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PMID: 17914059 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

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

Molecular biology of the cell ·Vol. 18 ·No. 12 ·2007-12-00 ·Pages 4957-68

Lynch KL, Gerona RR, Larsen EC, Marcia RF, Mitchell JC, Martin TF

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.

MeSH Terms
Animals Calcium/metabolism Cross-Linking Reagents Exocytosis 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 SNARE Proteins/metabolism Synaptosomal-Associated Protein 25/chemistry,metabolism Synaptotagmins/chemistry,genetics,metabolism
Chemicals
Cross-Linking Reagents Liposomes Phosphatidylinositol 4,5-Diphosphate Phosphatidylserines SNARE Proteins Synaptosomal-Associated Protein 25 Synaptotagmins Calcium
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Lynch K L
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Gerona R R L
Larsen E C
Marcia R F
Mitchell J C
Martin T F J
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e07-04-0368
Published
2007-12-00
Epub
2007-00-03
Pages
4957-68
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC2096586
Subset
IM
Grants
NIDDK NIH HHS · DK25861 · United States
NLM NIH HHS · 5T15LM007359-03 · United States
NLM NIH HHS · T15 LM007359 · United States
NIDDK NIH HHS · R37 DK025861 · United States
NIDDK NIH HHS · R01 DK025861 · United States
External Links
PubMed source DOI: 10.1091/mbc.e07-04-0368 PMC Full Text View journal details
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