Clathrin adaptor function at the trans Golgi network and endosomes

网格蛋白接头在反式高尔基体网络和内体中的功能

• 批准号: 7214809
• 负责人: Gregory S Payne
• 金额: $ 25.8万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214809
• 关键词: Adaptor Protein Complex 1; Address; Affect; Alleles; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Cell membrane; Cells; Chemicals; Class; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Defect; Development; Disease; Ear; Endosomes; Gene Deletion; Genes; Genetic; Goals; Heart Diseases; Hermanski-Pudlak Syndrome; I-Cell Disease; In Vitro; Individual; Inherited; Lead; Liposomes; Lysosomes; Malignant Neoplasms; Mediating; Membrane; Molecular; Molecular Genetics; Pathway interactions; Phosphatidylinositols; Process; Protein Sortings; Proteins; Relative (related person); Role; Saccharomyces cerevisiae; Sorting - Cell Movement; Temperature; Tertiary Protein Structure; Testing; Transcription Factor AP-1; Vesicle; Yeasts; base; chemical genetics; endosome membrane; genetic analysis; human disease; in vivo; inhibitor/antagonist; intracellular protein transport; mutant; novel; protein function; protein transport; small molecule; trafficking; trans-Golgi Network; yeast two hybrid system

DESCRIPTION (provided by applicant): A major sorting station in the secretory pathway is the trans Golgi network (TGN), where proteins are directed to the plasma membrane, endosomes, and lysosomes. Defects in sorting at the TGN can lead to inherited human diseases such as I-cell disease and Hermansky-Pudlak syndrome, and likely to contribute to more common, multigenic diseases such as cancer and heart disease. The long-term goal of this project is to define the molecular basis of protein sorting into vesicle-mediated pathways between the TGN and endosomes. Studies of Saccharomyces cerevisiae indicate that clathrin coated vesicles (ccv) participate in evolutionarily conserved protein transport between the TGN and endosomes. Two types of clathrin adaptors are implicated in these pathways, the AP-1 complex and monomeric Gga proteins. Analyses of the yeast adaptors has opened unique avenues to address ccv formation at the TGN/endosomes. A combination of genetic, molecular, biochemical, and cell biological strategies will be applied to achieve three specific aims. First, roles of Gga proteins and AP-1 in TGN/endosome clathrin-mediated trafficking pathways will be determined. Towards this end, roles for each adaptor in clathrin recruitment to membranes will be evaluated. Also, the relative distribution and function of each adaptor in different trafficking pathways between the TGN and endosomes will be defined. Second, the functions of two novel Gga2p-interacting proteins, Ent3p and Ent5p, will be determined. These proteins, which are required for clathrin-mediated traffic between the TGN and endosomes, are distinguished by the presence of ENTH-related domains. Function of these proteins in clathrin coat assembly and clathrin-mediated transport will be characterized in vivo using mutant cells and in vitro by development of a coat assembly assay using pure proteins and liposomes. Emphasis will be directed towards understanding the role of phosphoinositide binding by Ent3p and Ent5p ENTH-related domains. Additional TGN/endosome clathrin coat components will be identified using interaction and genetic approaches. Third, we will initiate a chemical genetic strategy to study AP-1 and Gga-mediated traffic by identifying and characterizing small molecule inhibitors. Together these studies are expected to provide significant advances in our understanding of the fundamental process of ccv formation and protein sorting at the TGN and endosomes.

描述（由申请人提供）：分泌途径中的一个主要分类站是反式高尔基网络（TGN），其中蛋白质被定向到质膜，内体和溶酶体。 TGN分类的缺陷会导致遗传性的人类疾病，例如I细胞疾病和Hermansky-Pudlak综合征，并可能导致更常见的多基因疾病，例如癌症和心脏病。该项目的长期目标是将蛋白质分类的分子基础定义为TGN和内体之间的囊泡介导的途径。 酿酒酵母的研究表明，网格蛋白涂层的囊泡（CCV）参与TGN和内体之间的进化保守蛋白质转运。两种类型的网格蛋白适配器与这些途径有关，即AP-1复合物和单体GGA蛋白。对酵母适配器的分析已经开放了独特的途径，以解决TGN/内体的CCV形成。遗传，分子，生化和细胞生物学策略的结合将用于实现三个特定目标。首先，将确定GGA蛋白和AP-1在TGN/内体旋转蛋白介导的运输途径中的作用。为此，将评估每个适配器在网格蛋白募集到膜中的作用。同样，将定义每个适配器在TGN和内体之间不同运输途径中的相对分布和功能。其次，将确定两个新型GGA2P相互作用蛋白ENT3P和ENT5P的功能。这些蛋白质是网格蛋白介导的TGN和内体之间所需的蛋白质，其区别在于存在与当中相关的域的存在。这些蛋白质在网格蛋白外套组装中的功能和网格蛋白介导的转运在体内将在体内表征，并通过使用纯蛋白和脂质体开发外套组装测定法。重点将致力于理解ENT3P和ENT5P中与Enth相关域的磷酸肌醇结合的作用。将使用相互作用和遗传方法来鉴定其他TGN/内体网蛋白外套成分。第三，我们将通过识别和表征小分子抑制剂来启动化学遗传策略，以研究AP-1和GGA介导的流量。预计这些研究将在我们对TGN和内体上CCV形成和蛋白质分类的基本过程的理解方面有重大进展。