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 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.