P-glycoprotein misfolds in Escherichia coli: evidence against alternating-topology models of the transport cycle.
P-glycoprotein (P-gp) is a drug transporter which pumps toxic hydrophobic compounds out of cells, conferring mutidrug resistance. P-gp is predicted to consist of 12 transmembrane alpha-helices and there is a strong body of experimental support for this model. However, a number of studies, including those on P-gp expressed in E. coli, have reported topologies with fewer than 12 transmembrane alpha-helices, leading to the hypothesis that the transmembrane topology of the protein changes during function. It is well established that P-gp undergoes conformational changes during its transport cycle and it has been recently shown that these changes are large in magnitude and could, potentially, reflect a changing transmembrane topology. One therefore, reassessed the transmembrane topology of P-gp expressed in E. coli and compared it directly with the topology of the protein expressed in mammalian cells. It was clear that the transmembrane topology of the protein was different in the different cell types and that the misfolding of P-gp in E. coli was due to the misrecognition of multiple P-gp sequences as topogenic signals. Thus, the alternative transmembrane topologies reported for P-gp in E. coli are artefacts of the heterologous expression system used, and models based on such data in which the transmembrane topology changes during drug transport are unlikely to be correct. Instead, the large conformational changes observed during the transport cycle are more likely due to changes in alpha-helix packing.
Linton
Kenneth J
KJ
MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, London, UK. kenneth.linton@ic.ac.uk
Higgins
Christopher F
CF
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics; D002461:Cell Line, Transformed / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases