PROTEOMIC ANALYSIS OF THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。核孔复合体(NPC)调节真核细胞中蛋白质和RNA在细胞质和核质之间的重要流动。它是一种超分子筛，横跨核膜的两层，形成连接细胞质和核质的40纳米水通道。小的代谢物在鼻咽癌中自由扩散，但大的蛋白质和RNA的流动是选择性的，需要货物中特定的运输信号。这些信号被称为核粘附素(也称为Importins、Exportins和Transportins)的移动受体识别，它们与NPC的蛋白质(核孔素；NUPS)相互作用，在细胞质和核质之间转运和运送货物。我们正在使用基于GST的亲和捕获分析与捕获的蛋白质的质谱学鉴定相结合的方法，获得酿酒酵母核质运输机制的54个组件之间的蛋白质相互作用图。这种方法可以在酵母抽提物中识别出数十种蛋白质，这些蛋白质与运输机械的单独组件(NUPS、KAPS、RAN及其效应器)特定地相互作用。我们目前正在研究在不同的细胞条件下，例如不同的细胞密度或温度，以及当细胞在细胞周期的不同阶段进行时，相互作用网络是如何调节的。这种方法提供了核质运输基础上的分子动力学的全球图景，以及机器的每个蛋白质组件进行的许多相互作用的详细地图。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The nuclear pore complex (NPC) regulates the vital flow of proteins and RNA between the cytoplasm and the nucleoplasm of eukaryotic cells. It is a supramolecular sieve that spans both membranes of the nuclear envelope to create a forty-nanometer aqueous channel that connects the cytoplasm and the nucleoplasm of cells. Small metabolites diffuse freely through the NPC, but the flow of large proteins and RNA is selective and requires specific transport signals in the cargo. The signals are recognized by mobile receptors termed karyopherins (also called importins, exportins and transportins), which interact with proteins of the NPC (nucleoporins; Nups) to translocate across and shuttle cargo between the cytoplasm and the nucleoplasm. We are obtaining a map of protein interactions between the 54 components of the nucleocytoplasmic transport machinery in S. cerevisiae using a GST based affinity-capture assay coupled to mass spectrometric identification of captured proteins. The approach has allowed the identification of dozens of proteins in yeast extracts that interact specifically with isolated components of the transport machinery (Nups, Kaps, Ran and its effectors). We are currently examining how the network of interactions is modulated under varying cellular conditions, such as different cell densities or temperature, and as cells progress through different stages of the cell cycle. The approach is providing a global picture of the molecular dynamics underlying nucleocytoplasmic transport, as well as a detailed map of the many interactions that each protein component of the machinery makes.