P‐Glycoprotein is localized in intermediate‐density membrane microdomains distinct from classical lipid rafts and caveolar domains

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Abstract
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, CHRB30, 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 GM1 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‐β‐cyclodextrin. The Brij‐96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X‐100‐based caveolin‐1/GM1 microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin‐1 and GM1 colocalized, while Pgp and caveolin‐1, or Pgp and GM1, 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.

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