How Karyopherins Move Across The Nuclear Pore Complex

描述（由申请人提供）：核丝蛋白介导的蛋白质通过核孔复合物（NPC）转运对真核细胞至关重要，但核丝蛋白通过NPC转运的机制尚未解决。肿瘤抑制蛋白、激素受体和细胞周期检查点控制蛋白是数百种必需的调节蛋白之一，它们需要通过NPC才能在细胞核中执行其功能。因此，更好地了解核胞质转运的一般机制对人类健康问题具有重要意义。这个项目的长期目标是在分子水平上理解核细胞蛋白如何利用它们与核孔蛋白的相互作用在NPC上移动，同时携带货物。实验策略是使用化学交联剂来识别作为NPC内Kap运动“垫脚石”的Nups，然后使用生化技术详细表征核柔蛋白和已识别的核孔蛋白之间的相互作用，最后使用从生化分析中获得的知识来设计和进行实验，以测试NPC内核柔蛋白运动的体内机制。酿酒酵母将作为本研究的真核生物模型。具体目标是：i)验证含有FG重复序列的核孔蛋白作为Kap95p在NPC上移动的连续“垫脚石”的假设，ii)验证核孔蛋白被专门安排在NPC内以显示Kap95p的“亲和梯度”以促进其在NPC上的移动的假设，iii)鉴定Kap95p中干扰其在不同Nups上停靠的能力的点突变并测试其在体内的影响。iv)对另外两种核细胞蛋白（输出蛋白Crm1 p和输入蛋白Kapi 04p）进行比较研究（如Kap95p所述），目的是揭示核细胞蛋白在NPC中相似和相反方向运动路径的一般和特定特征。

DESCRIPTION (provided by applicant): Karyopherin-mediated protein transport across the nuclear pore complex (NPC) is vital for eukaryotic cells yet the mechanisms of karyopherin translocation across the NPC are unsolved. Tumor suppressor proteins, hormone receptors, and cell-cycle checkpoint control proteins are among the hundreds of essential regulatory proteins that need access across the NPC before executing their function in the nucleus. Thus, it is important to human health issues that we achieve a better understanding of the general mechanisms of nucleocytoplasmic transport. The long-term goal of this project is to understand at a molecular level how karyopherins use their interaction with nucleoporins to move across the NPC while carrying cargo. The experimental strategy is to use chemical crosslinkers to identify Nups that function as "stepping stones" for Kap movement within the NPC, then to characterize in detail the interaction between karyopherins and identified nucleoporins using biochemical techniques, and finally to use the knowledge gained from biochemical analyses to design and conduct experiments that will test in vivo the mechanics of karyopherin movement within the NPC. The yeast S. cerevisiae will be used as a model eukaryote for this research. The specific aims are: i) to test the hypothesis that nucleoporins containing FG repeats function as sequential "stepping stones" in the movement of Kap95p across the NPC, ii) to test the hypothesis that nucleoporins are specifically arranged within the NPC to display a "gradient of affinities" for Kap95p that promotes its movement across the NPC, iii) to identify point mutations in Kap95p that interfere with its ability to dock at distinct Nups and test their effects in vivo, and iv) to conduct a comparative study (as delineated for Kap95p) for two additional karyopherins (the exportin Crm1 p, and the importin Kapi 04p) with the goal of uncovering general and specific features in the paths of karyopherin movement in similar and opposite directions across the NPC.