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阐明，从生物化学和细胞的角度，在内吞途径中调节蛋白质分选的机制。在即将到来的资助期内，工作的重点是从内体出口货物蛋白，内体是一组特征不佳的逆行运输途径，将货物分类到高尔基体。对于一些营养转运体，如酵母Fet3/Ftr1还原性铁转运体，这一步骤构成了内吞循环途径的初始阶段，将它们返回质膜进行再利用。包括营养转运蛋白、酸性水解酶分选受体和SNARE蛋白在内的模型货物蛋白已被建立来研究这些途径，并确定了其运输的遗传要求。拟议的实验将确定货物蛋白质的结构特征，这些结构特征是逆行分选所必需的，并确定识别这些信号的分选机制的组成部分。研究将集中于后转录物分选复合体的作用，这是一种进化上保守的复合体，由五种与核内体膜相关的蛋白质和分选连接蛋白组成，它们被认为在核内体输出途径中起着货物受体的作用。将使用的方法包括对酵母菌突变株的贩运进行遗传分析，对蛋白质周转率进行动力学分析，对货物与分拣机械之间以及分拣机械各部件之间的相互作用进行生化表征。这些研究将提供关键的见解，以了解货物蛋白如何在内体系统中被识别，识别如何转化为从内体输出货物，以及细胞如何利用这些分子相互作用来调节质膜的组成。

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.