Molecular studies of selective protein transport

描述(由申请人提供)：笼状蛋白包裹的囊泡(CCV)在将质膜蛋白分离到内吞途径以及在反高尔基网络(TGN)和内小体之间分离蛋白质方面发挥着重要作用。这些CCV介导的途径是真核细胞的基本保守成分；途径缺陷可能导致人类遗传性疾病，并可能导致癌症、心脏病和阿尔茨海默病等多基因疾病。此外，艾滋病毒等病原体利用这些途径感染细胞，避免免疫监视。该项目的总体目标是了解CCV在正常细胞中选择性蛋白运输的分子基础，为了解缺陷如何导致疾病提供基础。为了实现这一目标，我们在酿酒酵母中研究了CCV介导的蛋白质转运。在之前的资助期间，已经确定了一个由三种类型的网状蛋白适配器组成的网络，它们在TGN和内体之间起运输作用，包括AP-1复合体、GGA蛋白和内毒素相关蛋白。对酵母中这些接头的分析为解决CCV在TGN和内体形成的机制开辟了独特的途径。此外，还发现了内吞条域蛋白Rvs167P与泛素结合的新作用。这一发现为确定泛素结合在内吞作用中的功能提供了机会，而不是识别货物。将结合遗传、化学遗传、生化和活细胞成像策略来实现四个具体目标。首先，在野生型和突变型酵母菌株中，将使用内源表达的荧光适配器和笼状蛋白的延时活细胞成像来表征TGN/内体上的笼状蛋白外壳组装的机制。第二，补充生化策略将针对特定适配器在涂层组装、膜结合和变形以及货物选择中的作用进行定义。第三，前两个目标中的方法将被扩展以确定保守的TGN/内体辅助因子在CCV形成中的作用，并将识别新的辅助因子。第四，将确定Rvs167p在内吞作用中与泛素结合的机制和功能。总之，这些研究有望为CCV的形成和TGN与内吞体之间以及内吞作用过程中蛋白质分选的基本过程提供重要的见解，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。 与公共卫生相关：动物细胞结构和功能的一个基本方面涉及细胞内各隔室之间的蛋白质运输。这个项目将使用酵母作为真核细胞的模型，来研究一种特定类型的运输载体--笼蛋白包裹的囊泡所介导的转运机制。该项目提供的见解将有助于理解网状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.