Quantitative Analysis of Nucleocytoplasmic Trafficking

描述(申请人提供)：真核细胞将遗传物质隔离在细胞核中，通过在细胞核和细胞质之间选择性地转移蛋白质，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，蛋白质跨核膜的调节运输已被认为是越来越多的细胞过程中的关键一步。要阐明通过核孔调节蛋白质转运的机制，需要对标记核进出口的大分子复合体的信号进行详细的定义。跨核膜转位的最佳机制是蛋白质的输入，这依赖于“经典的”核定位信号或NLS。该NLS由一簇碱性残基(单部分)或两簇被10-12个残基分隔的碱性残基(二部分)组成。这一信号被由importin a和importin b组成的异二聚体导入受体复合体识别。我们最近开发了一种定量分析方法，用于测量导入受体与溶液中平衡的NLS序列的亲和力。利用这一荧光分析方法，我们已经开始在体外重建蛋白质进口的分子反应，以提供对易位反应的详细热力学描述。要完全理解核导入信号，就需要一个定量的导入反应模型，该模型将NLS氨基酸序列、体外相互作用能量和体内功能联系起来。对NLS序列的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，并为蛋白质输入的调控提供可能的模式。在这一建议中，我们通过定量分析、体外测量与体内功能的相关性以及描述经典输入途径的特异性来探索“经典”核输入的机制。

DESCRIPTION (provided by applicant): The 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 nuclear 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.