Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes.
The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.
Hagmann
Wolfgang
W
Abteilung Tumorbiochemie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Schubert
Jana
J
König
Jörg
J
Keppler
Dietrich
D
D000255:Adenosine Triphosphate / Q000502:physiology; D037281:Calnexin / Q000494:pharmacology*; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D024101:Mitochondrial Proteins; D011233:Precipitin Tests; D011422:Propionates / Q000494:pharmacology; D011510:Proteolipids / Q000737:chemistry*; D011804:Quinolines / Q000494:pharmacology; D012269:Ribosomal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D011422:Propionates; D011510:Proteolipids; D011804:Quinolines; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; D037281:Calnexin; D017997:Leukotriene C4; C059141:verlukast; D000255:Adenosine Triphosphate