Clathrin adaptor function at the trans Golgi network and endosomes

描述(申请人提供)：分泌途径中的一个主要分选站是跨高尔基网络(TGN)，其中蛋白质被定向到质膜、内小体和溶酶体。TGN的分选缺陷可能会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。该项目的长期目标是确定蛋白质分选到TGN和内小体之间的囊泡介导途径的分子基础。 对酿酒酵母的研究表明，包被蛋白的小泡(CCV)参与了TGN和内体之间进化保守的蛋白质运输。在这些途径中涉及两种类型的网状蛋白适配器，AP-1复合体和单体GGA蛋白。对酵母接头的分析开辟了独特的途径来解决在TGN/内吞体内形成CCV的问题。将综合运用遗传、分子、生化和细胞生物学策略来实现三个具体目标。首先，将确定GGA蛋白和AP-1在TGN/内吞体网状蛋白介导的转运途径中的作用。为此，将评估每个适配器在膜上网状蛋白募集中的作用。此外，还将定义每个接头在TGN和内体之间不同运输途径中的相对分布和功能。其次，将确定两个新的GGa2p相互作用蛋白Ent3p和Ent5p的功能。这些蛋白质是笼状蛋白介导的TGN和内体之间的运输所必需的，它们的区别在于存在Then相关结构域。这些蛋白质在分子筛膜组装和分子筛蛋白介导的转运中的功能将通过突变细胞在体内和体外通过建立一种使用纯蛋白质和脂质体的膜组装实验来表征。重点是了解Ent3p和Ent5p第10相关结构域结合肌醇磷脂的作用。将利用相互作用和遗传方法确定其他TGN/内体笼状蛋白外壳成分。第三，我们将启动一种化学遗传策略，通过识别和表征小分子抑制剂来研究AP-1和GGA介导的流量。总之，这些研究有望为我们理解CCV形成的基本过程以及TGN和内吞体内的蛋白质分选提供重大进展。

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.