Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions.
Misprocessed mutants of human P-glycoprotein accumulate as core-glycosylated intermediates in the endoplasmic reticulum and are rapidly degraded. Trypsin digestion was used to test for structural differences between mature and core-glycosylated forms of P-glycoprotein. We found that the core-glycosylated wild-type and mutant P-glycoproteins were both 100-fold more sensitive to trypsin compared with the mature form of the wild-type enzyme. This result suggested that the core-glycosylated forms of both wild-type and mutant P-glycoproteins have similar unfolded structures, whereas the mature enzyme is folded into a more compact structure. The core-glycosylated mutant P-glycoproteins could be converted to the mature trypsin-resistant form by synthesis in the presence of drug substrate. Addition of proteasome inhibitor MG-132 to stabilize the core-glycosylated intermediate resulted in the accumulation but not maturation of the mutant protein. Further analysis showed that the second transmembrane domain TMD2 also became more resistant to trypsin digestion only after coexpression with TMD1 in the presence of substrate. Taken together, these results suggest that simply stabilizing the core-glycosylated intermediate is not sufficient to promote maturation of the processing mutants and that drug substrates induce maturation by promoting superfolding of the transmembrane domains.
Loo
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TW
Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Clarke
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DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000502:physiology; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D014357:Trypsin / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008565:Membrane Proteins; D016572:Cyclosporine; D006596:Hexosaminidases; D014357:Trypsin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde