Abstract
Pdr5p is the yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Its high overproduction in Pdr1p transcription factor mutants allows us to study the molecular mechanism of multidrug transport and substrate specificity. We have developed new in vivo and in vitro assays of Pdr5p-mediated drug transport. We show that in spite of little sequence homology, and inverted topology in respect to that of mammalian P-glycoproteins, Pdr5p shares with them common substrates. Pdr5p extrudes rhodamines 6G and 123, from intact yeast cells in an energy-dependent manner. Plasma membrane preparations from a Pdr5p-overproducing strain exhibit ATP hydrolysis-dependent, osmotically sensitive rhodamine 6G fluorescence quenching. The quenching is competitively inhibited by micromolar concentrations of many anticancer drugs, such as vinblastine, vincristine, taxol, and verapamil, and of ionophoric peptides as well as steroids. In contrast, other anticancer drugs, like colchicine and some multidrug resistance modifiers, such as quinidine, exert noncompetitive inhibition. Our experimental system opens new possibilities for the analysis of structure-function relationship of multidrug transporter substrates and inhibitors.
Highlights
The growth of yeast cell shows little sensitivity to most of the anticancer agents [1], and it is not known whether any of the yeast ABC1 proteins exhibits the broad, yet limited multidrug transport specificity similar to that of the P-glycoproteins of the MDR type, which in mammalian cells are responsible for many failures in cancer chemotherapy [2,3,4]
Similar structural organization is found in other yeast ABC proteins constituting a new distinct family of yeast open reading frames sharing close homology with PDR5 [13, 14], including Snq2p which is involved in multidrug resistance [11, 15]
We show that Pdr5p shares with mammalian P-glycoproteins a series of common substrates and inhibitors, which were kinetically characterized in a new in vitro Pdr5p drug transport assay
Summary
Chemicals—Rhodamine 6G and sodium orthovanadate were from British Drug Chemicals, oligomycin from Janssen Chimica, 2 deoxy-Dglucose, progesterone, vinblastine, vincristine, taxol, tamoxifen, verapamil, puromycin, nifedipine, colchicine, yohimbine, quinidine, -estradiol, gramicidin D, nigericin, monensin, ATP disodium salt (A-3377), GTP, UTP, and sodium deoxycholate were from Sigma, ATP␥S tetralithium salt, CTP, ADP, chloramphenicol, Triton X-100, and n-octyl glucoside were from Boehringer Mannheim, valinomycin, FCCP, and CCCP were from Fluka, S13 was a kind gift from Dr P. Haman (Monsanto Co., St. Louis, MO), SF6847 was from Sumitomo Chemical Co. Ltd. Yeast Strains—The following S. cerevisiae strains were used in this study: US50-18C (MAT␣, PDR 1-3, ura 3, his-1) [18], US50-D5 (US5018C disrupted by pdr5-⌬3::URA3) [19], RW2802 (MATa, leu, met, ura, mak, KRE1), JG436 (RW2802 bearing PDR5::Tn5 insertion) [17], JG365-5C (MATa, pdr, PDR5::Tn5, met, ura3) [17] FY167928C (MATa, ura, trp1⌬63, leu2⌬1, his3⌬200, GAL2ϩ) Fairhead), FY1679-1D (MAT␣, ura, trp1⌬63, his3⌬200, GAL2ϩ) Fairhead), FYMKU-1/1 (FY1679-28C disrupted by pdr5⌬1::hisGURA3hisG) (this study), FYMKD-2/1 (diploid from cross between FYMKU-1/1 and FY1679-1D) (this study), FYMK-1/1 (FY167928C disrupted by pdr5-⌬1::hisG) (this study). The URA3 gene was removed by 5-fluoro-orotic acid treatment [21] of appropriate transformants
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