Sorting and trafficking in the endosomal system

描述（由申请人提供）：该项目的主要目标是从生物化学和细胞学角度阐明内吞途径中调节蛋白质分选的机制。在即将到来的资助期间，工作的重点是从内体输出货物蛋白，这是一组特征不佳的逆行运输途径，将货物分类到高尔基体。对于一些营养转运蛋白，如酵母Fet 3/Ftr 1还原铁转运蛋白，这一步骤构成了内吞再循环途径的初始阶段，使它们返回质膜以重新使用。模型货物蛋白，包括营养转运蛋白，酸性水解酶分选受体，和SNARE蛋白已被建立，以调查这些途径和他们的贩运的遗传要求已被确定。拟议的实验将确定货物蛋白质的结构特征是必要的逆行分选，并确定识别这些信号的分选机制的组成部分。该研究将重点关注retromer货物分选复合物的作用，这是一种与内体膜相关的五种蛋白质的进化保守复合物，以及分选连接蛋白，它们被认为是内体出口途径中的货物受体。拟使用的方法包括对酵母突变株贩运进行遗传分析、对蛋白质周转率进行动力学分析、对货物与分选机器之间以及分选机器各组成部分之间的相互作用进行生物化学表征。这些研究将提供关键的见解，货物蛋白如何在内体系统中识别，识别如何转化为货物从内体的出口，以及这些分子相互作用如何被细胞利用来调节质膜的组成。 公共卫生相关性：细胞生理学的许多方面直接依赖于质膜的蛋白质和脂质组成：营养物质获取，细胞-细胞识别和信号传导，细胞-基质粘附和细胞运动。质膜的分子组分与细胞内体处于动态平衡。拟议的研究将调查内体内的蛋白质分选机制，导致其再循环回质膜。

DESCRIPTION (provided by applicant): The broad goal of this project is to elucidate, in biochemical and cellular terms, a mechanism for regulated protein sorting within the endocytic pathway. The focus of the work in the upcoming funding period is on the export of cargo proteins from endosomes, a poorly characterized set of retrograde transport pathways that sort cargo to the Golgi apparatus. For some nutrient transporters, such as the yeast Fet3/Ftr1 reductive iron transporter, this step constitutes the initial stage of an endocytic recycling pathway that returns them to the plasma membrane for re-use. Model cargo proteins, including nutrient transporters, an acidic hydrolase sorting receptor, and SNARE proteins have been established to investigate these pathways and the genetic requirements for their trafficking have been determined. Proposed experiments will identify the structural features of cargo proteins that are necessary for retrograde sorting, and identify the components of the sorting machinery that recognize these signals. The research will focus on the roles of the retromer cargo sorting complex, an evolutionarily conserved complex of five proteins that are associated with endosome membranes, and sorting nexins, which are proposed to function as cargo receptors in endosome export pathways. Methodologies to be used include genetic analysis of trafficking in yeast mutant strains, kinetic analysis of protein turnover rates, biochemical characterization of interactions between cargo and the sorting machinery and between components of the sorting machinery. The studies will provide key insights into how cargo proteins are recognized in the endosomal system, how recognition is translated into export of cargo from endosomes, and how these molecular interactions are harnessed by the cell to modulate the composition of the plasma membrane. PUBLIC HEALTH RELEVANCE: Many aspects of cell physiology rely directly on the protein and lipid composition of the plasma membrane: nutrient acquisition, cell-cell recognition and signaling, cell-substrate adhesion, and cell motility. The molecular components of the plasma membrane are in a dynamic equilibrium with intracellular endosomes. The proposed studies will investigate mechanisms of protein sorting within endosomes that lead to their recycling back to the plasma membrane.