Dissecting the molecular machinery of nuclear transport

剖析核运输的分子机制

• 批准号: 7645723
• 负责人: MICHAEL P ROUT
• 金额: $ 27.89万
• 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/7645723
• 关键词: Amino Acids; Architecture; Automobile Driving; Behavior; Biochemical; Cataloging; Catalogs; Cell Nucleus; Cells; Coated vesicle; Complex; Computer software; Computing Methodologies; Cytoplasm; DNA; Data; Data Set; Databases; Electron Microscopy; Escherichia coli; Eukaryota; Evolution; Fruit; Goals; In Vitro; Individual; Instruction; Interphase Cell; Karyopherins; Kinetics; Learning; Life; 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; cancer cell; crosslink; improved; in vitro testing; 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）是跨核膜（NE）在核和细胞质区室之间进行交换的唯一介质。核质转运依赖于转运货物，其同源可溶性转运因子（许多称为Kaps）和NPC之间的相互作用。我们已经采取了一个全面的方法来定义NPC的功能架构的模式真核生物酵母菌（酵母）。我们确定了所有的酵母NPC蛋白质（NUPS），并绘制其在NPC的处置，这项工作使我们能够提出一个新的“虚拟门控”机制的核运输。我们还分配折叠类型的所有Nups和系统隔离Nup亚复合物，以确定他们的相互作用网络。然后，我们使用这些信息来计算NPC结构的3D图，足以解决整个NPC的分子组织。我们的工作暴露了一个简单的模块化的NPC的架构，此外，在包被囊泡和那些在NPC的结构之间的相似性表明它们共同的进化起源于一个祖先的“protocoatomer”。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，使我们能够分辨出其中Nup的形状。然后，我们将使用电子显微镜和交联研究重组Nup和Nup复合物，以揭示Nup及其复合物中折叠，结构域和蛋白质是如何组织的细节。接下来，我们将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，以重建Kaps及其货物在穿过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，理想情况下是原子分辨率，从而使我们能够在最基本的水平上了解NPC的起源，组装和机制。我们正在研究将材料运送到活细胞中的DNA的微小机器。这些被称为“核孔复合物”的机器允许DNA将其指令发送到细胞的其余部分，从而帮助调节细胞如何生活，发育并阻止自己犯下癌细胞中所见的错误。我们希望了解这些机器是如何工作的，以及它们是如何在生命的早期进化中出现的。