title: Syntaxin/Munc18 interactions in the late events during vesicle fusion and release in exocytosis.
abstract: The SNARE proteins, syntaxin, SNAP-25, and VAMP, form part of the core machinery for membrane fusion during regulated exocytosis. Additional proteins are required to account for the speed, spatial restriction, and tight control of exocytosis and a key role is played by members of the Sec1/Munc18 family of proteins that have been implicated either in vesicle docking or fusion itself through their interactions with the corresponding syntaxin. Using amperometry to assay the kinetics of single vesicle fusion/release events in adrenal chromaffin cells, the effect of expression of syntaxin 1A mutants was examined. Overexpression of wild-type syntaxin or its cytoplasmic domain had no effect on the kinetics of release during single exocytotic events although the cytoplasmic domain reduced the frequency of exocytosis. In contrast, expression of either an open syntaxin 1A or the I233A mutant resulted in increased quantal size and a slowing of the kinetics of release. The wild-type and mutant syntaxins were overexpressed to a similar extent and the only common defect shown by the syntaxin 1A mutants was reduced binding to Munc18-1. These results are consistent with a role for Munc18-1 in controlling the late stages of exocytosis by binding to and limiting the availability of syntaxin in its open conformation. Modification of the Munc18-1/syntaxin 1A interaction would therefore be a key mechanism for the regulation of quantal size.
authors.affiliation: Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Graham
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Barclay
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D051938:Munc18 Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D050827:Syntaxin 1; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C494212:STX1A protein, human; C497937:STXBP1 protein, human; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase; D014357:Trypsin