DISSECTING THE MOLECULAR MACHINERY OF NUCLEAR TRANSPORT

剖析核输运的分子机制

• 批准号: 7163524
• 负责人: MICHAEL P ROUT
• 金额: $ 11.47万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163524
• 关键词: Architecture; Binding Sites; Biochemical; Biological Assay; Cataloging; Catalogs; Cell Fractionation; Cell Nucleus; Cells; Chimera organism; Classification; Communication; Complex; Cytoplasm; Data; Defect; Development; Equipment and supply inventories; Eukaryota; Eukaryotic Cell; Genetic Materials; Genomics; In Vitro; Individual; Interphase Cell; Karyopherins; Knowledge; Label; Learning; Life; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Membrane; Methods; Modeling; Molecular; Molecular Structure; Movement; Nature; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Numbers; Oncogenic; Peptide Signal Sequences; Phase; Positioning Attribute; Proteins; Protocols documentation; Range; Rate; Reaction; Relative (related person); Reporter; Resolution; Role; Saccharomyces; Site; Staging; Structure; Suggestion; Techniques; Technology; Testing; Variant; Work; Yeasts; base; improved; in vivo; in vivo Model; member; novel; nucleocytoplasmic transport; protein protein interaction; reconstitution

Nuclear pore complexes (NPCs) are the sole mediators of nucleocytoplasmic exchange. They therefore define the contents of the nucleus. The pivotal role of the NPC in controlling communication between the genetic material and the rest of the cell is reflected in the many oncogenic and developmental defects directly associated with alterations in nucleocytoplasmic transport. A full understanding of how the NPC mediates transport is needed to discern the nature of these defects. In order to gain this understanding, we require a comprehensive inventory of the molecular components of the NPC, a knowledge of how each component contributes to the overall structure of this large molecular translocation machine, and information on the interactions its proteins make with components of the soluble phase. We have therefore taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast), in which we have produced a low resolution map of the yeast NPC ultrastructure and determined its protein composition. To preserve and study functionally relevant interactions, we are now developing new techniques for the subcellular fractionation and immunopurification of protein complexes from both the stationary and soluble phases of transport. The components of these complexes, and the molecular nature of their interactions, will be characterized using current and emerging mass spectrometric techniques. First, all the NPC components will be purified and the interactions each makes with other proteins will be thoroughly catalogued. We will then determine the position of the NPC proteins and their subcomplexes within our improved maps of the NPC molecular architecture. Members of the mobile phase will be studied in a similar fashion. We will follow the movement of particular transport factors across the NPC, determining the order and sites of interactions that the transport factors make with NPC proteins during nuclear transport. The rules governing transport will be investigated by comparing the data gained from the different transport factors. Our eventual aim is to integrate our ultrastructural and biochemical studies to understand the molecular basis of the translocation of different transport factors across the NPC. These studies should enable us to reconstitute key reactions of nucleocytoplasmic transport in vitro, and test possible mechanistic models in vivo.

核孔复合物（NPC）是核质交换的唯一介质。 因此，它们定义了原子核的内容。 NPC在控制遗传物质和细胞其余部分之间的通信中的关键作用反映在与核质转运改变直接相关的许多致癌和发育缺陷中。 需要充分了解全国人大如何调解运输，以辨别这些缺陷的性质。 为了获得这种理解，我们需要一个全面的清单的分子组成的NPC，每个组件如何有助于这个大的分子易位机器的整体结构的知识，和信息的相互作用，其蛋白质与可溶性相的组件。 因此，我们已经采取了一个全面的方法来定义的NPC在模型真核生物酵母菌（酵母）的功能架构，在其中，我们已经产生了一个低分辨率的地图酵母NPC超微结构，并确定其蛋白质组成。 为了保存和研究功能相关的相互作用，我们现在正在开发新的技术，用于从固定相和可溶性运输相的蛋白质复合物的亚细胞分级分离和免疫纯化。这些复合物的成分，以及它们相互作用的分子性质，将使用当前和新兴的质谱技术进行表征。 首先，所有的NPC成分将被纯化，每种成分与其他蛋白质的相互作用将被彻底编目。然后，我们将确定NPC蛋白及其亚复合物在我们改进的NPC分子结构图中的位置。 将以类似的方式研究移动的阶段的成员。 我们将跟踪特定转运因子在NPC中的运动，确定转运因子在核转运过程中与NPC蛋白相互作用的顺序和位点。 将通过比较从不同的迁移因素获得的数据来研究迁移的规律。 我们的最终目标是整合我们的超微结构和生化研究，以了解不同的转运因子跨NPC易位的分子基础。 这些研究将使我们能够在体外重建核质转运的关键反应，并在体内测试可能的机制模型。