Structure and function of the nuclear pore complex

描述(申请人提供)：核孔复合体的结构和功能对所有真核生物的细胞内组织至关重要，是通过核孔复合体(NPC)的功能实现的，大分子在细胞核和细胞质之间的准确运输。鼻咽癌是穿透核膜双层膜的巨大运输通道。然而，NPC是如何形成的，以及它们是如何插入核膜的，目前尚不清楚。核祖细胞在许多不同的运输活动中发挥着重要作用，包括信使RNA从细胞核到细胞质的输出。但是，尽管信使RNA具有基本的意义，但信使RNA通过鼻咽癌定向转移的途径仍然没有得到很好的界定。这项研究计划的目的是阐明NPC在核膜中形成和组装的机制，并表征NPC在信使RNA输出中的作用。本提案中描述的所有实验方法都利用了可用于单细胞真核生物酿酒酵母的蛋白质组、基因组和细胞生物学工具。酵母提供了一个很好的模型系统来表征NPC的组成和功能，研究活细胞中复杂的NPC组装过程，并开发新的重组分析方法。我们的目标是：(1)研究鼻咽癌成分Gle1和Dbp5在mRNA输出中的作用，并研究mRNAs如何通过鼻咽癌单向运输。(2)研究跨膜核孔蛋白NDc1在鼻咽癌组装和结构中的作用。(3)使用基于荧光的分析方法检测活细胞中NPC的生物合成，并使用已定义的成分在体外重建NPC的组装步骤。我们结合使用了创新的生化、遗传和细胞生物学方法来解决酿酒酵母的这三个特定目标。由于鼻咽癌是一个高度保守的结构，从这些研究中获得的机械性见解将直接与包括人类在内的所有真核生物相关。与公共健康相关：在所有真核生物中，通过核孔复合体调节大分子运输提供了一种基本的机制，信号转导途径和发育刺激通过该机制控制差异基因的表达。此外，许多病毒针对细胞核运输机制的组件以促进病毒传播，一些致癌易位涉及核孔复合体的组件以促进癌症的发展。因此，更好地了解介导核质运输的分子机制对于了解基本的细胞过程和开发新的抗病毒和抗癌治疗方法都是至关重要的。

DESCRIPTION (provided by applicant): STRUCTURE AND FUNCTION OF THE NUCLEAR PORE COMPLEX Of central importance to the intracellular organization of all eukaryotes is the accurate transport of macromolecules between the nucleus and the cytoplasm, which is achieved through the function of the nuclear pore complex (NPC). The NPC is an enormous transport channel that perforates the double membrane of the nuclear envelope. However, how NPCs form and how they are inserted into the nuclear membrane is not known. NPCs play an essential role in many diverse transport events including the export of messenger RNAs from the nucleus to the cytoplasm. But despite its fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC remains poorly defined. The goals of this research proposal are to elucidate the mechanism by which NPCs form and assemble in the nuclear envelope and to characterize the role of the NPC in messenger RNA export. All experimental approaches described in this proposal take advantage of the proteomic, genomic and cell biological tools available for use within the single cellular eukaryote Saccharomyces cerevisiae. Yeast provides an excellent model system to characterize the components and function of the NPC, to study the complex NPC assembly process in living cells, and to develop novel reconstitution assays. Our aims are: (1) To characterize the role of the NPC components Gle1 and Dbp5 in mRNA export and to investigate how mRNAs are unidirectionally transported across the NPC. (2) To study the role of the transmembrane nuclear pore protein Ndc1 in NPC assembly and structure. (3) To use fluorescence based assays to examine NPC biosynthesis in living cells and to reconstitute NPC assembly steps in vitro using defined components. We employ a combination of innovative biochemical, genetic and cell biological approaches to address these three specific aims in S. cerevisiae. Because the NPC is a highly conserved structure, the mechanistic insights obtained from these studies will be directly relevant to all eukaryotes, including humans. PUBLIC HEALTH RELEVANCE: In all eukaryotes, regulation of macromolecular transport through the nuclear pore complex provides an essential mechanism by which signal transduction pathways and developmental stimuli control differential gene expression. In addition, many viruses target components of the cellular nuclear transport machinery to facilitate viral propagation and several oncogenic translocations involve components of the nuclear pore complex to promote cancer development. Therefore, a better understanding of the molecular machinery that mediates nucleocytoplasmic transport is essential both for understanding fundamental cellular processes and the development of novel anti-viral and anti-cancer therapies.