title: ATP-dependent transport of organic anions in secretory vesicles of Saccharomyces cerevisiae.
abstract: Secretory mutants (sec1, sec6) of Saccharomyces cerevisiae accumulate large pools of secretory vesicles at the restrictive temperature (37 degrees C) because of a block in the delivery of vesicles to the cell surface. We report that secretory vesicles isolated from sec mutants exhibit ATP-dependent uptake of two classes of organic anions that are substrates for the canalicular carriers of mammalian liver. Transport of the bile acid taurocholate (TC) and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene (GS-DNP) into vesicles was temperature dependent and saturable and required ATP and Mg2+. Estimates of Km and Vmax were 177 microM and 1.2 nmol.min-1.mg-1 and 262 microM and 0.53 nmol.min-1.mg-1 for TC and GS-DNP, respectively. TC and GS-DNP did not complete for transport. TC transport was sensitive to vanadate and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited by glycocholate, and retained partial activity when UTP and GTP, but not nonhydrolyzable ATP analogues, replaced ATP. Dissipation of the electrochemical potential with a nitrate buffer and ionophores partially decreased (30-40%) the transport of both anions. Direct testing of the influence of membrane potential was performed in sec6-4 mutants, in which the expression of electrogenic [H+]ATPase activity is reduced by > 85% in glucose-containing medium. Vesicles from sec6-4 retained full activity for ATP-dependent TC and GS-DNP transport. These results indicate that the transporters operate independently of the membrane potential and that ATP is required. These findings reveal that yeast possess separate ATP-dependent transport mechanisms for elimination of bile acids and glutathione conjugates. The mechanisms are functionally similar to those present in mammalian systems.
authors.affiliation: Department of Physiology, Tufts University School of Medicine, Boston, MA 02111.
authors.forename: M V
authors.initials: MV
authors.lastname: St-Pierre
authors.forename: S
authors.initials: S
authors.lastname: Ruetz
authors.forename: L F
authors.initials: LF
authors.lastname: Epstein
authors.forename: P
authors.initials: P
authors.lastname: Gros
authors.forename: I M
authors.initials: IM
authors.lastname: Arias
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D012265:Ribonucleotides / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D013656:Taurocholic Acid / Q000378:metabolism*
chemical_list: D012265:Ribonucleotides; C030190:S-(2,4-dinitrophenyl)glutathione; D013656:Taurocholic Acid; D000255:Adenosine Triphosphate; D005978:Glutathione; D008274:Magnesium