How Karyopherins Move Across The Nuclear Pore Complex

描述(申请人提供)：核黄素介导的蛋白质跨核孔复合体(NPC)转运对真核细胞至关重要，但核粘附素跨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基本调节蛋白中的一部分，它们需要通过NPC才能在细胞核中执行其功能。因此，对于人类健康问题，更好地了解核质运输的一般机制是很重要的。这个项目的长期目标是在分子水平上了解核粘附素如何利用它们与核孔蛋白的相互作用在运载货物时穿过NPC。实验策略是使用化学交联剂来识别作为鼻咽癌内Kap运动的垫脚石的NUP，然后利用生化技术详细描述核粘附素与已识别的核孔蛋白之间的相互作用，最后利用从生化分析中获得的知识来设计和进行实验，以在体内测试鼻咽癌内核粘附素运动的机制。酿酒酵母将被用作本研究的真核生物模型。其具体目的是：i)检验含有FG重复序列的核孔素在Kap95p跨越NPC的移动中作为连续“垫脚石”作用的假设，ii)检验这样的假设，即在NPC内特定地安排核孔素以显示促进其跨越NPC移动的Kap95p的“亲和力梯度”，iii)识别Kap95p中干扰其在不同的核苷酸处停靠的能力的点突变，并在体内测试它们的作用，以及iv)对另外两种核粘蛋白(exportin CRM1，和Importin Kapi 04P)，目的是揭示核粘附素在鼻咽癌中以相似和相反方向移动的一般和特殊特征。

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.