FIGURE 5.

P-gp matures and retains activity when Phe-1086 is replaced with hydrophobic but not charged residues. A, F1086X mutants were expressed in the absence of drug substrates. B, processing mutants F1086D and F1086R were expressed in the presence (+) or absence (−) of cyclosporine A (Cyclo), and whole cell SDS extracts were subjected to immunoblot analysis. The positions of mature (170 kDa) and immature (150 kDa) P-gps are indicated. C, ATPase activities of histidine-tagged wild-type P-gp and F1086X mutants were measured in the presence of verapamil. Histidine-tagged mutants F1086D and F1086R were first expressed in the presence of cyclosporine A to promote maturation of P-gp before isolation of the protein by nickel-chelate chromatography. Each value is the mean ± S.D. (n = 3).