Loo T W, Clarke D M
Department of Medicine, University of Toronto, Ontario, Canada.
J Biol Chem. 1994 Nov 18;269(46):28683-9.
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.
人P-糖蛋白跨膜区段7内或紧邻其NH2末端的氨基酸发生突变，会使该蛋白丧失赋予细胞对细胞毒性药物抗性的能力。与野生型P-糖蛋白的170 kDa表观质量相比，这些突变型P-糖蛋白的表观质量均为150 kDa，且其表观质量会因内切糖苷酶H消化而改变。这些观察结果表明，这些突变蛋白加工不当，因而位于内质网中，未正确靶向至质膜。对于除Glu707→Ala和Tyr710→Ala之外的所有突变体，当细胞在26℃下生长时，150 kDa的P-糖蛋白加工为170 kDa成熟形式的酶的过程显著增加。在较低的生长温度下，成熟蛋白靶向至质膜，药物外排活性得以恢复。我们还分析了这些突变体可能存在的分子相互作用，这些相互作用可能导致其滞留在内质网中处于细胞内。我们发现，野生型和突变型P-糖蛋白的核心糖基化形式与分子伴侣钙连蛋白相关。然而，只有野生型酶能够摆脱与钙连蛋白的结合并靶向至质膜。突变体与钙连蛋白的长时间结合可能是由于蛋白错误折叠所致，这一点可由它们相对较短的半衰期（约3小时）得到证明，而野生型酶的半衰期为50小时。这些结果表明，钙连蛋白有助于一种质量控制机制，将错误折叠形式的P-糖蛋白滞留在内质网中。
