Zs. Li et al., THE YEAST CADMIUM FACTOR PROTEIN (YCF1) IS A VACUOLAR GLUTATHIONE S-CONJUGATE PUMP, The Journal of biological chemistry, 271(11), 1996, pp. 6509-6517
The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, wh
ich was isolated according to its ability to confer cadmium resistance
, encodes a 1,515-amino acid ATP-binding cassette (ABC) protein with e
xtensive sequence homology to the human multidrug resistance-associate
d protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and
Thiele, D. J, (1994) J. Biol. Chem. 269, 22853-22857). Direct compari
sons 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 nitroben
zene, as well as cadmium. Whereas membrane vesicles isolated from DTY1
65 cells contain two major pathways for transport of the model compoun
d S-(2,4-dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncou
pler-insensitive pathway and an electrically driven pathway. the corre
sponding membrane fraction from DTY167 cells is more than 90% impaired
for MgATP-dependent, uncoupler-insensitive DNP-GS transport. Of the t
wo DNP-GS transport pathways identified, only the MgATP-dependent, unc
oupler-insensitive pathway is subject to inhibition by glutathione dis
ulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-d
ependent, uncoupler-insensitive conjugate transport in vitro strictly
copurifies with the vacuolar membrane fraction. Intact DTY165 cells, b
ut not DTY167 cells, mediate vacuolar accumulation of the fluorescent
glutathione-conjugate, monochlorobimane-GS. Introduction of plasmid bo
rne, epitope-tagged gene encoding functional YCF1 into DTY167 cells al
leviates the 1-chloro-2,4-dinitrobenzene hypersensitive phenotype conc
omitant 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 insensi
tive vacuolar glutathione S-conjugate transporter. The energy requirem
ents, kinetics, substrate specificity, and inhibitor profile of YCF1-m
ediated transport demonstrate that the vacuolar glutathione conjugate
pump of yeast bears a strong mechanistic resemblance to the MRP1-encod
ed transporter of mammalian cells and the cognate, but as yet molecula
rly undefined, function of plant cells.