The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity.
P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity.
Loo
Tip W
TW
Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Bartlett
M Claire
MC
Clarke
David M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D016572:Cyclosporine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D006639:Histidine; D016572:Cyclosporine; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D007930:Leucine; D003078:Colchicine