Monitoring of histidine methylated Rpl3p

Abstract The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase Hpm1p mono-methylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extra-ribosomal phenotypes including improved growth rates in alternative carbon sources. Here we investigate how methylation of one histidine in one ribosomal protein results in diverse phenotypes, by combining targeted mass spectrometry, growth assays, quantitative proteomics and cross-linking mass spectrometry. We confirmed the localisation and stoichiometry of the H243 site, found unreported sensitivities of Δhpm1 yeast to non-ribosomal stressors, and identified thirty-one differentially-abundant proteins upon hpm1 knockout – most with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale cross-linking mass spectrometry for budding yeast, which resulted in the identification of 1,267 unique in vivo lysine-lysine crosslinks. By reproducibly monitoring over 350 of these in wild-type and Δhpm1, we detected changes to the ribosome, membrane protein structure, chromatin compaction, and mitochondrial protein-protein interactions. These changes occurred independently of changes in protein abundance. Taken together, these studies reveal a clear role for Hpm1p in the coordination of sugar metabolism, contextualise the deletion strain’s pleiotropy and illustrate how cross-linking mass spectrometry can generate mechanistic insights into complex cellular processes that are invisible to expression analysis of the proteome.

摘要 组氨酸甲基化是一类功能尚不明晰的翻译后修饰（post-translational modification）。甲基转移酶（methyltransferase）Hpm1p可对核糖体蛋白（ribosomal protein）Rpl3p的H243位点进行单甲基化修饰，同时也是酿酒酵母（Saccharomyces cerevisiae）中目前已知的唯一组氨酸甲基转移酶。值得注意的是，hpm1基因缺失菌株（deletion strain）表现出强烈的多效性，具备多种核糖体外表型，例如在替代碳源（alternative carbon sources）中生长速率提升。本研究结合靶向质谱（targeted mass spectrometry）、生长测定（growth assays）、定量蛋白质组学（quantitative proteomics）与交联质谱（cross-linking mass spectrometry）技术，探究单个核糖体蛋白上的单个组氨酸甲基化如何引发多样化表型。我们验证了H243位点的定位（localisation）与化学计量比（stoichiometry），发现Δhpm1酵母对未被报道过的非核糖体应激源（non-ribosomal stressors）存在敏感性，并鉴定出31种在hpm1敲除（knockout）后丰度发生变化的蛋白质——其中多数与糖代谢（sugar metabolism）调控存在明确关联。我们针对出芽酵母（budding yeast）优化了新兴的大规模定量交联质谱技术，最终鉴定出1267种独特的体内赖氨酸-赖氨酸交联（in vivo lysine-lysine crosslinks）产物。通过可重复地监测野生型（wild-type）与Δhpm1菌株中超过350种这类交联产物，我们检测到核糖体、膜蛋白结构、染色质压缩（chromatin compaction）以及线粒体蛋白质相互作用（mitochondrial protein-protein interactions）均发生了改变，且这些变化与蛋白质丰度（protein abundance）的变化无关。综上，本研究明确了Hpm1p在糖代谢调控中的核心作用，阐释了该基因缺失菌株多效性的分子背景，并展示了交联质谱技术如何为蛋白质组（proteome）表达分析无法揭示的复杂细胞过程提供机制层面的新见解。