Sorting and trafficking in the endosomal system

描述(由申请人提供)：这个项目的主要目标是从生化和细胞的角度阐明内吞途径中受调控的蛋白质分类的机制。在即将到来的资金期间，工作的重点是从内体出口货物蛋白质，这是一套特征不佳的逆行运输途径，将货物分拣到高尔基体。对于一些营养转运体，如酵母Fet3/Ftr1还原铁转运体，这一步骤构成了内吞循环途径的初始阶段，将它们返回质膜重新使用。已经建立了包括营养转运体、酸性水解酶分选受体和SNARE蛋白在内的模型货物蛋白来研究这些途径，并确定了它们运输的遗传要求。拟议的实验将确定逆行分拣所需的货物蛋白质的结构特征，并确定识别这些信号的分拣机械部件。这项研究将集中在逆转录-货物分类复合体和分类连接素的作用，逆转录-货物分类复合体是一种进化上保守的与内体膜相关的五种蛋白质的复合体，分类连接素被认为在内体输出途径中作为货物受体发挥作用。将使用的方法包括酵母突变菌株贩运的遗传分析、蛋白质周转率的动力学分析、货物与分拣机械之间以及分拣机械部件之间相互作用的生化特征。这些研究将提供关于内体系统如何识别货物蛋白质的关键见解，如何将识别转化为内体货物的输出，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。 与公共健康相关：细胞生理学的许多方面直接依赖于质膜的蛋白质和脂肪成分：营养获取、细胞-细胞识别和信号传递、细胞-底物黏附和细胞运动。质膜的分子组成与细胞内的内小体处于动态平衡状态。这项拟议的研究将调查内体内蛋白质分选的机制，这些机制导致它们循环回到质膜。

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.