Purification of the human apical conjugate export pump MRP2 - Reconstitution and functional characterization as substrate-stimulated ATPase

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conj ugate export pump in apical membranes of polarized cells and confers multid rug resistance. Purified MRP2 is essential for the detailed functional char acterization of this member of the family of ATP-binding cassette (ABC) tra nsporter proteins. In human embryonic kidney cells (HEK293), we have perman ently expressed MRP2 containing an additional C-terminal (His)(6)-tag. Immu noblot and immunofluorescence analyses detected the MRP2-(His)(6) overexpre ssing clones. Isolated membrane vesicles from the MRP2-(His)(6)-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C-4 and were photoaffinity-labelled with 8-azido-[alpha-P-32]A TP. MRP2-(His)(6) was solubilized from membranes of MRP2-(His)(6)-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)(6) was verified by MS analysis of tryptic p eptides. The purified MRP2-(His)(6) glycoprotein was reconstituted into pro teoliposomes and showed functional activity as ATPase in a protein-dependen t manner with a K-m for ATP of 2.1 mM and a V-max of 25 nmol ADP.mg MRP2(-1 ).min(-1). This ATPase activity was substrate-stimulated by oxidized and re duced glutathione and by S-decyl-glutathione. Future studies using pure MRP 2 reconstituted in proteoliposomes should allow further insight into the mo lecular parameters contributing to MRP2 transport function and to define it s intracellular partners for transport and multidrug resistance.