DISSECTING THE MOLECULAR MACHINERY OF NUCLEAR TRANSPORT

核孔复合体是核质交换的唯一媒介。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他部分之间的通讯方面的关键作用反映在许多与核质运输变化直接相关的致癌和发育缺陷上。要弄清这些缺陷的性质，就需要充分理解人大是如何调解交通的。为了获得这一理解，我们需要对NPC的分子成分进行全面的盘点，了解每个成分如何对这个大型分子转位机器的整体结构做出贡献，以及关于其蛋白质与可溶相成分相互作用的信息。因此，我们采取了一种全面的方法来定义真核生物酵母(酵母)中NPC的功能结构，在该模型中，我们制作了酵母NPC超微结构的低分辨率图谱，并确定了其蛋白质组成。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，用于从运输的固定和可溶阶段分离和免疫纯化蛋白质复合体。这些络合物的组成及其相互作用的分子性质将使用当前和新兴的质谱学技术进行表征。首先，所有鼻咽癌成分都将被提纯，每个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蛋白质及其亚复合体在我们改进的NPC分子结构图中的位置。移动阶段的成员将以类似的方式进行研究。我们将跟踪特定运输因子在鼻咽癌中的移动，确定运输因子在核运输过程中与鼻咽癌蛋白相互作用的顺序和位置。将通过比较从不同运输因素获得的数据来调查有关运输的规则。我们的最终目标是整合我们的超微结构和生化研究，以了解不同运输因子在鼻咽癌中转移的分子基础。这些研究应该使我们能够在体外重建核质运输的关键反应，并在体内测试可能的机制模型。

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.