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.