title: The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.
abstract: Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins.
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Daiki
authors.initials: D
authors.lastname: Kawada
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Tomita
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Nakata
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan; Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagano
authors.affiliation: Institute of Science and Technology Austria, Am Campus 1, A-3400 Klosterneuburg, Austria.
authors.forename: Daria Elisabeth
authors.initials: DE
authors.lastname: Siekhaus
authors.affiliation: Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan; Faculty of Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan. Electronic address: yama_jun@aoni.waseda.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, Katsushika-ku, Tokyo 125-8585, Japan; Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585, Japan. Electronic address: jtosiscb@rs.noda.tus.ac.jp.
authors.forename: Jiro
authors.initials: J
authors.lastname: Toshima
mesh_terms: D004705:Endocytosis; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000378:metabolism; D051964:Receptors, Mating Factor / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D020690:GTPase-Activating Proteins; C514182:Glo3 protein, S cerevisiae; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D051964:Receptors, Mating Factor; C083241:SNC1 protein, S cerevisiae; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: Arf-GAP; Endocytosis; Intracellular trafficking; Small GTPase; Vesicular trafficking