TY - JOUR
T1 - Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1
AU - Huang, Zhenhua
AU - Chang, Xiubao
AU - Riordan, John R.
AU - Huang, Youguo
N1 - Funding Information:
This work was funded by the project of National Natural Science Foundation of China (30230120). We are indebted to Prof. F.Y. Yang for his stimulating discussions and suggestions. We are grateful to Dr. A.W. Ford-Hutchinson of Merck Frosst Canada, for the gift of MK-571.
PY - 2004/1/28
Y1 - 2004/1/28
N2 - Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extrocytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.
AB - Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extrocytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.
KW - Floppase
KW - MRP1
KW - NBD-PC
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U2 - 10.1016/j.bbamem.2003.11.010
DO - 10.1016/j.bbamem.2003.11.010
M3 - Article
C2 - 14757231
AN - SCOPUS:1642534354
SN - 0005-2736
VL - 1660
SP - 155
EP - 163
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 1-2
ER -