Mutational analysis of the predicted first transmembrane segment of each homologous half of human P-glycoprotein suggests that they are symmetrically arranged in the membrane.
A recent study of P-glycoprotein membrane topology using phoA gene fusions provided evidence that the orientation of the first transmembrane segment of each homologous half of P-glycoprotein was different (Béjà, O., and Bibi, E. (1995) J. Biol. Chem. 270, 12351-12354). To test this hypothesis, we compared the functional consequences of mutations to residues in transmembrane segments (TMs) TM1 and TM7. Mutations to 3 residues occupying homologous positions in TM1 and TM7 resulted in mutant P-glycoproteins that were inactive. These mutants were found to be misprocessed. By contrast, mutations to other residues in TM1 resulted in functional P-glycoproteins. When putative TM1 was replaced with TM7 or TM7 with TM1 to yield TM7/TM7 or TM1/TM1 chimeras, both constructs yielded P-glycoproteins that were capable of conferring drug resistance in transfected cells. The purified P-glycoproteins from mutants TM7/TM7 and TM1/TM1 retained 59 and 28% of verapamil-stimulated ATPase activity, respectively. By contrast, interchanging TM6 and TM12 to yield TM6/TM6, TM12/TM12, or TM12/TM6 constructs resulted in P-glycoproteins that did not have any detectable ATPase activity and did not confer drug resistance in transfected cells. These results suggest that TM1 and TM7 likely have similar structural and functional roles in P-glycoprotein and that they have identical topologies.
Loo
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TW
Medical Research Council Group in Membrane Biology, Department of Medicine , University of Toronto, Ontario M5S 1A8, Canada.
Clarke
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DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D000818:Animals; D006596:Hexosaminidases; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013696:Temperature
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011993:Recombinant Fusion Proteins; D006596:Hexosaminidases