FUNCTIONAL STUDY OF NATIVE AND SYNTHETIC NUCLEI

我们研究的长期目标是了解 核孔控制着分子进出 原子核 近年来，人们对 在从质膜传递信号时发生的事件 到细胞核。 越来越多的信号转导问题， 发育决定、致癌转化和HIV-1病毒 传染性集中在核进口或出口上。 虽然我们现在有 对于核孔作用的概念框架，我们知之甚少 孔的分子性质或它如何控制这些无数的事件。 为了达到这一目的，在过去的赠款期间，我们开发了一个系统， 核孔本身可以被重建。 通过 免疫耗竭系统，生化改变核孔可以是 创建并分析功能。 利用这个系统， 鉴定核孔的亚基，p62-p58-p54复合物，和 表明该亚基是功能性核孔所必需的。 我们的研究主要集中在三个方面：1）详细的 对这种新发现的核孔亚基p62-58-54的分析 复杂. 具体来说，我们将确定的分子结构的 复杂，创造突变的复杂形式，并使用这些突变的复杂， 说明复合体是如何组装的。 从那里，我们会问， 复合物进一步组装到核孔中，一旦到了那里， 它在核运输中发挥作用。 2)第二个主要目标是启动一个 寻找新的核孔蛋白。 虽然核孔 估计含有约1000种蛋白质，可能少于或 其中60种是不同的，只有6种孔蛋白被 最终确定。 在对这种缺陷的生物化学方法中， 新鉴定的爪蟾孔蛋白，p200，将进行分析，以及 它的伴随蛋白，它们一起构成了第二个亚基， 核孔 这些实验的承诺是，随着每一个新的孔隙 蛋白质被识别，它可以立即用核孔进行测试， 重组系统的作用，孔功能。 强大的基因 还将进行筛选，以确定尚未发现的孔隙 酵母中的蛋白质 3)最后，核武器的拆卸和组装 孔将使用无细胞系统进行研究， 间期和有丝分裂。 一种新发现的磷酸化孔 在有丝分裂中发生的蛋白质将是本研究的重点。 总之，这些实验应该阐明的结构和功能， 核孔，它的调节方式，和机制， 它在有丝分裂时解体和重组。

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.