title: Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion.
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.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Kara L
authors.initials: KL
authors.lastname: Lynch
authors.forename: Roy R L
authors.initials: RR
authors.lastname: Gerona
authors.forename: Dana M
authors.initials: DM
authors.lastname: Kielar
authors.forename: Sascha
authors.initials: S
authors.lastname: Martens
authors.forename: Harvey T
authors.initials: HT
authors.lastname: McMahon
authors.forename: Thomas F J
authors.initials: TF
authors.lastname: Martin
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology*; D009154:Mutation; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; D002118:Calcium
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