Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme.
Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts. By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.
Morandat
Sandrine
S
Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Bâtiment Chevreul, Université Claude Bernard Lyon I, 43, boulevard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.
Bortolato
Muriel
M
Roux
Bernard
B
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008956:Models, Chemical; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry*; D013696:Temperature
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D011510:Proteolipids; C020485:proteoliposomes; D002784:Cholesterol; D000469:Alkaline Phosphatase