title: Munc18a scaffolds SNARE assembly to promote membrane fusion.
abstract: Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.
authors.forename: Travis L
authors.initials: TL
authors.lastname: Rodkey
authors.forename: Song
authors.initials: S
authors.lastname: Liu
authors.forename: Meagan
authors.initials: M
authors.lastname: Barry
authors.forename: James A
authors.initials: JA
authors.lastname: McNew
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; D051360:Cyclin-Dependent Kinase 5