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Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes
Wolfgang Hagmann1, Jana Schubert, Jörg König
1Abteilung Tumorbiochemie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Abstract:
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.
Insights
Researchers purified and reconstituted the apical multidrug resistance protein MRP2 (ABCC2) into proteoliposomes, demonstrating its active transport function for substrates like LTC4. This breakthrough enables further study of MRP2 interactions in polarized cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The apical multidrug resistance protein MRP2 (ABCC2) functions as an ATP-dependent export pump for anionic conjugates in polarized cells.
- MRP2 shares limited amino acid identity (48%) with its paralog, MRP1 (ABCC1).
Purpose of the Study:
- To reconstitute purified recombinant MRP2 (ABCC2) into proteoliposomes to demonstrate its functional activity in substrate transport.
- To characterize the kinetic parameters and inhibition of MRP2 transport.
- To investigate the interaction of MRP2 with the chaperone calnexin.
Main Methods:
- Purification of recombinant MRP2 (ABCC2).
- Reconstitution of purified MRP2 into proteoliposomes.
- Assay of substrate transport (LTC4) and ATP dependence.
- Determination of kinetic parameters (Km) and inhibition by MK571.
- Binding and co-reconstitution studies with calnexin.
Main Results:
- Purified recombinant MRP2 reconstituted in proteoliposomes exhibited active substrate transport.
- Kinetic analysis revealed Km values for ATP (560 µM) and LTC4 (450 nM).
- Transport activity was dependent on MRP2 protein amount and inhibited by MK571.
- MRP2 preferentially associates with calnexin, but calnexin is not essential for MRP2's LTC4 transport function.
Conclusions:
- This study reports the first successful reconstitution of transport-active purified recombinant MRP2.
- The characterized transport activity and inhibition provide a basis for further investigation.
- Purified MRP2 can be used to identify additional interacting proteins in the apical membrane of polarized cells.