Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 14, Pages 9639-9650Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.514273
Keywords
Exocytosis; Membrane Fusion; Membrane Reconstitution; Membrane Trafficking; Snare Proteins; SM Protein; VAMP2; Synaptobrevin; Synaptic Vesicle
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Funding
- German Research Foundation [SFB/TRR 83]
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Background: Munc18-1 is required for membrane fusion, but the underlying mechanism is unknown. Results: Distinct point mutations in domain 3a of Munc18-1 differentially affect the conformation of helix 12, VAMP2 binding, and membrane fusion. Conclusion: A conformational switch in helix 12 promotes SNAREpin assembly via the VAMP2 interaction. Significance: The Munc18-1-VAMP2 interaction may represent a general molecular mechanism of how SM proteins accelerate membrane fusion. Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended -helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.
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