The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates

被引:14
|
作者
Loo, TW
Clarke, DM
机构
[1] Univ Toronto, Dept Med, MRC, Grp Membrane Biol, Toronto, ON M5S 1A8, Canada
[2] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada
关键词
D O I
10.1021/bi982525y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 rest 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.
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收藏
页码:5124 / 5129
页数:6
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