ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins

The transport systems involved in the export of cellular reduced glutathion e (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including M RP1 and MRP2. The present study examined the hypothesis that the yeast orth ologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [H-3]GSH transp ort was measured in vacuolar membrane vesicles iso lated from a control str ain ol Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that l acks a functional Ycf1p, and in DTY167 transformed with a 2-mu m plasmid ve ctor containing YCF1, GSH transport in control vacuolar membrane vesicles w as mediated largely by an ATP-dependent, low affinity pathway (K-m = 15 +/- 4 mM). ATP-dependent [H-3]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene -2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 a nd MRP2, but was minimally affected by membrane potential or pH gradient un couplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a function al Ycf1p but was restored to near wild-type levels in the DTY167 strain tra nsformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the ot her hand, expression and functional activity of a bile acid transporter, Ba t1p, and of the V-type ATPase were similar in all three yeast strains. Thes e results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functi onal homology between Ycf1p and MRP1 and MRP2, these data support the hypot hesis that GSH efflux from mammalian cells is mediated by these membrane pr oteins.