title: Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.
abstract: 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.
authors.affiliation: Children's Cancer Research Group, Leeds Institute of Cancer and Pathology, St. James's University Hospital, Leeds, United Kingdom; and e.a.roundhill@leeds.ac.uk.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Roundhill
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Doug
authors.initials: D
authors.lastname: Turnbull
authors.affiliation: Children's Cancer Research Group, Leeds Institute of Cancer and Pathology, St. James's University Hospital, Leeds, United Kingdom; and.
authors.forename: Susan
authors.initials: S
authors.lastname: Burchill
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D017313:Fenretinide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D055785:Gene Knockdown Techniques; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051336:Mitochondrial Membranes / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D003609:Dactinomycin; D004317:Doxorubicin; D017313:Fenretinide; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011500:Protein Synthesis Inhibitors; D059005:Topoisomerase II Inhibitors; C493165:HSP90AB1 protein, human; C421109:multidrug resistance-associated protein 1
keywords: ABC transporter; Ewing’s sarcoma; doxorubicin; multidrug resistance