Structure and function of the nuclear pore complex

核孔复合体的结构和功能

• 批准号: 8215762
• 负责人: KARSTEN WEIS
• 金额: $ 37.17万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8215762
• 关键词: ATP phosphohydrolase; Address; Anabolism; Binding Proteins; Biochemical Genetics; Biogenesis; Biological; Biological Assay; Biological Models; Boxing; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Complex; Cytoplasm; Development; Eukaryota; Eukaryotic Cell; Event; Fluorescence; Funding; Genomics; Goals; Health; Human; In Vitro; Individual; Integral Membrane Protein; Life; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Microscopy; Molecular; Molecular Structure; Nuclear Envelope; Nuclear Outer Membrane; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Oncogenic; Pathway interactions; Phytic Acid; Play; Pore Proteins; Process; Proteins; Proteomics; Regulation; Research Proposals; Resolution; Role; Saccharomyces cerevisiae; Signal Transduction Pathway; Site; Stimulus; Structure; Testing; Tissue-Specific Gene Expression; Viral; Virus; Work; Yeasts; base; cancer therapy; in vivo; innovation; insight; mRNA Export; macromolecule; novel; nucleocytoplasmic transport; reconstitution; research study; scaffold; small molecule; tool

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.

描述（由申请人提供）：对所有真核生物的细胞内组织的核孔复合物的结构和功能是细胞核和细胞质之间的大分子的准确运输，这是通过核孔复合物（NPC）的功能实现的。 NPC是一个巨大的运输通道，可穿孔核包膜的双层膜。但是，尚不清楚如何形成NPC及其如何插入核膜。 NPC在许多多种运输事件中起着至关重要的作用，包括从细胞核出口到细胞质的Messenger RNA。但是，尽管具有根本的意义，但Messenger RNA定向通过NPC的途径仍然很差。这项研究建议的目标是阐明NPC在核包膜中形成和组装并表征NPC在Messenger RNA出口中的作用的机制。本提案中描述的所有实验方法都利用了可在单个细胞真核生物酿酒酵母中使用的蛋白质组学，基因组和细胞生物学工具。酵母提供了一个出色的模型系统，以表征NPC的组件和功能，研究活细胞中复杂的NPC组装过程，并开发新的重建测定法。我们的目标是：（1）表征NPC组件GLE1和DBP5在mRNA导出中的作用，并研究如何单向运输MRNA跨NPC。 （2）研究跨膜核孔蛋白NDC1在NPC组装和结构中的作用。 （3）使用基于荧光的测定法检查活细胞中的NPC生物合成，并使用定义的组件在体外重构NPC组装步骤。我们采用了创新的生化，遗传和细胞生物学方法的结合，以解决酿酒酵母中的这三个特定目标。由于NPC是一种高度保守的结构，因此从这些研究中获得的机械见解将与包括人类在内的所有真核生物直接相关。公共卫生相关性：在所有真核生物中，通过核孔复合物对大分子转运的调节提供了一种基本机制，通过该机制，信号转导途径和发育刺激控制差异基因表达。此外，许多病毒靶向细胞核转运机制的成分，以促进病毒传播，几种致癌易位涉及核孔复合物的成分，以促进癌症的发展。因此，更好地理解介导核细胞质转运的分子机制对于理解基本细胞过程以及新型抗病毒和抗癌疗法的发展至关重要。