Kolaczkowski M, van der Rest M, Cybularz-Kolaczkowska A, Soumillion J P, Konings W N, Goffeau A
Unité de Biochimie Physiologique, Université Catholique de Louvain, Place Croix du Sud 2/20, B-1348 Louvain la Neuve, Belgium.
J Biol Chem. 1996 Dec 6;271(49):31543-8. doi: 10.1074/jbc.271.49.31543.
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.
Pdr5p是酿酒酵母中的一种ATP结合盒转运蛋白，赋予酵母对多种不相关药物的抗性。在Pdr1p转录因子突变体中其高度过量表达使我们能够研究多药转运的分子机制和底物特异性。我们开发了新的体内和体外Pdr5p介导的药物转运检测方法。我们发现，尽管Pdr5p与哺乳动物P-糖蛋白的序列同源性较低，且拓扑结构相反，但它们具有共同的底物。Pdr5p以能量依赖的方式将若丹明6G和123从完整的酵母细胞中排出。来自Pdr5p过量表达菌株的质膜制剂表现出ATP水解依赖性、对渗透压敏感的若丹明6G荧光猝灭。许多抗癌药物，如长春碱、长春新碱、紫杉醇和维拉帕米，以及离子载体肽和类固醇的微摩尔浓度可竞争性抑制这种猝灭。相比之下，其他抗癌药物，如秋水仙碱和一些多药耐药调节剂，如奎尼丁，则产生非竞争性抑制作用。我们的实验系统为分析多药转运蛋白底物和抑制剂的结构-功能关系开辟了新的可能性。
