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

描述（由申请人提供）：通过核孔复合物（NPC）进行跨真核细胞核膜（将细胞核和细胞质分开）的转运。调节蛋白质转运发生在NPC的机制知之甚少。特别是，虽然对核蛋白输入进行了大量研究，但相对较少的工作研究了蛋白质从核到细胞质的输出。我们已经开发了一种动力学测定法来研究野生型酵母和缺乏特定NPC组分（Nups）的细胞中从细胞核转运出的相对速率，并且已经使用该测定法来鉴定有效核蛋白输出所需的Nups的子集。我们将研究哪些Nups和Nup结构域是底物在NPC中有效转运所需的，并确定不同方向的蛋白质转运是否需要不同的Nups子集。四个具体的目标将被用来研究特定的核蛋白在核质蛋白运输的作用。在具体目标1中，我们将确定从细胞核有效输出蛋白质所必需的Nups。具体目标2将通过在特定Nup区域产生突变来研究Nup亚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中特定Nup域的上下文对于传输活动是否重要。在具体目标3中，我们将研究不同的Nups和Nup结构域是否对不同转运因子介导的蛋白质输出和输入至关重要。使用调节核出口和小分子介导的蛋白质二聚化，我们将研究不同的基板在细胞中缺乏特定的NUP和NUP结构域的不同的运输蛋白的核进口和出口的动力学。最后，具体目标4将通过为中级实验室课程开发新的调查实验室组件以及在夏季和学年期间支持独立研究项目，将本科生纳入目标1 - 3所述的实验中。这项研究利用一种新的核蛋白输出测定法，提供了第一个全面的核蛋白输出的Nup功能的动力学分析。由此产生的数据将提供深入了解蛋白质易位在NPC中的机制。重要的是，我们将确定不同的Nups是否需要通过不同的转运因子进行输出，以及Nups的不同子集是否介导核蛋白的输入和输出。 为了正常运作，细胞必须仔细调节蛋白质进出细胞核的运动。如果不能做到这一点，就会导致细胞功能的改变，包括基因表达的改变、细胞生长失控和细胞过早死亡。该提案中描述的研究将深入了解细胞如何调节细胞质和细胞核之间的分子运动，以及这种调节如何影响细胞活性。

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.