Intracellular Role of Clathrin and Its Regulation

网格蛋白的细胞内作用及其调控

• 批准号: 7271716
• 负责人: THERESA Joyce O'HALLORAN
• 金额: $ 5.08万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7271716
• 关键词: Dictyostelium; cell cycle; clathrin; cytoplasm; electron microscopy; endocytosis; fluorescence microscopy; gene targeting; intracellular transport; molecular cloning; mutant; myosins; phenotype; protein purification; protein structure function; protein transport; tissue /cell culture

DESCRIPTION (provided by applicant): Our long-term objective is to define in molecular terms the cellular proteins that form, direct and regulate membrane traffic in eukaryotic cells. Our focus is on clathrin vesicles, a key transporter of receptors in eukaryotic cells. Clathrin coated vesicles play important cellular roles in the internalization of nutrients and signals from the extracellular milieu. Our experimental strategy is to use Dictyostelium as a model system. Our two specific aims are designed to answer the important questions of how and where clathrin lattices assemble in living cells. 1. We will determine the contribution of the clathrin light chain (CLC) to coated vesicle structure and function in living cells. (A) We will assess the in vitro assembly properties of CLC-deficient triskelions purified from CLC null cells. By determining the clathrin lattice structure and co-localization with Assembly Proteins in CLC null cells, the in vivo assembly properties of CLC-deficient triskelions on the plasma membrane will be defined. (B) Three independent approaches utilizing protein purification, two-hybrid analysis, and a high-copy genetic suppressor screen, will be used to identify proteins and genes that bind to or interact genetically with the clathrin light chain. The function and contribution of these binding and genetic partners to clathrin function will be determined. 2. We will determine the mechanism of regulation of clathrin lattice formation by the assembly protein CALM. CALM, clathrin assembly myeloid leukemia gene was identified as a gene whose disruption is associated with the disease myeloid leukemia. Possible roles for CALM as an obligate initiator of clathrin assembly on the plasma membrane or as a modulator of clathrin assembly will be distinguished. (A) Using phenotypic analysis of CALM-minus Dictyostelium, we will assess the contribution of CALM to trafficking of receptors that utilize clathrin-mediated pathways. Double mutant analysis of cells will test the possibility that CALM collaborates with proteins of similar domain structure to accomplish its intracellular role. (B) The requirement for CALM for the proper formation of clathrin assembly and vesicle formation will be assessed in clathrin-minus mutants. The order of CALM and clathrin assembly on the plasma membrane will be established. (C) Defined fragments of the CALM protein will be assessed for PIP2 and clathrin-binding. Expression of these domains in CALM minus cells will define the contribution of these domains to the proper localization and in vivo function of the CALM protein.

描述（由申请人提供）：我们的长期目标是用分子术语定义形成、指导和调节真核细胞膜运输的细胞蛋白。我们的重点是网格蛋白囊泡，它是真核细胞中受体的关键转运蛋白。网格蛋白包被的囊泡在营养物质的内化和来自细胞外环境的信号中发挥着重要的细胞作用。我们的实验策略是使用盘基网柄菌作为模型系统。我们的两个具体目标旨在回答网格蛋白晶格在活细胞中如何以及在何处组装的重要问题。 1. 我们将确定网格蛋白轻链 (CLC) 对活细胞中包被囊泡结构和功能的贡献。 (A) 我们将评估从 CLC 无效细胞纯化的 CLC 缺陷 triskelions 的体外组装特性。通过确定网格蛋白晶格结构以及与 CLC 无效细胞中组装蛋白的共定位，将定义质膜上 CLC 缺陷的 triskelions 的体内组装特性。 (B) 利用蛋白质纯化、双杂交分析和高拷贝遗传抑制筛选的三种独立方法将用于鉴定与网格蛋白轻链结合或遗传相互作用的蛋白质和基因。这些结合和遗传伴侣的功能和对网格蛋白功能的贡献将被确定。 2. 我们将确定组装蛋白CALM调节网格蛋白晶格形成的机制。 CALM（网格蛋白组装髓细胞白血病基因）被鉴定为其破坏与髓细胞白血病疾病相关的基因。 CALM 作为质膜上网格蛋白组装的专性引发剂或作为网格蛋白组装的调节剂的可能作用将被区分。 (A) 使用 CALM 减去盘基网柄菌的表型分析，我们将评估 CALM 对利用网格蛋白介导途径的受体运输的贡献。细胞的双突变分析将测试 CALM 与具有相似结构域结构的蛋白质协作以完成其细胞内作用的可能性。 (B) 将在网格蛋白缺失突变体中评估 CALM 对网格蛋白组装和囊泡形成的正确形成的要求。 CALM 和网格蛋白在质膜上的组装顺序将被确定。 (C) 将评估 CALM 蛋白的确定片段的 PIP2 和网格蛋白结合。这些结构域在 CALM 负细胞中的表达将定义这些结构域对 CALM 蛋白的正确定位和体内功能的贡献。