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

描述(申请人提供)：核孔复合体(NPC)是一个巨大的大分子通道，由称为核孔蛋白(NUPS)的蛋白质组成，使核膜的双向运输成为可能。全国人大的核心可以很容易地描述为被组织成同心层。这些层的组件不是彼此独立存在的，NPC的组件基于功能被分配给每一层。最外层由将鼻咽癌固定在核膜上的孔膜蛋白(POMS)组成。“外套”核芯形成下一个最内层，并将POMS连接到“适配器”层。在适配层中的一个关键角色是Nup93，它通过与适配层的其他蛋白质以及最内层的核孔蛋白相互作用形成一个中心枢纽，称为“运输通道”。四种核孔素组成了转运通道：Nup54、Nup58/45(Nup45是Nup58的剪接变体)和Nup62，它们都是细胞存活和核运输所必需的。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“中面”环。当Nup54与Nup58结合时，预计中面环的直径将增加一倍。此外，Nup54在运输通道内将Nup58和Nup62连接在一起，并包含两者的独立结合位点。虽然单个NUP的结构和运输因子已经被很好地描述，但NPC内不同的环层如何相互作用以及中面环的直径是如何被调制的还知之甚少。这项建议的目的是确定适配器层和传输通道之间相互作用的基础，以及它们之间的相互作用如何调节中面环的打开和关闭。目的1：确定Nup93与Nup54、Nup58、Nup62三元配合物的晶体结构。我将使用标准结晶技术筛选Nup93与Nup54、Nup58和Nup62的复合体晶体，并已经可以使用尺寸排除层析分离这四个NupP的复合体。解决这个复合体的结构将揭示适配器层如何与NPC的传输层相互作用，以及Nup93和适配器层如何调节Nup58环的打开和关闭。目的2：从功能上表征转接器层和运输通道之间的相互作用，以确定转接器层如何调节环的打开和闭合。我将使用与核定位序列-GFP融合蛋白偶联的金颗粒半渗透性细胞核导入试验来研究破坏Nup93与Nup54、Nup58和Nup62之间相互作用的突变体是否影响核导入。此外，我将使用尺寸排除层析和等温滴定量热法来表征突变对Nup93与运输通道相互作用的影响。最后，使用蛋白质印迹和质谱仪，我将探索适配器层和运输通道之间的额外相互作用。

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.