Synaptotagmin C2A Loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

K. L. Lynch, R. R.L. Gerona, E. C. Larsen, R. F. Marcia, J. C. Mitchell, T. F.J. Martin

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

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 Ca 2+-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 Ca 2+-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.

Original languageEnglish
Pages (from-to)4957-4968
Number of pages12
JournalMolecular Biology of the Cell
Volume18
Issue number12
DOIs
StatePublished - Dec 2007
Externally publishedYes

Funding

FundersFunder number
National Institute of Diabetes and Digestive and Kidney DiseasesR01DK025861
U.S. National Library of MedicineT15LM007359

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