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Vol. 18, Issue 12, 4957-4968, December 2007

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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^*,, and T.F.J. Martin^*

Departments of *Biochemistry and Mathematics, University of Wisconsin, Madison, WI 53706

Submitted April 24, 2007; Revised September 21, 2007; Accepted September 26, 2007
Monitoring Editor: Patrick Brennwald

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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

Address correspondence to: T.F.J. Martin (tfmartin{at}wisc.edu).

Abbreviations used: SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNAP25, synaptosomal associated protein of 25 kDa; VAMP, vesicle-associated membrane protein; PS, phosphatidylserine; PIP2, phosphatidylinositol-4,5-bisphosphate; TIRF, total internal reflection fluorescence; ANF-EGFP, atrial naturetic factor-enhanced green fluorescent protein; CgB, chromogranin B.