Roundhill Elizabeth, Turnbull Doug, Burchill Susan
Children's Cancer Research Group, Leeds Institute of Cancer and Pathology, St. James's University Hospital, Leeds, United Kingdom; and
Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, United Kingdom.
FASEB J. 2016 May;30(5):1712-23. doi: 10.1096/fj.15-283408. Epub 2015 Dec 31.
Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.
质膜多药耐药相关蛋白1（MRP-1）在尤因肉瘤（ES）中的过表达预示着预后不良。MRP-1也在线粒体中表达，我们研究了MRP-1在线粒体亚结构中的定位，并探究了MRP-1的转运机制以及该细胞器在对阿霉素反应中的作用。通过差速离心和用洋地黄皂苷溶解膜来检测ES细胞系中MRP-1的线粒体定位。通过免疫沉淀、免疫荧光显微镜检查以及使用小发夹RNA和抑制剂（阿朴唑、17-AAG和NVPAUY）敲除热休克蛋白（HSPs）来研究MRP-1是否由热休克蛋白陪伴。通过计数活细胞数量来研究破坏线粒体MRP-1依赖性外排活性对阿霉素细胞毒性作用的影响。线粒体MRP-1是糖基化的，定位于线粒体外膜，在那里它与HSP90共表达。MRP-1与HSP90和HSP70都结合，尽管只有抑制HSP90β会降低线粒体中MRP-1的表达。破坏线粒体MRP-1依赖性外排显著增加了阿霉素的细胞毒性作用（联合指数，<0.9）。我们首次证明线粒体MRP-1在线粒体外膜中表达，并且是HSP90β的客户蛋白，在那里它可能在阿霉素诱导的ES耐药中起作用。-朗希尔，E.，特恩布尔，D.，伯奇尔，S.伴侣蛋白HSP90β将MRP-1定位于线粒体外膜
