ATP-DEPENDENT TRANSPORT OF ORGANIC-ANIONS IN SECRETORY VESICLES OF SACCHAROMYCES-CEREVISIAE

Secretory mutants (sec1, sec6) of Saccharomyces cerevisiae accumulate large pools of secretory vesicles at the restrictive temperature (37 d egrees C) because of a block in the delivery of vesicles to the cell s urface. We report that secretory vesicles isolated from sec mutants ex hibit ATP-dependent uptake of two classes of organic anions that are s ubstrates for the canalicular carriers of mammalian liver. Transport o f the bile acid taurocholate (TC) and the glutathione conjugate of 1-c hloro-2,4-dinitrobenzene (GS-DNP) into vesicles was temperature depend ent and saturable and required ATP and Mg2+. Estimates of K-m and V-ma x were 177 mu M and 1.2 nmol.min(-1) mg(-1) and 262 mu M and 0.53 nmol min(-1) mg(-1) for TC and GS-DNP, respectively. TC and GS-DNP did not compete for transport. TC transport was sensitive to vanadate and 4,4 '-diisothiocyanostilbene-2,2'-disulfonate inhibited by glycocholate, a nd retained partial activity when UTP and GTP, but not nonhydrolyzable ATP analogues, replaced ATP. Dissipation of the electrochemical poten tial with a nitrate buffer and ionophores partially decreased (30-40%) the transport of both anions. Direct testing of the influence of memb rane potential was performed in sec6-4 mutants, in which the expressio n of electrogenic [H+]ATPase activity is reduced by >85% in glucose co ntaining medium. Vesicles from sec6-4 retained full activity for ATP-d ependent TC and GS-DNP transport. These results indicate that the tran sporters operate independently of the membrane potential and that ATP is required. These findings reveal that yeast possess separate ATP-dep endent transport mechanisms for elimination of bile acids and glutathi one conjugates. The mechanisms are functionally similar to those prese nt in mammalian systems.