Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 18, Issue 12, Pages 4957-4968Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E07-04-0368
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Funding
- NIDDK NIH HHS [R01 DK025861, R37 DK025861, DK25861] Funding Source: Medline
- NLM NIH HHS [5T15LM007359-03, T15 LM007359] Funding Source: Medline
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Synaptotagmins contain tandem C2 domains and function as Ca2+ sensors for vesicle exocytosis but the mechanism for coupling Ca2+ rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca2+-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca2+-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca2+-dependent synaptotagmin-1 binding to SNAREs without affecting Ca2+-dependent membrane binding. The mutants failed to confer Ca2+ regulation on SNARE-dependent liposome fusion and failed to restore Ca2+-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca2+-dependent SNARE binding by synaptotagmin is essential for Ca2+-triggered vesicle exocytosis and that Ca2+-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca2+-dependent SNARE and membrane binding occur simultaneously.
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