SORTING OF LIPID-PROTEIN COMPLEX IN EPITHELIAL CELLS

上皮细胞中脂质-蛋白质复合物的分类

• 批准号: 6330981
• 负责人: DEBORAH Ann BROWN
• 金额: $ 7.47万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6330981
• 关键词: G protein; Golgi apparatus; SDS polyacrylamide gel electrophoresis; apical membrane; biological signal transduction; caveolins; cell membrane; electron microscopy; electron spin resonance spectroscopy; epithelium; gel electrophoresis; glycosphingolipids; immunoprecipitation; intracellular transport; laboratory mouse; laboratory rat; lipid structure; lipid transport; membrane lipids; membrane proteins; phosphatidylinositols; protein kinase; protein purification; protein structure function; protein transport; tissue /cell culture

Detergent-resistant membranes (DRMs) isolated from mammalian cells have been implicated in a diverse set of functions, ranging from intracellular sorting in polarized epithelial cells, to cell-surface signal transduction, to the structure of non-clathrin-coated plasma membrane pits called caveolae. Each of these roles will be examined, and a model explaining he structure of these membranes will be tested. Cholesterol-rich model membranes contain distinct domains; a novel phase called the liquid-ordered phase (Io) phase in addition to the liquid-crystalline (Ic) phase. Io phase lipids are packed more tightly than those in the Ic phase, but have more mobility than gel phase lipids. DRMs isolated from cells appear to be present in a phase similar to the Io phase. The long-term goal of this work is to determine how these domains function in cells. Many proteins that can be isolated from cells in DRMs are modified with saturated acyl chains (myristate and/or palmitate), that fit well into ordered domains. Three such proteins will be purified and incorporated into model membranes that contain Io phase domains; the caveolar coat protein caveolin, the Src- family kinase Yes, and the G protein Gi. The ability of these proteins to associate with DRMs from these liposomes in the absence of other proteins will be determined, as will the dependence of this behavior on protein acylation. Possible effects of Io phase domains on the function of these proteins will also be explored. These studies could have important health- related implications, as signaling proteins such as Src-family kinases can be oncogenic. Previous studies suggest that DRMs can be isolated from intracellular membranes as well as plasma membranes. DRMs will be isolated from the Golgi apparatus. Proteins found to be enriched in Golgi-derived DRMs may be important in sorting and trafficking along the secretory pathway, and will be further characterized. Complementary studies using cells and motel membranes will test our model that DRMs exist in cells as discrete domains. Recent results suggest that lipids with high acyl chain melting temperatures, such as sphingolipids, promote the formation of the Io phase in the presence of cholesterol. The lipid composition of DRMs from cells depleted of sphingolipid and/or cholesterol, and of model membranes that form DRMs, will be examined to test the correlation between formation of an Io-like phase and detergent-insolubility. Additional methods for examining the Io-like phase in model membranes and possibly in cell membranes will be used. Finally, the ability of the sphingolipid-poor inner leaflet of the plasma membrane to form DRMs will be tested using inside-out erythrocyte ghosts.

从哺乳动物细胞中分离的耐洗涤剂膜（DRM）具有 与各种功能有关，范围从细胞内 在极化上皮细胞中分类，以转导细胞表面信号转导， 到称为非粘蛋白涂层的质膜凹坑的结构称为 小屋。 这些角色中的每一个都将进行检查，并解释了他的模型 这些膜的结构将进行测试。 富含胆固醇的模型 膜包含不同的域；一个称为液体排序的新型阶段 除液晶（IC）相外，相位（IO）相。 io 相比IC相位的脂质更紧密，但具有 比凝胶相脂质更多。 与细胞分离的DRM似乎是 以类似于IO阶段的阶段存在。 这个长期目标 工作是确定这些域在细胞中的作用。 许多蛋白质 可以从DRM中的细胞中分离出来的饱和酰基 链（肉豆蔻酸酯和/或棕榈酸酯），非常适合有序域。 三种这样的蛋白质将被纯化并掺入模型膜中 包含IO相域； Caveolar涂层蛋白可爱素，Src- 家族激酶是的，G蛋白GI。 这些蛋白质的能力 在没有其他蛋白质的情况下，与这些脂质体的DRM相结合 将确定这种行为对蛋白质的依赖性 酰化。 IO相域对这些功能的可能影响 还将探索蛋白质。 这些研究可能具有重要的健康 - 相关含义，因为信号蛋白（例如SRC家庭激酶）可以 致癌。 先前的研究表明，DRM可以与 细胞内膜以及质膜。 DRM将被隔离 来自高尔基体。 发现蛋白质富含高尔基 DRM在分泌的分类和贩运方面可能很重要 路径，并将进一步表征。 互补的研究使用 细胞和汽车旅馆膜将测试我们的模型，即DRM在细胞中存在于细胞中 离散域。 最近的结果表明，具有高酰基链的脂质 熔融温度（例如鞘脂）促进了形成 IO阶段在胆固醇的存在下。 DRM的脂质组成 从剥离鞘脂和/或胆固醇的细胞以及模型 将检查形成DRM的膜以测试 形成类似IO的相位和洗涤剂 - 不溶性。 额外的 用于检查模型膜中IO样相的方法 将使用细胞膜。 最后，鞘脂贫乏的能力 将使用质膜的内部小叶进行DRM的形成DRM 内而外的红细胞幽灵。