A FUNCTIONAL STUDY OF NATIVE AND SYNTHETIC NUCLEI

我们研究的长期目标是了解 核孔控制着进出核孔的分子交通。 原子核。近些年来，人们对生物多样性的认识呈爆炸式增长。 发生在质膜信号转导过程中的事件 到原子核。越来越多的信号转导问题， 发育决定、致癌转化与HIV-1病毒 传染性集中在核进口或出口上。尽管我们现在有 关于核孔活动的概念框架，我们知之甚少 毛孔的分子性质或它是如何控制这些无数事件的。 为了达到这种性质，在过去的赠款期间，我们开发了一个系统，通过 核孔本身是可以重组的。穿过 免疫系统耗尽，生化改变的核孔可 为函数创建和分析。使用该系统，这已经成为可能 为了鉴定核孔的一个亚单位，p62-p58-p54复合体，以及 表明这个亚基是功能性核孔所必需的。 我们在这项建议中的研究集中在三个大的领域：1)详细的 新发现的核孔亚基p62-58-54的分析 很复杂。具体来说，我们将确定该分子的分子结构 复合体，创造突变复合体形式，并利用这些突变复合体 阐述建筑群是如何组装的。从那时起，我们将会问， 复合体进一步聚集到核孔中，一旦到达那里，会起到什么作用 它在核运输中发挥着作用。2)第二个主要目标是发起 寻找核孔的新蛋白质。虽然核孔 估计含有约1000个总蛋白质，可能少于或 相当于60个不同的，只有6个孔道蛋白 最终确定了。在解决这一缺陷的生化方法中，一种 将分析新发现的非洲爪哇毛孔蛋白P200，以及 它的伴生蛋白，这些蛋白共同组成了 核孔。这些实验的前景是，当每一个新的毛孔 蛋白质被鉴定后，可以立即用核孔进行测试 重建系统及其在毛孔功能中的作用。一种强大的基因 还将进行筛查，以确定尚未发现的毛孔 酵母中的蛋白质。3)最后，核的拆卸和组装 毛孔将使用无细胞系统进行研究，该系统在 间期和有丝分裂。一种新发现的毛孔磷酸化 发生在有丝分裂中的蛋白质将是本研究的重点。 综上所述，这些实验应该阐明细胞的结构和功能。 核孔，它的调节方式，以及核孔的机制 它在有丝分裂时的分解和重塑。

The long term objective of our research is to understand the way in which the nuclear pore controls the molecular traffic entering and exiting the nucleus. In recent years, there has been an explosion in knowledge of the events which occur in the transduction of a signal from the plasma membrane to the nucleus. Increasingly, questions of signal transduction, developmental determination, oncogenic transformation, and HIV-1 viral infectivity are focusing on nuclear import or export. Although we now have a conceptual framework for the action of the nuclear pore, we know little of the molecular nature of the pore or how it controls these myriad events. To get at that nature, in the past grant period we developed a system by which the nuclear pore itself can be reconstituted. Through immunodepletion of the system, biochemically altered nuclear pores can be created and analyzed for function. Using the system, it has been possible to identify a subunit of the nuclear pore, the p62-p58-p54 complex, and show that this subunit is required for functional nuclear pores. Our studies in this proposal focus on three broad areas: 1) A detailed analysis of this newly discovered nuclear pore subunit, the p62-58-54 complex. Specifically, we will determine the molecular structure of the complex, create mutant complex forms, and use those mutant complexes to address how the complex is assembled. From there, we will ask how the complex further assembles into the nuclear pore, and once there, what role it plays in nuclear transport. 2) A second major goal is to initiate a search for new proteins of the nuclear pore. Although the nuclear pore would be estimated to contain ~1000 total proteins, perhaps less than or equal to 60 of which are different, only 6 pore proteins have been conclusively identified. In a biochemical approach to this deficit, a newly identified Xenopus pore protein, p200, will be analyzed, as well as its companion proteins, which together comprise a second subunit of the nuclear pore. The promise of these experiments is that as each new pore protein is identified, it can immediately be tested with the nuclear pore reconstitution system for its role in pore function. A powerful genetic screen will also be carried out to identify as yet undiscovered pore proteins in yeast. 3) Lastly, the disassembly and assembly of the nuclear pore will be studied using a cell-free system which cycles between interphase and mitosis. A newly discovered phosphorylation of pore proteins which occurs at mitosis will be the focus of this study. Together, these experiments should elucidate the structure and function of the nuclear pore, the way in which it is regulated, and the mechanism of its disassembly and reformation at mitosis.