The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump

• Published in: The Journal of biological chemistry 1996-03, Vol.271 (11), p.6509-6517
• Main Authors: Li, Z S, Szczypka, M, Lu, Y P, Thiele, D J, Rea, P A
• Format: Article
• Language: English
• Subjects: ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Base Sequence; Biological Transport, Active; cadmio; cadmium; Cadmium - pharmacology; Carrier Proteins - genetics; Carrier Proteins - metabolism; cell membranes; detoxificacion metabolica; detoxification metabolique; Dinitrochlorobenzene - pharmacology; DNA Primers - genetics; DNA, Fungal - genetics; Drug Resistance, Microbial - genetics; Fungal Proteins - genetics; Fungal Proteins - metabolism; gene; genes; Genes, Fungal; glutathion; glutathione; Glutathione - metabolism; glutation; Humans; Kinetics; membranas celulares; membrane cellulaire; Membrane Transport Proteins; metabolic detoxification; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Plasmids; proteinas; proteine; proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Uncoupling Agents - pharmacology; vacuola; vacuole; vacuoles; Vacuoles - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.11.6509

The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515-amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J. (1994) J. Biol. Chem. 269, 22853-22857). Direct comparisons between S. cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild type strain, DTY165, demonstrate that YCF1 is required for increased resistance to the toxic effects of the exogenous glutathione S-conjugate precursor, 1-chloro-2,4-dinitrobenzene, as well as cadmium. Whereas membrane vesicles isolated from DTY165 cells contain two major pathways for transport of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway, the corresponding membrane fraction from DTY167 cells is more than 90% impaired for MgATP-dependent, uncoupler-insensitive DNP-GS transport. Of the two DNP-GS transport pathways identified, only the MgATP-dependent, uncoupler-insensitive pathway is subject to inhibition by glutathione disulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-dependent, uncoupler-insensitive conjugate transport in vitro strictly copurifies with the vacuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the fluorescent glutathione-conjugate, monochlorobimane-GS. Introduction of plasmid borne, epitope-tagged gene encoding functional YCF1 into DTY167 cells alleviates the 1-chloro-2,4-dinitrobenzene-hypersensitive phenotype concomitant with restoration of the capacity of vacuolar membrane vesicles isolated from these cells for MgATP-dependent, uncoupler-insensitive DNP-GS transport. On the basis of these findings, the YCF1 gene of S. cerevisiae is inferred to encode an MgATP.