Quantitative Analysis of Nucleocytoplasmic Trafficking

描述（由申请人提供）：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有力的机制。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步骤。阐明通过核孔调节蛋白质易位的机制需要详细定义标记核输入或输出的大分子复合物的信号。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由一簇碱性残基（单分的）或由10-12个残基分开的两簇碱性残基（双分的）组成。该信号被包含输入蛋白a和输入蛋白B的异二聚体输入受体复合物识别。我们最近开发了一种定量测定，测量在溶液中平衡时输入受体对NLS序列的亲和力。通过这种荧光分析，我们已经开始在体外重建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。一个完整的理解核输入信号需要一个定量模型的输入反应，相关的NLS氨基酸序列，在体外的相互作用能，和在体内的功能。一个NLS序列的能量需求的详细描述将有助于识别这些序列在蛋白质的一级结构，以及建议可能的模式，用于调节蛋白质的进口。在这个建议中，我们探讨了“经典”核进口的机制，通过定量分析，在体外测量与体内功能的相关性，并划定经典进口途径的特异性。

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.