MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

这一建议的总体目标是研究NTF2介导的RAN导入的机制。RAN GTP酶是所有真核细胞中一个必不可少的过程--核运输的中心组成部分。本实验室和其他实验室的结果表明，小GTP酶RAN的核进口是由核运输因子2(NTF2)介导的，定义了一条新的RAN导入途径。在这里，我们将基于我们的RAN导入模型测试三个假设。我们将利用体外生物化学和通透性细胞实验，研究NTF2截留对RAN导入的影响以及RanGDP对核孔的靶向性。此外，我们将使用定量交换分析和一种新的渗透性细胞分析来验证RCC1交换活性是从核孔释放RAN所必需的预测。最后，我们将研究Nup 153在RAN运输中的功能，Nup 153是一种候选NTF2：RanGDP核孔对接蛋白，被认为在其他运输途径中发挥功能。对这些假说的研究可能会揭示NTF2介导的RAN运输所需的分子相互作用的新信息，无论是在运输的可溶性阶段还是在核孔的静止阶段。这些新数据可能会为我们进一步了解核运输提供关键信息，核运输是所有真核细胞中生命绝对需要的动态过程。

The overall goal of this proposal is to investigate the mechanism of NTF2-mediated Ran import. The Ran GTPase is a central component of an essential process in all eukaryotic cells -- nuclear transport. Results from this laboratory and others demonstrate that nuclear import of the small GTPase Ran is mediated by Nuclear Transport Factor 2 (NTF2), defining a novel Ran import pathway. Here, we will test three hypotheses based on our model for Ran import. Using in vitro biochemistry and a permeabilized cell assay, we will investigate the effect on Ran import of NTF2 sequestration and targeting of RanGDP to the nuclear pores. Additionally, we will test the prediction that RCC1 exchange activity is required for release of Ran from nuclear pores using quantitative exchange assays and a novel permeabilized cell assay. Finally, we will investigate the function in Ran transport of Nup 153, a candidate NTF2:RanGDP nuclear pore docking protein that is proposed to function in other transport pathways. Investigation of these hypotheses is likely to reveal new information as to the molecular interactions required for NTF2-mediated Ran transport, both in the soluble phase of transport and in the stationary phase at the nuclear pore. These new data may provide information critical to our further understanding of nuclear transport, a dynamic process absolutely required for life in all eukaryotic cells.