KARYOPHERIN X, A NOVEL TRANSPORT RECEPTOR

蛋白质和RNA在细胞质和细胞核之间的易位是由一系列可溶性转运受体介导的，其中只有9个在哺乳动物中被发现。关键调节蛋白的不适当定位与诸如癌变等疾病状态相关的研究结果强调了理解这些因素的重要性。此外，一些转运蛋白在病毒（如HIV）的生命周期和发病机制中起着不可或缺的作用。拟议的研究计划是基于鉴定和克隆一种新的人类运输受体核细胞蛋白X。目标是生物化学表征核细胞蛋白X，鉴定其货物和绘制其识别和结合货物序列中的基序。为实现这些目标，将追求两个具体目标。目的1是确定这种核粘蛋白是否具有界定进口蛋白- β样转运受体家族成员的生化特性。目标二是验证这个蛋白质是一个真正的转运体的假设，通过识别它的货物，并表明它通过核孔介导货物的易位。目的1实验测试Ran（一种小GTPase）结合的转运体，抑制影响Ran核苷酸状态的因素，以及与核孔蛋白的相互作用。Aim 2的实验方法包括固定化转运蛋白和细胞裂解物的拉下试验，以及通透化细胞的体外转运试验。从长远来看，工作将集中在绘制为核细胞蛋白x介导的运输分类货物的靶向序列上。

The translocation of proteins and RNA between the cytoplasm and nucleus is mediated by a family of soluble transport receptors, of which only nine have so far been identified in mammals. The importance of understanding these factors is underscored by findings that correlate the improper localization of key regulatory proteins to disease states such as carcinogenesis. In addition, several transporters play integral roles in the life cycle and pathogenesis of viruses (e.g., HIV). The proposed research plan is based on the identification and cloning of a novel, human transport receptor, karyopherin X. The objectives are to biochemically characterize karyopherin X, identify its cargo and map the motif it recognizes and binds to within the cargo sequence. To realize these goals, two specific aims will be pursued. Aim number 1 is to determine if this karyopherin shares the biochemical properties that define the members of the importin-beta-like transport receptor family. Aim number 2 is to test the hypothesis that this protein is a bona fide transporter, by identifying its cargo and showing that it mediates the translocation of this cargo across the nuclear pore. Aim number 1 experiments test the transporter for Ran (a small GTPase) binding, inhibition of the factors that influence the nucleotide state of Ran, and interactions with nuclear pore proteins. Experimental approaches for Aim number 2 include pulldown assays with immobilized transporter and cell lysates and in vitro transport assays in permeabilized cells. In the longer term, efforts will focus on mapping the targeting sequence(s) that sorts cargo for karyopherin X-mediated transport.