Functional reconstitution of substrate transport by purified multidrug resistance protein MRP1 (ABCC1) in phospholipid vesicles.
The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69-82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.
Mao
Q
Q
Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Deeley
R G
RG
Cole
S P
SP
D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D018432:Drug Resistance, Multiple; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010743:Phospholipids / Q000378:metabolism*; D011510:Proteolipids; D014316:Tritium; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D005978:Glutathione; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010743:Phospholipids; D011510:Proteolipids; D014316:Tritium; D014750:Vincristine; C020485:proteoliposomes