Cai J, Daoud R, Georges E, Gros P
Department of Biochemistry and Institute of Parasitology, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec, Canada H3G 1Y6.
Biochemistry. 2001 Jul 27;40(28):8307-16. doi: 10.1021/bi010093c.
Overexpression of the multidrug resistance-associated protein (MRP1) causes multidrug resistance in cultured cells. MRP1 transports a large number of glutathione, glucuronide, and sulfate-conjugated organic anions by an ATP-dependent efflux mechanism. Six other MRP proteins exist (MRP2-7), and mutations in some of these genes cause major pathological conditions in humans. A detailed characterization of the structure and mechanism of action of these proteins requires an efficient expression system from which large amounts of active protein can be obtained. We report the expression of a recombinant MRP1 in the methylotrophic yeast Pichia pastoris. The protein is expressed in the membrane fraction of these cells, as a stable and underglycosylated 165 kDa peptide. Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants. MRP1 expressed in P. pastoris binds 8-azido[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion, which can be competed by a molar excess of ADP and ATP. Under hydrolysis conditions (at 37 degrees C), orthovanadate induces trapping of the 8-azido[alpha-(32)P]nucleotide in MRP1, which can be further modulated by known MRP1 ligands. MRP1 is also labeled by a photoactive analogue of rhodamine 123 (IAARh123) in P. pastoris/MRP1 membranes, and this can be competed by known MRP1 ligands. Finally, MRP1-positive membrane vesicles show ATP-dependent uptake of LTC(4). Thus, MRP1 expressed in P. pastoris is active and shows characteristics of MRP1 expressed in mammalian cells, including drug binding, ligand-modulated formation of the MRP1-MgADP-P(i) intermediate (ATPase activity), and ATP-dependent substrate transport. The successful expression of catalytically active and transport-competent MRP1 in P. pastoris should greatly facilitate the efficient production and isolation of the wild type or inactive mutants of MRP1, or of other MRP proteins for structural and functional characterization.
多药耐药相关蛋白1（MRP1）的过表达会导致培养细胞产生多药耐药性。MRP1通过一种ATP依赖的外排机制转运大量谷胱甘肽、葡糖醛酸和硫酸酯结合的有机阴离子。还存在其他六种MRP蛋白（MRP2 - 7），其中一些基因的突变会导致人类出现主要的病理状况。要详细表征这些蛋白的结构和作用机制，需要一个高效的表达系统，从中可获得大量活性蛋白。我们报道了重组MRP1在甲基营养型酵母毕赤酵母中的表达。该蛋白在这些细胞的膜部分表达，为一种稳定且低糖基化的165 kDa肽。表达水平非常高，比多药耐药的HeLa/MRP1转染细胞中的表达水平高30倍。在毕赤酵母中表达的MRP1以Mg(2+)依赖且对EDTA敏感的方式结合8 - 叠氮基[α-(32)P]ATP，这可被摩尔过量的ADP和ATP竞争。在水解条件下（37摄氏度），正钒酸盐诱导8 - 叠氮基[α-(32)P]核苷酸在MRP1中捕获，这可被已知的MRP1配体进一步调节。在毕赤酵母/MRP1膜中，MRP1也被罗丹明123的光活性类似物（IAARh123）标记，这可被已知的MRP1配体竞争。最后，MRP1阳性膜囊泡显示出ATP依赖的白三烯C4摄取。因此，在毕赤酵母中表达的MRP1具有活性，并表现出在哺乳动物细胞中表达的MRP1的特征，包括药物结合、配体调节的MRP1 - MgADP - P(i)中间体形成（ATP酶活性）以及ATP依赖的底物转运。在毕赤酵母中成功表达具有催化活性和转运能力的MRP1应极大地促进MRP1野生型或无活性突变体或其他MRP蛋白的高效生产和分离，用于结构和功能表征。
