P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains.
P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.
Radeva
Galina
G
Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada.
Perabo
Jocelyne
J
Sharom
Frances J
FJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D021941:Caveolae / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003505:Cyclodextrins / Q000494:pharmacology; D005732:Gangliosides / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D005732:Gangliosides; D002784:Cholesterol