Clathrin adaptor function at the trans Golgi network and endosomes

描述（由申请人提供）：分泌途径中的一个主要分选站是反式高尔基体网络（TGN），其中蛋白质被定向至质膜、内体和溶酶体。TGN的分选缺陷可能导致遗传性人类疾病，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，如癌症和心脏病。这个项目的长期目标是确定蛋白质分选到TGN和内体之间的囊泡介导的通路的分子基础。 对酿酒酵母的研究表明，网格蛋白包被的囊泡（cCV）参与了TGN和内体之间进化上保守的蛋白质转运。两种类型的网格蛋白衔接子涉及这些途径，AP-1复合物和单体Gga蛋白。酵母衔接子的分析已经开辟了解决TGN/内体处的ccv形成的独特途径。遗传，分子，生物化学和细胞生物学策略的组合将被应用于实现三个具体目标。首先，将确定Gga蛋白和AP-1在TGN/内体网格蛋白介导的运输途径中的作用。为此，将评价每种衔接子在网格蛋白向膜募集中的作用。此外，将定义每个衔接子在TGN和内体之间的不同运输途径中的相对分布和功能。其次，两个新的Gga 2 p相互作用蛋白，Ent 3 p和Ent 5 p的功能，将被确定。这些蛋白质，这是所需的网格蛋白介导的交通之间的TGN和内涵体，区分的存在下ENTH相关的结构域。这些蛋白质在网格蛋白外壳组装和网格蛋白介导的运输中的功能将在体内使用突变细胞进行表征，并在体外通过开发使用纯蛋白质和脂质体的外壳组装试验进行表征。重点将针对理解的作用，磷酸肌醇结合的Ent 3 p和Ent 5 p ENTH相关的结构域。将使用相互作用和遗传方法鉴定其他TGN/内体网格蛋白外壳组分。第三，我们将启动一个化学遗传策略，通过识别和表征小分子抑制剂来研究AP-1和Gga介导的交通。总之，这些研究有望为我们理解TGN和内体的ccv形成和蛋白质分选的基本过程提供重大进展。

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.