Abstract
Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the presynaptic membrane. Chaperones Munc18-1 and Munc13-1 cooperatively catalyze SNARE assembly via an intermediate 'template' complex containing Syntaxin-1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 'linker' region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~ 1 : 1 stoichiometry by the self-assembled Munc13-1 nanoclusters.
© 2020 Federation of European Biochemical Societies.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Binding Sites
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Lipid Bilayers / chemistry
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Lipid Bilayers / metabolism
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Liposomes / chemistry
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Liposomes / metabolism*
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Mice
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Models, Molecular
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Nerve Tissue Proteins / chemistry
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Optical Tweezers
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Phosphatidylcholines / chemistry
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Phosphatidylcholines / metabolism
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Phosphatidylethanolamines / chemistry
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Phosphatidylethanolamines / metabolism
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Phosphatidylserines / chemistry
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Phosphatidylserines / metabolism
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Polyethylene Glycols / chemistry
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Polyethylene Glycols / metabolism
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Synaptosomal-Associated Protein 25 / chemistry
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Synaptosomal-Associated Protein 25 / genetics
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Synaptosomal-Associated Protein 25 / metabolism*
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Syntaxin 1 / chemistry
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Syntaxin 1 / genetics
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Syntaxin 1 / metabolism*
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Vesicle-Associated Membrane Protein 2 / chemistry
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Vesicle-Associated Membrane Protein 2 / genetics
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Vesicle-Associated Membrane Protein 2 / metabolism*
Substances
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Lipid Bilayers
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Liposomes
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Nerve Tissue Proteins
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Phosphatidylcholines
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Phosphatidylethanolamines
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Phosphatidylserines
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Recombinant Fusion Proteins
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Snap25 protein, mouse
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Synaptosomal-Associated Protein 25
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Syntaxin 1
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Unc13a protein, mouse
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Vesicle-Associated Membrane Protein 2
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dioleoylphosphatidylethanolamine-polyethyleneoxide
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vesicle-associated membrane protein 2, mouse
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Polyethylene Glycols
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1,2-dioleoylphosphatidylserine
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1,2-oleoylphosphatidylcholine