title: Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process.
abstract: Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.
authors.forename: Kirk W
authors.initials: KW
authors.lastname: Donovan
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853 apb5@cornell.edu.
authors.forename: Anthony
authors.initials: A
authors.lastname: Bretscher
mesh_terms: D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D018995:Myosin Heavy Chains / Q000378:metabolism; D024701:Myosin Type V / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D055571:Secretory Pathway; D022142:Secretory Vesicles / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: C077512:MYO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D024701:Myosin Type V; C064320:SEC4 protein, S cerevisiae; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins