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 transm embrane protein belonging to the ATP-binding cassette transporter superfami ly,In addition to conferring resistance to various antineoplastic agents, M RP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C-4 (LTC4). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be st imulated 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 w e show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC4 uptake by MRP1 proteoliposomes was osmoticall y sensitive and could be inhibited by two MRP1-specific monoclonal antibodi es. LTC4 uptake was also markedly reduced by the competitive inhibitor, S-d ecyl-glutathione, as well as by the MRP1 substrates 17 beta -estradiol 17-b eta-(D-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K-m for ATP and LTC4 were 357 +/- 184 muM and 3 66 +/- 38 nM, respectively, and 2.14 +/- 0.75 muM for 17 beta -estradiol 17 -beta-(D-glucuronide). Transport of vincristine required the presence of bo th ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport compet ent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.