Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains.

Glycosylphosphatidylinositol (GPI)-anchored proteins can be isolated from both cells and sphingolipid and cholesterol-rich liposomes (SCRLs) in association with detergent-insoluble membranes. We found previously that detergent insolubility of lipids was characteristic of phases in which lipid acyl chains are ordered. We presented evidence that GPI-anchored proteins are insoluble because they associate with cholesterol and sphingolipid-rich lipid domains with properties similar to the liquid-ordered phase. Here, this model was tested by a variety of approaches. First, we demonstrated that saponin, which removes cholesterol from cell membranes and allows solubilization of GPI-anchored proteins by Triton X-100, had the same effect on the GPI-anchored protein alkaline phosphatase (PLAP) in SCRLs of appropriate lipid composition. The similarity of saponin action in cells and simple liposomes suggests that the compound disrupts protein-lipid interactions. However, direct interactions between PLAP and cholesterol were not needed for insolubility, because the protein was also insoluble in cholesterol-free liposomes containing lipid in an ordered phase. Instead, cholesterol acted by greatly enhancing the formation of a detergent-insoluble phase by sphingolipids, which have a tendency to form ordered phases. We propose that saponin solubilizes GPI-anchored proteins because the lipid composition of cell membranes (and the SCRLs used above) supports ordered phase formation in the presence but not the absence of cholesterol. Supporting this model, saponin did not promote Triton X-100 solubilization of PLAP in SCRLs with sphingolipid levels high enough to allow ordered phase formation in the absence of cholesterol. We also showed that two additional GPI-anchored proteins are detergent-insoluble in SCRLs and that detergent does not artifactually create ordered domains or cause components of solubilized membranes to associate with detergent-resistant membranes present in separate bilayers in the same lysate. We conclude that the ordered domain model explains the behavior of detergent-resistant membranes in liposomes and cells.