PROTEOMIC ANALYSIS OF THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY

核质运输机器的蛋白质组学分析

• 批准号: 7724162
• 负责人: MICHAEL F REXACH
• 金额: $ 2.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724162
• 关键词: Affinity; Biological Assay; Cell Cycle Stage; Cell Density; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; Coupled; Cytoplasm; Diffuse; Eukaryotic Cell; Exportins; Funding; Glutathione S-Transferase; Grant; Importins; Institution; Karyopherins; Maps; Membrane; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Protein Interaction Mapping; Protein-Protein Interaction Map; Proteins; Proteomics; RNA; Research; Research Personnel; Resources; Running; Saccharomyces cerevisiae; Signal Transduction; Source; Temperature; United States National Institutes of Health; Yeasts; aqueous; base; molecular dynamics; nanometer; nucleocytoplasmic transport; receptor

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 核孔复合物（NPC）调节了蛋白质和RNA的重要流动，而真核细胞的细胞质和核质之间的重大流动。这是一个超分子筛，它跨越了核包膜的两个膜，以创建一个连接细胞质和细胞核质的40纳米水通道。 小型代谢产物通过NPC自由扩散，但是大蛋白和RNA的流动是选择性的，需要在货物中特定的传输信号。 这些信号被称为核蛋白的移动受体（也称为导入蛋白，导蛋白和运输蛋白），它们与NPC的蛋白质（核孔蛋白； NUP）相互作用，以在细胞质和核质量之间跨越货物。 我们正在使用基于GST的基于GST的亲和力扣除测定法与捕获的蛋白质的质谱鉴定，获得了酿酒酵母中核细胞质运输机械的54个成分之间的蛋白质相互作用图。 该方法允许在酵母提取物中鉴定数十种蛋白质，这些蛋白质与运输机械的分离成分（NUP，KAPS，RAN及其效应子）专门相互作用。 我们目前正在研究如何在不同的细胞条件（例如不同的细胞密度或温度）以及细胞通过细胞周期的不同阶段进行的相互作用网络调节。 该方法提供了核细胞质转运的分子动力学的全局图片，以及机械中每个蛋白质成分所产生的许多相互作用的详细图。