Analysis of the role of FG nucleoporins in nuclear protein export.

描述（由申请人提供）：真核细胞通过核孔复合物（NPCs）通过分离细胞核和细胞质的核膜进行转运。调控蛋白在npc间转运的机制尚不清楚。特别是，虽然对核蛋白输入进行了大量研究，但对蛋白质从细胞核向细胞质的输出进行了相对较少的研究。我们开发了一种动力学试验来研究野生型酵母和缺乏特定NPC成分（Nups）的细胞中细胞核外转运的相对速率，并使用该试验确定了有效核蛋白输出所需的Nups子集。我们将研究哪些Nups和Nup结构域是跨NPC的底物有效易位所必需的，并确定蛋白质货物在不同方向上的易位是否需要不同的Nups子集。四个特定的目标将用于研究特定的Nups在核细胞质蛋白运输中的作用。在特异性目标1中，我们将确定从细胞核中有效输出蛋白质所必需的Nups。特异性目标2将通过在特定的Nup区域产生突变来研究Nup子结构域在易位中的功能。我们还将在Nup之间交换域，以确定NPC内特定Nup域的上下文对运输活动是否重要。在Specific Aim 3中，我们将研究不同的Nup和Nup结构域是否对由不同运输因子介导的蛋白质进出口至关重要。利用受调节的核输出和小分子介导的蛋白质二聚化，我们将研究在缺乏特定Nups和Nup结构域的细胞中，不同转运蛋白对不同底物的核输入和输出的动力学。最后，具体目标4将把本科生纳入目标1 - 3中描述的实验中，通过为中级水平的实验课程开发一个新的研究性实验室组件，并在夏季和学年期间支持独立的研究项目。本研究利用一种新颖的核蛋白输出实验，首次对Nup在核蛋白输出中的功能进行了全面的动力学分析。由此产生的数据将为人们对鼻咽癌蛋白质易位的知之甚少的机制提供见解。重要的是，我们将确定不同的Nups是否需要通过不同的运输因子输出，以及不同的Nups子集是否介导核蛋白的输入和输出。

DESCRIPTION (provided by applicant): Transport across the nuclear envelope separating the nucleus and cytoplasm of eukaryotic cells occurs through nuclear pore complexes (NPCs). The mechanisms by which regulated protein transport occurs across the NPCs are poorly understood. In particular, while much research has been done on nuclear protein import, relatively little work has examined the export of proteins from the nucleus to the cytoplasm. We have developed a kinetic assay to investigate the relative rate of transport out of the nucleus in wild type yeast and in cells lacking specific NPC components (Nups) and have used this assay to identify a subset of Nups necessary for efficient nuclear protein export. We will investigate which Nups and Nup domains are required for efficient translocation of substrate across the NPC and determine if different subsets of Nups are required for translocation of protein cargos in different directions. Four specific aims will be used to investigate the role of specific Nups in nucleocytoplasmic protein transport. In Specific Aim 1, we will identify the Nups necessary for efficient protein export from the nucleus. Specific Aim 2 will investigate the function of Nup subdomains in translocation by generating mutations in specific Nup regions. We will also swap domains between Nups to determine if the context of specific Nup domains within the NPC is important for transport activity. In Specific Aim 3, we will examine whether distinct Nups and Nup domains are essential for protein export and import mediated by different transport factors. Using regulated nuclear export and small molecule-mediated protein dimerization, we will examine the kinetics of nuclear import and export of distinct substrates by different transport proteins in cells lacking specific Nups and Nup domains. Finally, Specific Aim 4 will incorporate undergraduate students into the experiments described in Aims 1 - 3, through both the development of a new investigative lab component for an intermediate-level lab course and the support of independent research projects during the summer and academic year. This research utilizes a novel nuclear protein export assay to provide the first comprehensive kinetic analysis of Nup function in nuclear protein export. The resulting data will provide insights into the poorly understood mechanism for protein translocation across the NPC. Importantly, we will identify whether distinct Nups are necessary for export by different transport factors and if different subsets of Nups mediate nuclear protein import and export. PUBLIC HEALTH RELEVANCE: In order to function properly, cells must carefully regulate the movement of proteins into and out of the nucleus. Failure to do so results in changes in cell function, including alterations in gene expression, uncontrolled cell growth, and premature cell death. The research described in this proposal will provide insights into how cells regulate the movement of molecules between the cytoplasm and nucleus and how that regulation impacts cellular activity.