A Structure and Function Study: The Transport Channel of the Nuclear Pore Complex

结构与功能研究：核孔复合体的运输通道

• 批准号: 8456423
• 负责人: RICHARD A WING
• 金额: $ 5.22万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456423
• 关键词: Adaptor Signaling Protein; Affect; Bilateral; Binding; Binding Sites; Biological Assay; Caliber; Calorimetry; Cell Nucleus; Cell Survival; Cells; Chimeric Proteins; Complex; Corpus striatum structure; Crystallization; Cytoplasm; Cytoplasmic Filaments; Goals; Gold; Immunoelectron Microscopy; Individual; Integral Membrane Protein; Laboratories; Link; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Membrane Proteins; Methods; Molecular Sieve Chromatography; Mutation; Necrosis; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Peripheral; Primary biliary cirrhosis; Process; Proteins; RNA Splicing; Role; Structure; Syndrome; Techniques; Titrations; Variant; Vertebrates; Virus Diseases; Western Blotting; Work; Yeasts; base; cooking; human disease; infancy; man; mutant; nucleocytoplasmic transport; particle; public health relevance; scaffold; trafficking

DESCRIPTION (provided by applicant): The nuclear pore complex (NPC) is a massive macromolecular gateway, composed of proteins called nucleoporins (Nups), which makes bidirectional transport across the nuclear membrane possible. The core of the NPC can be readily described as being organized into concentric layers. The components of these layers do not exist independently of one another and the components of the NPC are assigned to each layer based upon function. The outermost layer consists of pore membrane proteins (POMs) that anchor the NPC to the nuclear membrane. The "coat" Nups form the next innermost layer and connect the POMs to the "adaptor" layer. A key player within the adaptor layer is Nup93, which forms a central hub by interacting with the other proteins of the adaptor layer as well as the nucleoporins of the innermost layer called the "transport channel". Four nucleoporins, all of which are essential for cell viability and nuclear transport, comprise the transport channel: Nup54, Nup58/45 (Nup45 is a splice variant of Nup58), and Nup62. Based on previous work in the Blobel laboratory, Nup58 is proposed to form the central "midplane" ring of the transport channel through which cellular traffic passes. The diameter of the midplane ring is predicted to double in diameter upon binding of Nup54 to Nup58. Additionally, Nup54 links Nup58 and Nup62 together within the transport channel and contains independent binding sites for both. Although the structures of individual nups and transport factors are well characterized, how different ring layers within the NPC interact and how the diameter of the midplane ring is modulated is poorly understood. The goal of this proposal is to determine the basis for interaction between the adaptor layer and the transport channel and how their interaction might modulate the opening and closing of the midplane ring. Aim 1: To determine the crystal structure of a complex of Nup93 with the triple complex of Nup54, Nup58, and Nup62. I will screen for crystals of a complex of Nup93 with Nup54, Nup58, and Nup62 using standard crystallization techniques and can already isolate a complex of these four nups using size-exclusion chromatography. Solving the structure of this complex will reveal how the adaptor layer interacts with the transport layer of the NPC and how Nup93 and the adaptor layer regulate the opening and closing of the Nup58 ring. Aim 2: To functionally characterize the interactions between the adaptor layer and the transport channel to determine how the adaptor layer may modulate ring opening and ring closing. I will use semi-permeabilized cell nuclear import assays with gold particles conjugated to nuclear localization sequence-GFP fusion proteins to investigate whether or not mutants that disrupt the interaction between Nup93 and Nup54, Nup58, and Nup62 affect nuclear import. Additionally, I will characterize the effect of the mutations on the interaction of Nup93 with the transport channel using size-exclusion chromatography and isothermal titration calorimetry. Finally, using western blotting and mass spectrometry, I will probe for additional interactions between the adaptor layer and transport channel.

描述（由申请人提供）：核孔复合物（NPC）是一个巨大的大分子网关，由称为核孔蛋白（Nups）的蛋白质组成，它使得穿过核膜的双向运输成为可能。 NPC 的核心可以简单地描述为组织成同心层。这些层的组件并不是彼此独立存在的，NPC 的组件根据功能分配给每一层。最外层由孔膜蛋白 (POM) 组成，将 NPC 固定在核膜上。 “外套”Nups 形成下一个最内层，并将 POM 连接到“适配器”层。衔接层中的关键角色是 Nup93，它通过与衔接层的其他蛋白质以及最内层（称为“运输通道”）的核孔蛋白相互作用形成中心枢纽。四种核孔蛋白对细胞活力和核运输都至关重要，它们组成了运输通道：Nup54、Nup58/45（Nup45 是 Nup58 的剪接变体）和 Nup62。基于 Blobel 实验室之前的工作，Nup58 被提议形成蜂窝流量通过的传输通道的中央“中平面”环。预计 Nup54 与 Nup58 结合后，中平面环的直径将增加一倍。此外，Nup54 在传输通道内将 Nup58 和 Nup62 连接在一起，并包含两者的独立结合位点。尽管各个nups的结构和传输因子已得到很好的表征，但NPC内不同环层如何相互作用以及中面环的直径如何调节却知之甚少。该提案的目标是确定适配器层和传输通道之间交互的基础以及它们的交互如何调制中板环的打开和关闭。目标 1：确定 Nup93 与 Nup54、Nup58 和 Nup62 三重复合物的复合物的晶体结构。我将使用标准结晶技术筛选 Nup93 与 Nup54、Nup58 和 Nup62 的复合物晶体，并且已经可以使用尺寸排阻色谱分离这四个 nup 的复合物。解决这个复合体的结构将揭示适配器层如何与NPC的传输层交互以及Nup93和适配器层如何调节Nup58环的打开和关闭。目标 2：从功能上表征适配器层和传输通道之间的交互，以确定适配器层如何调制环打开和环关闭。我将使用与核定位序列-GFP 融合蛋白缀合的金颗粒进行半透化细胞核输入测定，以研究破坏 Nup93 与 Nup54、Nup58 和 Nup62 之间相互作用的突变体是否影响核输入。此外，我将使用尺寸排阻色谱法和等温滴定量热法来表征突变对 Nup93 与转运通道相互作用的影响。最后，我将使用蛋白质印迹和质谱法来探究衔接层和传输通道之间的其他相互作用。