title: Munc18-1 regulates early and late stages of exocytosis via syntaxin-independent protein interactions.
abstract: Sec1/Munc18 (SM) proteins are involved in various intracellular membrane trafficking steps. Many SM proteins bind to appropriate syntaxin homologues involved in these steps, suggesting that SM proteins function as syntaxin chaperones. Organisms with mutations in SM genes, however, exhibit defects in either early (docking) or late (fusion) stages of exocytosis, implying that SM proteins may have multiple functions. To gain insight into the role of SM proteins, we introduced mutations modeled on those identified in Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae into mammalian Munc18-1. As expected, several mutants exhibited reduced binding to syntaxin1A. However, three mutants displayed wild-type syntaxin binding affinities, indicating syntaxin-independent defects. Expression of these mutants in chromaffin cells either increased the rate and extent of exocytosis or altered the kinetics of individual release events. This latter effect was associated with a reduced Mint binding affinity in one mutant, implying a potential mechanism for the observed alteration in release kinetics. Furthermore, this phenotype persisted when the mutation was combined with a second mutation that greatly reduced syntaxin binding affinity. These results clarify the data on the function of SM proteins in mutant organisms and indicate that Munc18-1 controls multiple stages of exocytosis via both syntaxin-dependent and -independent protein interactions.
authors.affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
authors.forename: Leonora F
authors.initials: LF
authors.lastname: Ciufo
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Barclay
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
authors.forename: Alan
authors.initials: A
authors.lastname: Morgan
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology; D000483:Alleles; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology; D004594:Electrophysiology; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D051938:Munc18 Proteins; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C497937:STXBP1 protein, human; C497939:Stxbp1 protein, rat; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase