Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae.
Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae.
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DOI：
10.1016/j.abb.2010.05.023
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发表时间：
2010-08-15
影响因子：
3.9
通讯作者：
Clarke, Steven G.
中科院分区：
文献类型：
--
作者：
Lipson, Rebecca S.;Webb, Kristofor J.;Clarke, Steven G.
关键词：
真核细胞延伸因子1A（eEF 1A 1）是酿酒酵母（Saccharomycescerevisiae）中丰富的胞浆蛋白，在不同物种中高度保守。该蛋白质经历多个翻译后修饰，包括四个侧链赖氨酸残基的N-甲基化。然而，负责催化这些修饰的酶仍然难以捉摸。在这里，我们表明，通过完整的蛋白质质谱，删除两个基因编码的推定甲基转移酶的结果在质量损失的eEF 1A。YHL 039 W基因是SET结构域亚家族的成员，包括细胞色素c和核糖体蛋白赖氨酸甲基转移酶，其缺失导致对应于单个甲基的eEF 1A质量损失。删除YIL 064 W/SEE 1基因，编码一个非常保守的7 β链甲基转移酶序列，以前已被证明会影响囊泡运输，在这项工作中，我们表明，删除导致两个甲基的损失相当于从eEF 1A。我们发现，删除35个其他推定的和已建立的SET结构域和7个β链甲基转移酶对eEF 1A的质量没有影响。最后，我们表明，野生型提取物，而不是YIL 064 W/SEE 1突变体提取物，可以催化S-腺苷甲硫氨酸依赖的体外甲基化的低甲基化eEF 1A。我们认为，YHL 039 W（现在命名为EFM 1的延伸因子甲基转移酶1）和YIL 064 W/SE 1编码不同的eEF 1A甲基转移酶，分别单甲基化和二甲基化这个蛋白质的赖氨酸残基。
Eukaryotic elongation factor 1A (eEF1A1) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.
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