Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.
Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.
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DOI：
10.1016/j.cub.2007.03.064
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发表时间：
2007-05-01
期刊：
影响因子：
9.2
通讯作者：
Andersen, Jens S.
中科院分区：
文献类型：
--
作者：
Lam, Yun Wah;Lamond, Angus I.;Mann, Matthias;Andersen, Jens S.
核仁是一种亚核细胞器，其中rRNA被转录、加工，并与核糖体蛋白组装成核糖体亚基。利用质谱技术结合精氨酸和赖氨酸稳定同位素的脉冲掺入，对新合成的内源性蛋白质在哺乳动物核仁中出现的速率进行了定量且无偏的全局分析。
新合成的核糖体蛋白在核仁中的积累速度比其他核仁组分更快。对表达荧光蛋白标记核仁因子的稳定HeLa细胞系进行的延时荧光显微镜研究也表明，核糖体蛋白比其他组分积累得更快。光漂白和质谱实验表明，只有一部分新合成的核糖体蛋白被组装成核糖体并输出到细胞质。蛋白酶体抑制导致核糖体蛋白在细胞核中积累，但不在细胞质中积累。在蛋白酶体抑制之前抑制rRNA转录进一步增加了核质中核糖体蛋白的积累。
核糖体蛋白的表达水平高于核糖体亚基正常生产速率所需的水平，并且在核仁中的积累速度比所有其他核仁组分都快。这通过核质中未组装的核糖体蛋白的持续降解来平衡，从而为哺乳动物细胞提供了一种机制，以确保核糖体蛋白水平永远不会对核糖体亚基的高效组装产生速率限制。本研究中使用的双重延时策略，将蛋白质组学和成像相结合，为定量分析新合成蛋白质通过细胞器的通量提供了一种强有力的方法。
The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli. Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm. Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure that ribosomal protein levels are never rate limiting for the efficient assembly of ribosome subunits. The dual time-lapse strategy used in this study, combining proteomics and imaging, provides a powerful approach for the quantitative analysis of the flux of newly synthesized proteins through a cell organelle.
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影响因子：
14.8
作者：
Ong, Shao-En;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
9.2
作者：
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通讯作者：
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7
作者：
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影响因子：
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作者：
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影响因子：
5.3
作者：
GRITZ, L;ABOVICH, N;ROSBASH, M
通讯作者：
ROSBASH, M