Prolonged association of temperature-sensitive mutants of human P-glycoprotein with calnexin during biogenesis.
Mutation of amino acids located within or immediately NH2-terminal to transmembrane segment 7 of human P-glycoprotein abolished the ability of the protein to confer resistance to cytotoxic drugs. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein, and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were processed improperly, so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. Processing of the 150-kDa P-glycoprotein to the 170-kDa mature form of the enzyme for all of the mutants, except Glu707-->Ala and Tyr710-->Ala, was dramatically increased when the cells were grown at 26 degrees C. At the lower growth temperature, the mature protein was targeted to the plasma membrane, and drug efflux activity was restored. We also analyzed the mutants for possible molecular interactions that may contribute to their intracellular retention. We found that core-glycosylated forms of the wild-type and mutant P-glycoproteins were associated with the molecular chaperone calnexin. Only wild-type enzyme, however, was able to escape association with calnexin and be targeted to the plasma membrane. Prolonged association of the mutants with calnexin may be due to misfolding of the protein as evidenced by their relative short half-life of about 3 h, compared with 50 h for the wild-type enzyme. These results suggest that calnexin contributes to a quality control mechanism to retain misfolded forms of P-glycoprotein in the endoplasmic reticulum.
Department of Medicine, University of Toronto, Ontario, Canada.
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Loo
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Clarke
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational; D013696:Temperature
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D037281:Calnexin