Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p

• Published in: Molecular & cellular proteomics 2022-07, Vol.21 (7), p.100249, Article 100249
• Main Authors: Bartolec, Tara K., Hamey, Joshua J., Keller, Andrew, Chavez, Juan D., Bruce, James E., Wilkins, Marc.R.
• Format: Article
• Language: English
• Subjects: cross-linking mass spectrometry; histidine methylation; interactomics; mass spectrometry; methylation; methyltransferase; pleiotropy; post-translational modification; proteomics; ribosome; Saccharomyces cerevisiae
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2022.100249

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates 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 extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential cross-linking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture cross-linking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine–lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein–protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how cross-linking MS will be an essential tool for understanding complex phenotypes. [Display omitted] •Hpm1p-mediated histidine methylation of Rpl3p localized to H243 by mass spectrometry.•Novel extraribosomal functional phenotypes found on loss of Hpm1p.•Large-scale in vivo quantitative and comparative crosslinking analysis performed.•Changes detected in structures and interactions, independent of protein abundance.•Phenotypes could be explained or predicted through crosslinking analysis. The yeast histidine methyltransferase Hpm1p targets a ribosomal substrate but has many unexplained extraribosomal phenotypes. To understand these, we used protein expression analysis and quantitative cros