Ca²⁺-dependent synaptotagmin binding to SNAP-25 is essential for Ca²⁺-triggered exocytosis

Synaptotagmin is a proposed Ca²⁺ sensor on the vesicle for regulated exocytosis and exhibits Ca²⁺-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca²⁺ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca²⁺ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca²⁺-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca²⁺-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca²⁺-dependent fusion step. These results indicate that the Ca²⁺-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca²⁺-dependent triggering of membrane fusion.