SORTING OF A 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: 3307308
• 负责人: DEBORAH Ann BROWN
• 金额: $ 13.48万
• 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: 1992-09-30 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307308
• 关键词: apical membrane; cell membrane; electron microscopy; epithelium; gel electrophoresis; glycosphingolipids; immunoprecipitation; intracellular transport; laboratory rat; lipid structure; lipid transport; membrane lipids; membrane proteins; phosphatidylinositols; protein biosynthesis; protein purification; protein structure; protein transport

The plasma membrane of epithelial cells is divided by tight junctions into two distinct domains, apical and basolateral, that differ markedly in their protein and lipid composition. A central question in cell biology is to determine how this asymmetry is generated and maintained. Viruses that cause influenza, measles, and other diseases depend on intracellular protein sorting pathways for their propagation. Understanding how intracellular sorting operates may provide new approaches to combating these diseases. Plasma membrane proteins and some membrane lipids are sorted in the trans Golgi network in certain cultured epithelial cells, and are packaged into transport vesicles destined for the apical or basolateral surface. Proteins anchored in membranes by glycosyl phosphatidyl inositol (GPI) are transported apically. GPI anchorage can cause this targeting. Glycosphingolipids are also delivered primarily to the apical surface. It has been proposed that GPI-anchored proteins may associate with glycosphingolipids inside the cell, and that the two may be sorted co-ordinatedly into the apical transport pathway. Our goal is to test this theory, and to isolate and characterize the lipid-protein complexes. Membrane complexes that contain both glycolipids and GPI-anchored proteins have been isolated, using their surprising insolubility in non-ionic detergents. The aim of this application is to learn whether these complexes contain the domains of associated GPIanchored proteins and glycolipids from the Golgi that were proposed above. These domains would be likely to contain sorting proteins, since they form in the sorting compartment and contain two classes of molecules that are targeted to the same surface. Isolation of these sorting proteins is the long-range goal of this work. Biochemical and ultrastructural techniques will be used to characterize the complexes. The effects of altering the cellular lipid composition and the conditions of detergent lysis on complex formation will be determined, as will the behavior of GPI-anchored proteins reconstituted into vesicles of defined lipid composition. The intracellular site(s) of origin of the complexes will be determined by subcellular fractionation and by membrane labelling prior to isolation of complexes. The next aim is to determine the protein profile of the membrane complexes, to identify candidate proteins that may be important in sorting. Finally, the possible inter-relationship of glycolipid and GPI-anchored protein sorting will be determined using specific inhibitors.

上皮细胞的质膜由紧密连接 分为两个不同的域，顶端和基底外侧，它们明显不同 在其蛋白质和脂质成分中。 细胞中的一个核心问题 生物学将确定如何生成和维持这种不对称性。 引起流感，麻疹和其他疾病的病毒取决于 细胞内蛋白质分类途径的繁殖。 了解细胞内分类如何提供新的 打击这些疾病的方法。 质膜蛋白和某些膜脂质在反式中分类 某些培养的上皮细胞中的高尔基网络，并包装到 用于顶端或基底外侧表面的运输囊泡。 糖基磷脂酰肌醇（GPI）锚定在膜上的蛋白质 以顶端运输。 GPI锚定可能导致此目标。 糖磷脂也主要传递到顶部表面。 有人提出，GPI锚定蛋白可能与 细胞内的糖磷脂，并且两者可以分类 协调进入顶端运输途径。 我们的目标是测试 该理论，并隔离和表征脂质 - 蛋白质复合物。 包含糖脂和GPI锚定的膜络合物 蛋白质是孤立的，使用它们令人惊讶 非离子洗涤剂。 该应用程序的目的是了解是否 这些复合物包含相关GPIANCHIDER蛋白的域 以及上面提出的高尔基体的糖脂。 这些域 可能包含分类蛋白，因为它们在 分类室并包含两类的分子 针对同一表面。 这些分类蛋白的隔离是 这项工作的远程目标。 生化和超微结构技术将用于表征 复合体。 改变细胞脂质组成的影响 并且对复合形成的洗涤剂裂解的条件将是 确定，GPI锚定蛋白的行为也会重构 进入定义的脂质组成的囊泡。 细胞内部位 复合物的起源将通过亚细胞分馏来确定 并通过在分离复合物之前进行膜标记。 下一个目标 是确定膜复合物的蛋白质谱， 确定可能在分类中很重要的候选蛋白质。 最后， 糖脂和GPI锚定蛋白的可能相互关系 分类将使用特定抑制剂确定。