STRUCTURE/FUNCTION OF THE GOLGI MEMBRANE SKELETON

高尔基膜骨架的结构/功能

• 批准号: 6520018
• 负责人: KENNETH A BECK
• 金额: $ 15.8万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520018
• 关键词: Golgi apparatus; MDCK cell; SDS polyacrylamide gel electrophoresis; ankyrins; binding proteins; electron microscopy; fluorescence microscopy; genetic library; immunoelectron microscopy; light microscopy; membrane activity; membrane structure; molecular cloning; northern blottings; nucleic acid sequence; photomicrography; polymerase chain reaction; protein biosynthesis; protein isoforms; protein protein interaction; protein structure function; protein transport; spectrin; tissue /cell culture; yeast two hybrid system

The spectrin membrane skeleton is a ubiquitous, submembranous cytoskeleton that associates with the cytoplasmic surface of membranes. Genetic defects and deficiencies in erythrocyte isoforms of membrane skeleton components give rise to hemolytic disorders in humans. Two functions have been identified for the spectrin membrane skeleton: the maintenance of plasma membrane structural integrity and the formation of discrete membrane domains enriched in classes of membrane proteins. The recent identification of Golgi localized isoforms of membrane skeleton proteins reveals an even broader potential role for the membrane skeleton in the regulation of cellular architecture. The goal of this proposal is to characterize this Golgi membrane skeleton and to determine its function. It is hypothesized that the Golgi isoform of the membrane skeleton serves two critical functions which are analogous to the functions proposed for the plasma membrane isoforms: the maintenance of Golgi membrane structural identify and the formation of distinct membranes involved in membrane protein sorting within the Golgi complex. The specific questions that will be addressed are: 1. Are Golgi membrane skeleton proteins unique isoforms specialized for Golgi function? The precise identify of Golgi membrane skeleton proteins spectrin and ankyrin will be identified by molecular cloning. These studies will be essential for subsequent aims. 2. Will the identification of membrane skeleton bind proteins reveal critical features of the structure and function of the Golgi membrane skeleton? It is proposed that cytoplasmic binding proteins will be regulators of membrane skeleton assembly and integral membrane proteins that interact with the membrane skeleton assembly and integral membrane proteins that interact with the membrane skeleton will be "substrate proteins" acted on my membrane skeleton. The identification of these proteins will therefore provide important information regarding the assembly of the membrane skeleton as well as its protein. 3. Will dominant negative perturbations of the Golgi membrane skeleton illustrate a role for the Golgi membrane skeleton in the maintenance of Golgi structure and the formation of distinct membrane domains? Dominant negative disruption of the Golgi membrane skeleton will be performed in intact cells to directly test the proposed functions of the novel cytoskeletal structure.

血影蛋白膜骨架是一种普遍存在的膜下细胞骨架，与膜的细胞质表面结合。遗传缺陷和膜骨架成分的红细胞亚型缺陷会导致人类溶血性疾病。已确定血影蛋白膜骨架有两个功能：维持质膜结构完整性和形成富含膜蛋白类别的离散膜域。最近对膜骨架蛋白高尔基体局部亚型的鉴定揭示了膜骨架在细胞结构调节中更广泛的潜在作用。该提案的目标是描述高尔基体膜骨架的特征并确定其功能。据推测，膜骨架的高尔基亚型具有两个关键功能，这些功能类似于质膜亚型的功能：维持高尔基膜结构的识别和形成参与高尔基复合体内膜蛋白分类的不同膜。将要解决的具体问题是： 1. 高尔基体膜骨架蛋白是专门用于高尔基体功能的独特亚型吗？高尔基膜骨架蛋白血影蛋白和锚蛋白的精确鉴定将通过分子克隆来鉴定。这些研究对于后续目标至关重要。 2. 膜骨架结合蛋白的鉴定能否揭示高尔基体膜骨架结构和功能的关键特征？据提议，细胞质结合蛋白将是膜骨架组装的调节剂，与膜骨架组装相互作用的整合膜蛋白和与膜骨架相互作用的整合膜蛋白将是作用于我的膜骨架的“底物蛋白”。因此，这些蛋白质的鉴定将提供有关膜骨架及其蛋白质组装的重要信息。 3. 高尔基膜骨架的显性负扰动是否说明高尔基膜骨架在维持高尔基结构和形成不同膜域中的作用？将在完整细胞中对高尔基体膜骨架进行显性负破坏，以直接测试新型细胞骨架结构的拟议功能。