The transmembrane domains of the human multidrug resistance P-glycoproteinare sufficient to mediate drug binding and trafficking to the cell surface

The human multidrug resistance P-glycoprotein (P-gp) is organized in two ta ndem repeats with each repeat consisting of an N-terminal hydrophobic domai n containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the d eletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembra ne domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutan t was sensitive to trypsin and endoglycosidase H. Expression in the presenc e of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. T he mutant was then detected at the cell surface and was sensitive to digest ion by endoglycosidase F, By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surfac e. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the ce ll surface.