A novel SET domain methyltransferase modifies ribosomal protein Rpl23ab in yeast
A novel SET domain methyltransferase modifies ribosomal protein Rpl23ab in yeast
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DOI：
10.1074/jbc.m507672200
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发表时间：
2005-10-14
影响因子：
4.8
通讯作者：
Clarke, S
中科院分区：
文献类型：
--
作者：
Porras-Yakushi, TR;Whitelegge, JP;Clarke, S
体内研究表明，酿酒酵母（Saccharomyces cerevisiae）的核糖体大亚基蛋白L23a （Rpl23ab）在赖氨酸残基处甲基化。然而，编码负责修饰的甲基转移酶的基因尚未被确定。我们在这里发现酵母YPL208w基因产物，甲基转移酶SET结构域家族的成员，催化了该反应，我们现在将其命名为Rkm1（核糖体赖氨酸(K)甲基转移酶1）。在体内用s -腺苷- l[甲基- h -3]蛋氨酸标记含有候选SET结构域基因缺失突变的酵母菌株。[H-3]]裂解物经SDS凝胶电泳分离后测定甲基放射性。与亲本菌株或其他候选缺失菌株相比，rkm1 （Delta ypl208w）敲除菌株中缺失了一个放射性标记的15 kda物种。用RNase或蛋白酶K处理野生型细胞提取物表明，甲基接受底物是一种蛋白质。用高盐蔗糖梯度对亲本和敲除菌株的细胞裂解物进行分离。SDS凝胶电泳的梯度分析表明，15 kda的甲基接受底物与大的核糖体亚基洗脱。用纯化核糖体进行的体外甲基化实验证实，接受甲基的底物是一种核糖体蛋白。对15 kDa多肽的氨基酸分析表明，该多肽含有epsilon- [H-3]二甲基赖氨酸残基。15kda多肽的质谱分析鉴定为Rpl23ab。电喷雾质谱法分析了大核糖体亚基蛋白的完整质量，证实底物是Rpl23ab，并且它在两个不同的位点被Rkm1特异性地二甲基化。这些结果表明SET结构域甲基转移酶可以参与翻译作用以及先前描述的转录作用。
In vivo studies have shown that the ribosomal large subunit protein L23a (Rpl23ab) in Saccharomyces cerevisiae is methylated at lysine residues. However, the gene encoding the methyltransferase responsible for the modification has not been identified. We show here that the yeast YPL208w gene product, a member of the SET domain family of methyltransferases, catalyzes the reaction, and we have now designated it Rkm1 ( ribosomal lysine ( K) methyltransferase 1). Yeast strains with deletion mutations in candidate SET domain-containing genes were in vivo labeled with S-adenosyl-L[ methyl-H-3] methionine. [H-3]] Methyl radioactivity was determined after lysates were fractionated by SDS gel electrophoresis. When compared with the parent strain or other candidate deletion strains, a loss of a radiolabeled 15-kDa species was observed in the rkm1 (Delta ypl208w) knock-out strain. Treatment of wild-type cell extracts with RNase or proteinase K demonstrated that the methyl-accepting substrate is a protein. Cellular lysates from parent and knockout strains were fractionated using high salt sucrose gradients. Analysis of the gradient fractions by SDS gel electrophoresis demonstrated that the 15-kDa methyl-accepting substrate elutes with the large ribosomal subunit. In vitro methylation experiments using purified ribosomes confirmed that the methyl-accepting substrate is a ribosomal protein. Amino acid analysis of the in vivo labeled 15 kDa polypeptide showed that it contains epsilon- [H-3] dimethyllysine residues. Mass spectrometry of tryptic peptides of the 15 kDa polypeptide identified it as Rpl23ab. Analysis of the intact masses of the large ribosomal subunit proteins by electrospray mass spectrometry confirmed that the substrate is Rpl23ab and that it is specifically dimethylated at two distinct sites by Rkm1. These results show that SET domain methyltransferases can be involved in translational roles as well as in the previously described transcriptional roles.