PROTEOMIC ANALYSIS OF THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。核孔复合物（NPC）调节真核细胞细胞质和核质之间蛋白质和RNA的重要流动。它是一种超分子筛，跨越核膜的两层膜，形成一个40纳米的水通道，连接细胞质和核质。小的代谢物通过鼻咽癌自由扩散，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。这些信号被称为核丝蛋白（也称为输入蛋白、输出蛋白和运输蛋白）的移动受体识别，并与NPC蛋白（核孔蛋白；Nups）相互作用，在细胞质和核质之间进行转运和运送货物。我们正在使用基于GST的亲和捕获试验与捕获蛋白质的质谱鉴定相结合，获得酿酒酵母核胞质转运机制54个组分之间蛋白质相互作用的图谱。该方法可以鉴定酵母提取物中的数十种蛋白质，这些蛋白质与运输机制的分离成分（Nups, Kaps， Ran及其效应器）特异性相互作用。我们目前正在研究在不同的细胞条件下，如不同的细胞密度或温度，以及细胞在细胞周期的不同阶段如何调节相互作用的网络。该方法提供了核细胞质转运分子动力学的全局图景，以及该机制中每个蛋白质成分所产生的许多相互作用的详细图谱。

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.