Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation
Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation
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DOI：
10.1074/jbc.m112.400564
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发表时间：
2012-11-02
影响因子：
4.8
通讯作者：
de la Cruz, Jesus
中科院分区：
文献类型：
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作者：
Fernandez-Pevida, Antonio;Rodriguez-Galan, Olga;de la Cruz, Jesus
大多数核糖体蛋白在核糖体生物发生和功能中发挥重要作用。在这里，我们研究了必需核糖体蛋白 L40 在酿酒酵母这些过程中的贡献。 RPL40A 或 RPL40B 基因的缺失以及 L40 的体内耗竭都会损害 60S 核糖体亚基的生物发生。多聚体谱分析揭示了半聚体的积累和游离 60 S 核糖体亚基的适度减少。 L40 耗尽菌株中的脉冲追踪、Northern 印迹和引物延伸分析清楚地表明，L40 并不是前体 rRNA (pre-rRNA) 加工反应所严格必需的，但有助于最佳的 27 SB pre-rRNA 成熟。此外，L40 的消耗阻碍了 60S 前核糖体颗粒的核质输出。重要的是，所有这些缺陷很可能是细胞质前 60S 核糖体亚基 Nmd3 和 Rlp24 释放受损及其无效再循环回核的直接后果。一致地，我们表明血凝素表位标记的 L40A 在细胞质中组装成几乎成熟的前 60 S 核糖体颗粒。最后，我们发现血凝素表位标记的 L40A 赋予对 sodarin 的抗性，sordarin 是一种损害真核延伸因子 2 功能的翻译抑制剂，而 rpl40a 和 rpl40b 无效突变体对此抗生素过敏。我们得出的结论是，L40 在很晚的阶段组装成前 60S 核糖体亚基，并且其并入 60S 核糖体亚基是亚基连接的先决条件，并且可以确保易位过程的正常运行。
Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.