The yeast cadmium factor (YCF1) gene encodes an MgATP-energized glutathione S-conjugate transporter responsible for the vacuolar sequestration of organic compounds after their S-conjugation with glutathione. However, while YCF1 was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd 2؉ nor the relationship between this process and organic glutathione-conjugate transport are known. Here we show through direct comparisons between vacuolar membrane vesicles purified from Saccharomyces cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd⅐glutathione complexes. The substrate requirements, kinetics and Cd 2؉ ͞ glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd⅐GS 2 ). On the basis of these results-the Cd 2؉ hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd⅐GS 2 formation-this new pathway is concluded to contribute substantially to Cd 2؉ detoxification.A new class of ATP-binding cassette (ABC) transporter responsible for MgATP-energized transport of organic compounds after their conjugation with glutathione (GSH) has recently been discovered. Formerly designated the GS-X pump (1), this transporter, or family of transporters, has been implicated in the extrusion of a broad range of S-conjugated compounds from the cytosol.To date, two closely related GS-X pumps have been identified molecularly. These are the human multidrug resistanceassociated protein (MRP1) (2, 3) and the yeast cadmium factor (YCF1) protein (4, 5). MRP1 and YCF1 are 43% identical (63% similar) at the amino acid level, possess nucleotide binding folds with an equivalent spacing of conserved residues, and contain two subclass-specific structures, a central truncated cystic fibrosis transmembrane conductance regulatorlike ''regulatory'' domain, rich in charged amino acids, and an Ϸ200-amino acid residue N-terminal extension (2, 4). MRP1 catalyzes the MgATP-energized transport of leukotriene C 4 and related GSH S-conjugates (GS-conjugates) across the plasma membrane of mammalian cells (3, 6, 7). YCF1 catalyzes the transport of organic GS-conjugates into the vacuole of Saccharomyces cerevisiae (5).Given the participation of both of these integral membrane proteins in the transport of organic GS-conjugates and their implied role in the elimination and͞or sequestration of cytotoxic drugs, it is intriguing that the YCF1 gene was initially identified by screening a yeast genomic library for the ability of multicopy DNA fragments to confer resistance to cadmium salts in the growth medium (4). The question of how the vacuolar sequestration of organic GS-conjugates by YCF1 is related to Cd 2ϩ resistance therefore arises. Specifically, is the detox...
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-di-nitrobenzene, 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-insensive 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 acuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the quorescent 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-energized, uncoupler-insensitive vacuolar glutathione S-conjugate transporter. The energy requirements, kinetics, substrate specificity, and inhibitor profile of YCF1-mediated transport demonstrate that the vacuolar glutathione conjugate pump of yeast bears a strong mechanistic resemblance to the MRP1-encoded transporter of mammalian cells and the cognate, but as yet molecularly undefined, function of plant cells.
Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.
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