Intracellular Role of Clathrin and Its Regulation

描述(申请人提供)：我们的长期目标是用分子术语定义在真核细胞中形成、引导和调节细胞膜运输的细胞蛋白。我们的重点是网状蛋白囊泡，这是真核细胞中受体的关键转运体。笼状蛋白包裹的囊泡在细胞外营养物质和信号的内化过程中起着重要的细胞作用。我们的实验策略是使用Dictyostelials作为一个模型系统。我们的两个具体目标是为了回答网状蛋白晶格在活细胞中如何以及在哪里组装的重要问题。 1.我们将在活细胞中确定笼状蛋白轻链(CLC)对包被囊泡结构和功能的贡献。(A)我们将评估从CLC缺失细胞中纯化的CLC缺陷型三聚体的体外组装特性。通过确定CLC缺失细胞中的笼状蛋白晶格结构和与组装蛋白的共定位，可以确定CLC缺陷的三离子在质膜上的体内组装特性。(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，将用于识别与笼状蛋白轻链结合或与之遗传相互作用的蛋白质和基因。这些结合和遗传伙伴对网状蛋白功能的作用和贡献将被确定。 2.我们将确定组装蛋白Calm对笼状蛋白晶格形成的调控机制。Calm，clathrin组装髓系白血病基因被鉴定为一种基因，其中断与髓系白血病相关。作为胞膜上网状蛋白组装的必要引发者或作为网状蛋白组装的调节器的CALAME可能扮演的角色将被区分。(A)使用Calm-减去Dictyostelials的表型分析，我们将评估Calm对利用网状蛋白介导的途径的受体运输的贡献。对细胞的双重突变分析将测试Calm与结构相似的蛋白质合作完成其细胞内作用的可能性。(B)将评估笼状蛋白缺失突变体正确形成笼状蛋白组装和囊泡所需的平静条件。质膜上的CAMAL和分子筛蛋白的组装顺序将被建立。(C)将评估确定的Calm蛋白片段是否与PIP2和笼蛋白结合。这些结构域在Calm负细胞中的表达将决定这些结构域对Calm蛋白的正确定位和体内功能的贡献。

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.