Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1.
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 (extracytoplasmic). 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.
Huang
Zhenhua
Z
National Laboratory of Biomacromolecules, Center for Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Chang
Xiubao
X
Riordan
John R
JR
Huang
Youguo
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D003630:Daunorubicin / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D011510:Proteolipids / Q000737:chemistry
D009327:4-Chloro-7-nitrobenzofurazan; D000255:Adenosine Triphosphate; D003630:Daunorubicin; D005978:Glutathione; D008051:Lipid Bilayers; D008274:Magnesium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010713:Phosphatidylcholines; D011510:Proteolipids; C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; C020485:proteoliposomes; C421109:multidrug resistance-associated protein 1