Functional expression of multidrug resistance protein 1 in Pichia pastoris

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-d ependent efflux mechanism. Six other MRP proteins exist (MRP27), and mutati ons in some of these genes cause major pathological conditions in humans. A detailed characterization of the structure and mechanism of action of thes e proteins requires an efficient expression system from which large amounts of active protein can be obtained. We report the expression of a recombina nt MRP1 in the methylotrophic yeast Pichia pastor-is. The protein is expres sed in the membrane fraction of these cells, as a stable and underglycosyla ted 165 kDa peptide. Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants. MRP1 express ed in P. pastoris binds 8-azido[alpha-P-32]ATP in a Mg2+-dependent and EDTA -sensitive fashion, which can be competed by a molar excess of ADP and ATP. Under hydrolysis conditions (at 37 degreesC), orthovanadate induces trappi ng of the 8-azido[a-32P]nucleotide in MRP1, which can be further modulated by known MRP1 ligands. MRP1 is also labeled by a photoactive analogue of rh odamine 123 (IAARh123) in P. pastoris/MRP1 membranes, and this can be compe ted by known MRP1 ligands. Finally, MRP1-positive membrane vesicles show AT P-dependent uptake of LTC4. Thus, MRP1 expressed in P. pastoris is active a nd shows characteristics of MRP1 expressed in mammalian cells, including dr ug binding, ligand-modulated formation of the MRP1-MgADP-P-i intermediate ( ATPase activity), and ATP-dependent substrate transport. The successful exp ression of catalytically active and transport-competent MRP1 in P. pastoris should greatly facilitate the efficient production and isolation of the wi ld type or inactive mutants of MRP1, or of other MRP proteins for structura l and functional characterization.