How Karyopherins Move Across The Nuclear Pore Complex

描述（由申请人提供）：核转运蛋白介导的蛋白质转运穿过核孔复合体（NPC）对于真核细胞至关重要，但核转运蛋白转运穿过NPC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，它们在细胞核中执行功能之前需要穿过NPC。因此，它是重要的人类健康问题，我们实现了更好地了解核质转运的一般机制。该项目的长期目标是在分子水平上了解核转运蛋白如何利用它们与核孔蛋白的相互作用在携带货物时穿过NPC。实验策略是使用化学交联剂来识别Nups，其作为NPC内Kap运动的“垫脚石”，然后使用生物化学技术来详细表征核转运蛋白和识别的核孔蛋白之间的相互作用，最后使用从生物化学分析获得的知识来设计和进行实验，该实验将在体内测试NPC内核转运蛋白运动的力学。酵母S.酿酒酵母将被用作本研究的模式真核生物。具体目标是：i）检验含有FG重复的核孔蛋白在Kap95 p穿过NPC的移动中充当顺序“垫脚石”的假设，ii）检验核孔蛋白在NPC内特异性排列以显示对Kap95 p的“亲和力梯度”的假设，所述亲和力梯度促进其穿过NPC的移动，iii）鉴定Kap95 p中干扰其停靠在不同Nup上的能力的点突变，并测试其体内效果，及iv）进行比较研究（如Kap95p所述），用于两种额外的核转运蛋白（输出Crm1 p和输入Kapi 04 p），目的是揭示核转运蛋白在NPC中以相似和相反方向移动的路径中的一般和特异性特征。

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.