca2+-dependent neuroexocytosis in PC12 cells. to this interaction functional in an We Fasshauer and Rizo for their of the Sx1a and Munc18-1 Martin for critical of an of the for with for in of the Munc18-1 and for on the We also to and for and with the with

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