Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
Biochemistry. 2012 Jun 26;51(25):5091-104. doi: 10.1021/bi300186g. Epub 2012 Jun 15.
We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation of Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.
我们使用质谱分析和氨基酸分析，对酵母酿酒酵母中的三个小核糖体亚基蛋白 Rps2、Rps3 和 Rps27a 的翻译后甲基化进行了特征描述。我们发现 Rps2 在精氨酸-10 处被 Rmt1 甲基转移酶亚化学计量修饰。我们通过质谱和定点突变分析证明，Rps3 在精氨酸-146 处被 ω-单甲基化修饰。用丙氨酸取代位置 146 处的精氨酸与细胞生长缓慢有关，这表明该位点的氨基酸身份可能影响核糖体功能和/或生物发生。酿酒酵母中 Rps3 的三维结构分析表明，精氨酸-146 与小亚基 rRNA 接触。对编码潜在酵母甲基转移酶的缺失突变体的筛选表明，YOR021C 基因的缺失导致 Rps3 甲基化缺失。我们证明，当用 S-腺苷甲硫氨酸和从 YOR021C 缺失菌株制备的低甲基化核糖体孵育时，重组 Yor021c 催化 ω-单甲基精氨酸的形成。有趣的是，Yor021c 属于 SPOUT 甲基转移酶家族，迄今为止，该家族仅被证明可以修饰 RNA 底物。我们的发现表明 SPOUT 甲基转移酶在自然界中具有更广泛的作用。最后，我们在 Rps27a 的位置 39 处的锌-半胱氨酸簇中发现了一个存在化学计量甲基化的半胱氨酸残基。在小核糖体亚基内发现的这三个新的蛋白质修饰位点，现在将允许对其在翻译和可能的其他细胞过程中的功能作用进行分析。
