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...

Reestablishment of the resting state after stimulus-coupled elevations of cytosolic-free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. Here we describe the isolation of two genes, CAXI and CAX2, from Arabidopsis thaliana that suppress a mutant of Saccharomyces cerevisiae that has a defect in vacuolar Ca2+ accumulation. (20). RNA was purified and analyzed as described (21). Degenerate PCR was performed using tomato genomic DNA as template. The sequences of the CAX primers were 5'-GCTTTTATCAGTRYCATCTTGCTTCC-3', where R = A or G and Y = C or T, and 5'-GTCCATCTKTTGMC-CCATCCAWCCAAT-3', where K = T or G, M = C or A, and W = A or T. Amplification was for 50 cycles using the following thermal profile: 94°C for 1 min, 55°C for 1.5 min, 72°C for 1 min, followed by a final 5 min of extension at 72°C. The PCR product was isolated, cloned into pGEM-T vector (Promega) and sequenced.

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Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m(-3) nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m(-3) (n=19) and 41.8 ± 2.6 mol · m(-3) (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m(-3) (n = 4) and 72.8 ± 8.4 mol · m(-3) (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m(-3) (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m(-3) (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m(-3), while the vacuole accumulated nitrate to 30-50 mol · m(-3). The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.