Dissecting the molecular machinery of nuclear transport

剖析核运输的分子机制

• 批准号: 7317588
• 负责人: MICHAEL P ROUT
• 金额: $ 27.72万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7317588
• 关键词: Amino Acids; Architecture; Automobile Driving; Behavior; Cataloging; Catalogs; Cell Nucleus; Cells; Coated vesicle; Complex; Computer software; Computing Methodologies; Cytoplasm; DNA; Data; Data Set; Databases; Electron Microscopy; Escherichia coli; Eukaryota; Eukaryotic Cell; Evolution; Fruit; Goals; In Vitro; Individual; Instruction; Interphase Cell; Karyopherins; Kinetics; Learning; Life; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Molecular Structure; Morphology; Motion; Movement; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Phase; Positioning Attribute; Proteins; Reaction; Recombinants; Relative (related person); Research Personnel; Resolution; Route; Saccharomyces; Shapes; Structural Models; Structure; Techniques; Tertiary Protein Structure; Testing; Work; Yeasts; abstracting; biochemical model; cancer cell; crosslink; improved; in vivo; in vivo Model; kinematics; mutant; nucleocytoplasmic transport; progenitor; programs; reconstitution; research study; restraint; trafficking; vector; virtual

DESCRIPTION (provided by applicant): ABSTRACT Nuclear pore complexes (NPCs) are the sole mediators of exchange across the nuclear envelope (NE) between the nuclear and cytoplasmic compartments. Nucleocytoplasmic transport depends on the interplay between transport cargoes, their cognate soluble transport factors (many termed Kaps), and NPCs. We have taken a comprehensive approach to defining the functional architecture of the NPC in the model eukaryote Saccharomyces (yeast). We identified all the yeast NPC proteins (Nups) and plotted their disposition in the NPC; this work allowed us to propose a new "virtual gating" mechanism for nuclear transport. We also assigned fold types to all the Nups and systematically isolated Nup subcomplexes to determine the network of interactions they make. We then used this information to compute a 3D map of the NPC architecture, sufficient to resolve the molecular organization of the entire NPC. Our work exposed a simple modularity in the architecture of the NPC; moreover, similarities between structures in coated vesicles and those in the NPC suggest their common evolutionary origin in a progenitor "protocoatomer". Our goal is now to produce high resolution dynamic maps of the NPC. First, we will use additional "low-fruit, high-payoff' immunopurification and immunolocalization experiments to rapidly improve our NPC map, to the point at which we can discern the shapes of the Nups in it. We will then study recombinant Nups and Nup complexes using electron microscopy and crosslinking, to reveal fine details on how the folds, domains, and proteins are organized within the Nups and their complexes. Next, we will reconstitute key reactions of nucleocytoplasmic transport in vitro, and test possible mechanistic models in vivo, in order to reconstruct the movements Kaps and their cargos make on crossing the NPC. We will finally synergistically decode this information and convert it into dynamic, 3D representations of the NPC and nuclear transport, ideally at atomic resolution, thereby allowing us to understand the origin, assembly and mechanism of the NPC at the most fundamental level. LAY SUMMARY We are studying the tiny machines that shuttle materials to the DNA in living cells. These machines, called "nuclear pore complexes", allow the DNA to send its instructions to the rest of the cell, and so help regulate how a cell lives, develops, and stops itself from making the kinds of mistakes seen in cancer cells. We wish to understand how these machines work and how they arose in the early evolution of life.

描述（由申请人提供）： 摘要 核孔复合物（NPC）是核膜和细胞质区室之间跨核膜（NE）交换的唯一介体。核细胞质运输取决于运输货物、其同源可溶性运输因子（许多称为 Kaps）和 NPC 之间的相互作用。我们采用了一种综合方法来定义真核生物酵母（酵母）模型中 NPC 的功能结构。我们鉴定了所有酵母 NPC 蛋白 (Nups) 并绘制了它们在 NPC 中的分布；这项工作使我们能够提出一种新的核运输“虚拟门控”机制。我们还为所有 Nup 分配了折叠类型，并系统地分离了 Nup 子复合体，以确定它们之间的相互作用网络。然后，我们使用这些信息来计算 NPC 架构的 3D 图，足以解析整个 NPC 的分子组织。我们的工作揭示了 NPC 架构中的简单模块化；此外，包被囊泡的结构与NPC中的结构之间的相似性表明它们在祖细胞“原包衣体”中具有共同的进化起源。我们现在的目标是制作 NPC 的高分辨率动态地图。首先，我们将使用额外的“低成果、高回报”免疫纯化和免疫定位实验来快速改进我们的 NPC 图谱，直到我们可以辨别其中 Nups 的形状。然后，我们将使用电子显微镜和交联研究重组 Nups 和 Nup 复合物，以揭示 Nups 中的折叠、结构域和蛋白质如何组织及其它们的详细信息。 复合物。接下来，我们将在体外重建核细胞质运输的关键反应，并在体内测试可能的机制模型，以重建Kaps及其货物在穿越NPC时的运动。我们最终将协同解码这些信息，并将其转换为 NPC 和核运输的动态 3D 表示，最好是原子分辨率，从而使我们能够了解起源、组装和 全国人大的最基层机制。简单总结 我们正在研究将物质运送到活细胞中 DNA 的微型机器。这些机器被称为“核孔复合体”，允许 DNA 将指令发送到细胞的其余部分，从而帮助调节细胞的生存、发育并阻止自身犯癌细胞中常见的错误。我们希望了解这些机器是如何工作的以及它们是如何在早期进化中出现的 生活。