Schroeder R J, Ahmed S N, Zhu Y, London E, Brown D A
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
J Biol Chem. 1998 Jan 9;273(2):1150-7. doi: 10.1074/jbc.273.2.1150.
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.
糖基磷脂酰肌醇（GPI）锚定蛋白可从细胞以及与去污剂不溶性膜相关的富含鞘脂和胆固醇的脂质体（SCRLs）中分离出来。我们之前发现脂质的去污剂不溶性是脂质酰基链有序排列阶段的特征。我们提供的证据表明，GPI锚定蛋白不溶是因为它们与具有类似于液晶相性质的富含胆固醇和鞘脂的脂质结构域相关联。在此，通过多种方法对该模型进行了测试。首先，我们证明了皂苷可从细胞膜中去除胆固醇，并使Triton X - 100能够溶解GPI锚定蛋白，对于具有适当脂质组成的SCRLs中的GPI锚定蛋白碱性磷酸酶（PLAP）也有同样的效果。皂苷在细胞和简单脂质体中的作用相似，这表明该化合物破坏了蛋白质 - 脂质相互作用。然而，PLAP的不溶性并不需要其与胆固醇直接相互作用，因为该蛋白在含有处于有序相脂质的无胆固醇脂质体中同样不溶。相反，胆固醇通过极大地增强鞘脂形成去污剂不溶性相的能力来发挥作用，鞘脂倾向于形成有序相。我们提出，皂苷可溶解GPI锚定蛋白是因为细胞膜（以及上述使用的SCRLs）的脂质组成在有胆固醇存在时支持有序相形成，而在无胆固醇时则不然。支持该模型的是，皂苷不会促进Triton X - 100对鞘脂水平高到足以在无胆固醇时形成有序相的SCRLs中的PLAP进行溶解。我们还表明，另外两种GPI锚定蛋白在SCRLs中对去污剂不溶，并且去污剂不会人为地形成有序结构域或导致溶解膜的成分与同一裂解物中单独双层中存在的去污剂抗性膜相关联。我们得出结论，有序结构域模型解释了脂质体和细胞中去污剂抗性膜的行为。
