Quantitative Analysis of Nucleocytoplasmic Trafficking

核细胞质贩运的定量分析

• 批准号: 6839920
• 负责人: ANITA H. CORBETT
• 金额: $ 26.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6839920
• 关键词: Quantitative; Analysis; Nucleocytoplasmic; Trafficking

EXCEED THE SPACE PROVIDED. IThe sequestering of genetic material in the nucleus by eukaryotic cells provides a powerful mechanism for the regulation of gene expression and other cellular processes through the selective translocation of proteins between the nucleus and the cytoplasm. Recently, the regulated transport of proteins across the nuc ear envelope has been recognized as a crucial step in an increasing number of cellular processes. Elucidation of the mechanisms of regulated protein translocation through nuclear pores requires a detailed definition of the signals that mark a macromolecular complex for nuclear import or export. The best-characterized mechanism for translocation across the nuclear envelope is for protein import that depends on the 'classical' nuclear localization signal or NLS. This NLS consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10-12 residues (bipartite). This signal is recognized by the heterodimeric import receptor complex comprising importin a and importin b. We have recently developed a quantitative assay that measures the affinity of the import receptors for an NLS sequence at equilibrium in solution. With this fluorescence assay, we have begun to reconstitute the molecular reactions of protein import in vitro to provide a detailed thermodynamic description of the translocation reaction. A complete understanding of nuclear import signals requires a quantitative model for the import reaction that correlates NLS amino acid sequence, in vitro interaction energies, and in vivo functionality. A detailed description of the energetic requirements for an NLS sequence will facilitate the recognition of these sequences in protein primary structure as well as suggest possible modes for the regulation of protein import. In this proposal, we explore the mechanism of 'classical' nuclear import through quantitative analysis, correlation of in vitro measurements with in vivo function, and delineation of the specificity of the classical import pathway. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。真核细胞将遗传物质隔离在细胞核中，通过蛋白质在细胞核和细胞质之间的选择性易位，为基因表达和其他细胞过程的调节提供了强大的机制。最近，蛋白质穿过核耳膜的调节运输已被认为是越来越多的细胞过程中的关键步骤。阐明通过核孔调节蛋白质易位的机制需要对标记核输入或输出的大分子复合物的信号进行详细定义。跨核膜易位的最佳表征机制是依赖于“经典”核定位信号或 NLS 的蛋白质输入。该 NLS 由一组碱性残基（单部分）或由 10-12 个残基分隔的两个碱性残基簇（二部分）组成。该信号被包含输入蛋白 a 和输入蛋白 b 的异二聚输入受体复合物识别。我们最近开发了一种定量测定法，可测量溶液中平衡状态下 NLS 序列输入受体的亲和力。通过这种荧光测定，我们开始重建体外蛋白质输入的分子反应，以提供易位反应的详细热力学描述。对核输入信号的完整理解需要一个与 NLS 氨基酸序列、体外相互作用能量和体内功能相关的输入反应的定量模型。对 NLS 序列能量需求的详细描述将有助于识别蛋白质一级结构中的这些序列，并提出调节蛋白质输入的可能模式。在本提案中，我们通过定量分析、体外测量与体内功能的相关性以及经典输入途径的特异性的描述来探索“经典”核输入的机制。表演网站==========================================章节结束===============================================