The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

Abstract

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCFI) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione Sconjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.Members of the adenosine 5'-triphosphate (ATP)-binding cassette family (ABC) of transporter proteins play a central role in cellular detoxification processes in animals (1), fungi (2, 3), and plants (4,5). This is illustrated by multidrug resistant cancer cells, resistant against a range of drugs with different chemical structures and cellular targets following exposure to a single cytotoxic drug (1). At least two ABC transporters can contribute to multidrug resistance in human cancer cells: The MDR1 P-glycoprotein (for review see ref. 1) and the multidrug resistance-associated protein (MRP) (6). Both are plasma membrane glycoproteins that can lower the intracellular concentration of natural product drugs (1,7,8).Another cellular detoxification mechanism with broad substrate specificity is the enzymatic (transferase-mediated) or nonenzymatic association of compounds with the thiol group of glutathione (GSH 11). These transporters have not only been found in many mammalian cell types (9), but also in yeast (3) and plant cells (4, 12). They have a relatively broad substrate specificity, but prefer substrates containing a large hydrophobic moiety and two negative charges (9, 10). GS-X pumps are also involved in the detoxification of heavy metals (13-15).Several recent studies indicate that the human MRP is one of these elusive GS-X pumps: (i) Overexpression of MRP in human cancer cells resulted in an increased ATP-dependent transport of GSH S-conjugates into isolated plasma membrane vesicles (16,17). (ii) Transfection of an MRP cDNA in pig kidney epithelial cells increased the basolateral export of a GSH S-conjugate (18). (iii) The export of anti-cancer drugs by MRP requires intracellular GSH (19,20). (iv) Overexpression of MRP increased the efflux of GSH from human lung canc...

Transport of the glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP

“…2 and 3). These values are similar to those for DNP-[3H]SG [5,6], suggesting that EA-SG and DNP-SG are transported with equal efficiencies by MRP. Unmodified EA (10 ~tM) was taken up into membrane vesicles of lung cancer cells, but uptake was independent of MgATP and was not increased in the MRP-overexpressing cell lines, indicating that unmodified EA is not a substrate of MRP (results not shown).…”

“…We recently showed that expression of human MRP cDNA in a S. cerevisiae mutant strain with a disrupted YCF1 gene (strain DTY168 [25]) was able to restore the uptake of DNP-SG in microsomal vesicles isolated from DTYI68 [6]. Vesicles from DTY 168 cells expressing MRP cDNA (strain DTY168(MRP) [6]) also showed time-and MgATP-dependent uptake of [14C]EA-SG (Fig.…”

“…This cell line was isolated by a multistep selection of GLC4 cells up to 1152 nM doxorubicin [3,5]. The yeast strain DTY168 (Mato~, ura3-52, ycflA::his, leu2-3, -112, his6) [25] was used for heterologous expression of the MRP cDNA [6], (109 cells) were washed twice, scraped into ice-cold phosphate-buffered saline (PBS) and collected by centrifugation at 1000×g for 10 rain at 4°C, GLC4 and GLC4/ADR cells were harvested by centrifugation and washed twice with PBS. The cell pellets were resuspended in 50 ml of a hypotonic buffer (1 mM Tris-HC1, pH 7.0) and stirred gently for 2 h on ice in the presence of 400 units of recombinant Serratia marcescens nuclease (Benzonase, grade I, protease-free; Merck Darmstadt, Germany) and the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), leupeptide (2 ~tg/ml), pepstatin (1 gg/ml), and aprotinin (2 gg/ml).…”

“…Membrane vesicles from S. cerevisiae cells were prepared as described by Tommasini et al [6]. In short, cells were grown overnight in SD medium (0.7% (w/v) yeast nitrogen base without amino acids (Difco), 0.5% (w/v) casamino acids (Difco), 2% (w/v) glucose) to an OD600 of approx.…”

“…(iii) Drug transport by MRP is relatively insensitive to classic reversal agents of Pgp-mediated multidrug resistance (MDR), such as verapamil and cyclosporin (analogues) [2,3], but is modulated by inhibitors of organic anion transport, such as probenecid and sulfinpyrazone [6,7,9,10]. These data suggest that MRP is a GS-X pump [I1] and link MRP-mediated MDR to drug resistance associated with GSH and GSH S-transferases (GSTs) [5,8,12].…”