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

Multiple topologies have been detected for the COOH-terminal half of the hu man multidrug resistance P-glycoprotein (P-gp), In one topology, the predic ted 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-l ength molecule. When the halves of P-gp are expressed as separate polypepti des, 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 t opologies can fold into an active enzyme, we assayed for interaction betwee n the N- and C-Halves of P-gp since functional P-gp requires interaction be tween both halves. In a mutant P-gp (E875C) that gave about equal amounts o f both topologies, only the C-Half(CL3-cyt) could be recovered by nickel ch romatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed tog ether, exhibited verapamil- and vinblastine-stimulated ATPase activities th at 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 enhance d expression of C-Half(CL3;cyt). By contrast, interaction of C-Half (CL3-ex t) 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.