title: Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion.
abstract: The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca(2+) and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca(2+). In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3-9 min) that was required for subsequent Ca(2+)-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.
authors.affiliation: Department of Neuroscience, Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.forename: Huisheng
authors.initials: H
authors.lastname: Liu
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Gu
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D008561:Membrane Fusion; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050863:Synaptotagmin I / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; D050600:SNARE Proteins; D050863:Synaptotagmin I; D002118:Calcium