ABSTRACT
Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-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 Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-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 Ca(2+)-dependent SNARE and membrane binding occur simultaneously.
Subject(s)
Calcium/metabolism , Exocytosis , SNARE Proteins/metabolism , Synaptotagmins/metabolism , Animals , Cross-Linking Reagents , Liposomes/metabolism , Mass Spectrometry , Models, Biological , Models, Molecular , Mutation/genetics , PC12 Cells , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylserines/metabolism , Protein Binding , Protein Structure, Quaternary , Rats , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmins/chemistry , Synaptotagmins/geneticsABSTRACT
Chemical synapses contain specialized pre- and postsynaptic structures that underlie rapid synaptic transmission and its modulation. Studies of postsynaptic organization have revealed a network of interacting proteins that enable rapid synaptic responses and their modulation. Recent genetic and electrophysiological studies on two active zone proteins-RIM and Munc13-reveal important roles in priming vesicles for Ca(2+)-triggered fusion and in mediating the regulation of this process. This work sheds new light on how presynaptic structure provides speed and plasticity to synaptic transmission.
