Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior.

Abstract

Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-T. lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol lposomes. These findings may explain the behavior of GPIanchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.Membranes that contain GPI-anchored proteins have been isolated from cultured epithelial cells (1, 2), fibroblasts (2), and lymphocytes (3) on the basis of their surprising insolubility in nonionic detergents such as Triton X-100. These membranes are rich in sphingolipids and cholesterol (1) and contain several proteins in addition to those anchored by GPI (2,4,5). These include caveolin, a marker for 50-nm-diameter non-clathrin-coated plasma membrane invaginations called caveolae (6). This and other studies have led to the suggestion that caveolae can be isolated from cells by detergent extraction (7, 8).It is not known how the lipids and proteins in these membranes allow them to remain associated with each other in the presence of detergent. Bilayers consisting of phospholipids and glycosphingolipids contain compositionally distinct domains (reviewed in ref. 9). It has been suggested that the same interactions that cause glycolipids to form clusters may cause their detergent resistance (1, 10). There is evidence to suggest that hydrogen bonds between glycosphingolipids may help form membrane domains (11). Alternatively, or in addition, acyl chain interactions may be important in this process, as glycolipid-rich domains can be present in a gel-like state (9). We have tested these models by determining the detergent solubility of artificial liposomes, with or without ...

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