RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identica l with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi ct al. Biochemistry 39, 9327-9334), Multidru g resistance-associated protein (MRP1) belonging to the family of the ABC-t ransporters has also been suggested to be a GS-E transporter in human eryth rocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocyte s, In Western blot analyses using antibodies against RLIP76, a strong expre ssion of RLIP76 was observed in erythrocytes. Immunohistochemical studies u sing a fluorescent probe showed association of RLIP76 with erythrocyte memb rane, which was consistent with its transport function. Neither MRP1 nor Pg p were detected in erythrocytes when the antibodies against MRP1 or Pgp wer e used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies ag ainst RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C-4, and the GSH conjugate of 4-hydroxynonenal, was observed with a maxima l inhibition of about 70%, On the contrary, in the IOVs coated with the ant ibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP 76 is the major ATP-dependent transporter of GS-E and DOX in human erythroc ytes, (C) 2001 Academic Press.