The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates.
Multiple topologies have been detected for the COOH-terminal half of the human multidrug resistance P-glycoprotein (P-gp). In one topology, the predicted third cytoplasmic loop (CL3) is on the cytoplasmic side (P-gp-CL3-cyt) of the membrane. In an alternate topology, CL3 is on the extracellular side of the membrane (P-gp-CL3-ext). It is not known if both forms of P-gp are active because it is difficult to distinguish either topology in the full-length molecule. When the halves of P-gp are expressed as separate polypeptides, the two topologies of the C-Half are readily distinguished on SDS-PAGE, because only the C-Half (CL3-ext) is glycosylated. To test whether both topologies can fold into an active enzyme, we assayed for interaction between the N- and C-Halves of P-gp since functional P-gp requires interaction between both halves. In a mutant P-gp (E875C) that gave about equal amounts of both topologies, only the C-Half (CL3-cyt) could be recovered by nickel chromatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed together, exhibited verapamil- and vinblastine-stimulated ATPase activities that were similar to the wild-type enzyme. We also found that biosynthesis of mutant E875C C-Half in the presence of the N-Half P-gp resulted in enhanced expression of C-Half (CL3-cyt). By contrast, interaction of C-Half (CL3-ext) with N-Half P-gp was not detected. These results show that the topology of the C-Half portion of P-gp greatly influences its interactions with the amino-terminal half of the molecule.
Loo
T W
TW
Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Ontario, Canada.
Clarke
D M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000235:genetics; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D010446:Peptide Fragments; D014700:Verapamil; D000251:Adenosine Triphosphatases