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首页> 外文期刊>Journal of Molecular Biology >On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.
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On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

机译：关于小鼠S5核糖体蛋白从细胞质向核仁的细胞内运输。

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摘要

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.
机译：哺乳动物核糖体蛋白的非核糖体功能最近引起了全世界的关注。源自核糖体材料的小鼠核糖体蛋白S5（rpS5）是组装的非磷酸化蛋白。但是，游离形式的rpS5蛋白会发生磷酸化。在这项研究中，我们（a）研究了磷酸化在rpS5蛋白转运到细胞核然后进入核仁中的潜在作用，以及（b）确定了rpS5的哪些结构域参与了这种细胞内运输。小鼠rpS5 cDNA的体外PCR诱变，以及随后克隆和表达与绿色荧光蛋白融合产生的rpS5截短重组形式的补充，允许使用共聚焦显微镜和Western blot分析对HeLa细胞中rpS5细胞内运输的研究。我们的结果表明以下内容：（a）rpS5蛋白通过区域38-50aa进入细胞核，该区域形成了一个随机线圈，如分子动力学模拟所揭示。 （b）用酪蛋白激酶II免疫沉淀rpS5和固定化的金属亲和层析分析，并辅以体外激酶测定，发现rpS5的磷酸化似乎是其从细胞核到核仁运输不可或缺的;进入细胞核后，Thr-133磷酸化通过酪蛋白激酶II触发Ser-24磷酸化，从而促进rpS5进入核仁。 rpS5 N末端区域的另一个重要作用是调节蛋白质的细胞水平。在细胞生长的静止后期而不是对数早期，重复出现在整个rpS5以下的卫星C末端带，这表明特定的降解可能平衡了未组装的核糖体蛋白分子与有效组装的核糖体蛋白分子核糖体亚基。总体而言，这些数据提供了有关rpS5分子内有助于其细胞功能的结构和功能域的新见解。

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