title: In vitro reconstitution of Rab GTPase-dependent vesicle clustering by the yeast lethal giant larvae/tomosyn homolog, Sro7.
abstract: Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.
authors.affiliation: From the Departments of Cell Biology and Physiology and.
authors.forename: Guendalina
authors.initials: G
authors.lastname: Rossi
authors.affiliation: From the Departments of Cell Biology and Physiology and.
authors.forename: Kelly
authors.initials: K
authors.lastname: Watson
authors.affiliation: From the Departments of Cell Biology and Physiology and.
authors.forename: Mallory
authors.initials: M
authors.lastname: Demonch
authors.affiliation: Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
authors.forename: Brenda
authors.initials: B
authors.lastname: Temple
authors.affiliation: From the Departments of Cell Biology and Physiology and pjbrennw@med.unc.edu.
authors.forename: Patrick
authors.initials: P
authors.lastname: Brennwald
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D001692:Biological Transport; D005089:Exocytosis; D015966:Gene Expression Regulation, Fungal; D006056:Golgi Apparatus / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D050767:Qc-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D050767:Qc-SNARE Proteins; C060862:SEC15 protein, S cerevisiae; C090117:SEC9 protein, S cerevisiae; C116430:SRO7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: Exocytosis; Membrane Reconstitution; Rab; Trafficking; Vesicles