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

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.