Peking University, Beijing, China.
Cell Res. 2010 Aug;20(8):948-62. doi: 10.1038/cr.2010.89. Epub 2010 Jun 22.
Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin beta directly or via the adaptor importin alpha to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin beta. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.
中心体蛋白是一种保守的中心体成分，它为许多中心体蛋白提供了结构支架，因此在中心体和有丝分裂纺锤体的组织和功能中起着至关重要的作用。尽管已经表明中心体蛋白定位于细胞质中，并报道对莱普霉素 B（LMB）敏感，LMB 是 Crm1 的特异性抑制剂，但作为核内外运输信号的中心体蛋白的区域尚未确定。在这项研究中，我们鉴定了中心体蛋白中五个具有不同输出活性的新的核输出信号（NES）。当介导中心体蛋白的核输出时，这五个 NES 都可以以 LMB 敏感的方式与 Crm1 结合。我们还证明，中心体蛋白的 8-42 位氨基酸区域包含一个由三个碱性氨基酸簇组成的三部分核定位信号（NLS）。中心体蛋白的 NLS 直接或通过衔接蛋白 importin α 与 importin β 结合形成导入复合物，该复合物可被 RanQ69L 破坏，RanQ69L 是一种具有高亲和力与 importin β 结合的显性失活的 Ran GTPase。此外，我们发现全长中心体蛋白中 NES 的突变导致核质共定位，而 NLS 的突变则会阻止中心体蛋白的核导入。基于这些结果，我们认为 NES 和 NLS 是中心体蛋白在细胞周期中进行亚细胞定位和核质转运所必需的。
