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Mol Biol Cell. 2008 Dec;19(12):5093-103. doi: 10.1091/mbc.E08-03-0235. Epub 2008 Sep 17.

Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion.

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  • 1Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

Abstract

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca(2+) is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca(2+) also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca(2+)-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca(2+)-dependent SNARE binding or in Ca(2+)-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca(2+)-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca(2+)-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca(2+)-dependent membrane insertion and SNARE binding to drive fusion pore expansion.

PMID:
18799625
[PubMed - indexed for MEDLINE]
PMCID:
PMC2592635
Free PMC Article
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