title: Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy.
abstract: Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsusika-ku, Tokyo 125-8585, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Kawamura
authors.affiliation: Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsusika-ku, Tokyo 125-8585, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagano
authors.affiliation: Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsusika-ku, Tokyo 125-8585, Japan; Faculty of Science and Engineering, Waseda University, Wakamatsu 2-2, Shinjuku-ku, Tokyo 162-8480, Japan. Electronic address: yama_jun@aoni.waseda.ac.jp.
authors.forename: Junko Y
authors.initials: JY
authors.lastname: Toshima
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsusika-ku, Tokyo 125-8585, Japan; Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsusika-ku, Tokyo 125-8585, Japan. Electronic address: jtosiscb@rs.noda.tus.ac.jp.
authors.forename: Jiro
authors.initials: J
authors.lastname: Toshima
mesh_terms: D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; C079894:GYP6 protein, S cerevisiae; C094135:Rab6 protein; D029701:Saccharomyces cerevisiae Proteins; D020559:Monomeric GTP-Binding Proteins; C499307:YPT6 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: Budding yeast; N-terminal tagging; Rab/Ypt; Vesicle transport; Ypt6p