Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis.
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.
Lynch
K L
KL
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Gerona
R R L
RR
Larsen
E C
EC
Marcia
R F
RF
Mitchell
J C
JC
Martin
T F J
TF
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005089:Exocytosis; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism; D050857:Synaptotagmins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D002118:Calcium