Molecular studies of selective protein transport

描述（由申请人提供）：网格蛋白包被囊泡（ccv）在将质膜蛋白分选入内吞途径以及在反式高尔基体网络（TGN）和内体之间分选蛋白质方面发挥重要作用。这些ccv介导的通路是真核细胞的基本保守元件;通路缺陷可导致遗传性人类疾病，并可能导致多基因疾病，如癌症，心脏病和阿尔茨海默病。此外，HIV等病原体利用这些途径感染细胞并避免免疫监视。这个项目的总体目标是了解正常细胞中ccv选择性蛋白质转运的分子基础，为理解缺陷如何导致疾病提供基础。为了实现这一目标，ccv介导的蛋白质转运已在酵母酿酒酵母中表征。在之前的资助期间，已经定义了在TGN和内体之间转运中起作用的三种类型的网格蛋白衔接子的网络，其由AP-1复合物、Gga蛋白和胰蛋白酶相关蛋白组成。对酵母中这些衔接子的分析已经开辟了独特的途径来解决在TGN和内体处形成ccv的机制。此外，一个新的作用泛素结合的内吞BAR结构域蛋白，Rvs 167 p，已被发现。这一发现提供了一个机会，以确定功能的泛素结合的内吞作用以外的货物识别。遗传学、化学遗传学、生物化学和活细胞成像策略的组合将被应用于实现四个具体目标。首先，将使用内源性表达的荧光衔接子和网格蛋白的延时活细胞成像在野生型和突变酵母菌株中表征网格蛋白外壳在TGN/内体处组装的机制。第二，互补的生化策略将针对定义特定衔接子在外壳组装、膜结合和变形以及货物选择中的作用。第三，前两个目标的方法将被扩展到确定保守的TGN/内体辅助因子在ccv形成中的功能，并将鉴定新的辅助因子。第四，将确定内吞过程中Rvs 167 p结合泛素的机制和功能。总之，这些研究有望为TGN和内体之间以及内吞过程中ccv形成和蛋白质分选的基本过程提供重要见解，从而有助于为理解这些过程在人类疾病中的作用奠定基础。 公共卫生相关性：动物细胞结构和功能的一个基本方面涉及细胞内隔室之间的蛋白质运输。本研究将以酵母为模型真核细胞，探讨由特定类型的转运载体网格蛋白包被囊泡介导的转运机制。该项目提供的见解将有助于了解网格蛋白介导的运输缺陷如何导致疾病。

DESCRIPTION (provided by applicant): Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells; pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer, heart disease, and Alzheimer's disease. Also, pathogens such as HIV take advantage of these pathways to infect cells and avoid immune surveillance. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. During the previous funding period a network of three types of clathrin adaptors that function in transport between the TGN and endosomes has been defined, consisting of the AP-1 complex, Gga proteins, and epsin-related proteins. Analysis of these adaptors in yeast has opened unique avenues to address the mechanism of ccv formation at the TGN and endosomes. Additionally, a novel role for ubiquitin binding by an endocytic BAR domain protein, Rvs167p, has been uncovered. This finding provides an opportunity to define functions for ubiquitin binding in endocytosis other than cargo recognition. A combination of genetic, chemical genetic, biochemical, and live cell imaging strategies will be applied to achieve four specific aims. First, the mechanism of clathrin coat assembly at the TGN/endosomes will be characterized in wild-type and mutant yeast strains using time-lapse live cell imaging of endogenously expressed fluorescent adaptors and clathrin. Second, complementary biochemical strategies will be directed at defining roles for particular adaptors in coat assembly, membrane binding and deformation, and cargo selection. Third, approaches in the first two aims will be extended to determine the functions of conserved TGN/endosome accessory factors in ccv formation, and new accessory factors will be identified. Fourth, the mechanism and function of ubiquitin binding by Rvs167p during endocytosis will be determined. Together these studies are expected to provide significant insights into the fundamental process of ccv formation and protein sorting in pathways between the TGN and endosomes, and during endocytosis, thereby helping to establish a foundation for understanding the roles of these processes in human disease. PUBLIC HEALTH RELEVANCE: A fundamental aspect of animal cell structure and function involves protein transport between compartments within the cell. This project will employ yeast as a model eukaryotic cell to address the mechanism of transport mediated by a specific type of transport carrier, clathrin coated vesicles. Insights provided by this project will help to understand how defects in clathrin-mediated transport contribute to disease.