Dataset Information

Novel roles for ribosomal histidine methylation revealed by proteomics and large-scale cross-linking mass spectrometry

ABSTRACT: The methylation of histidine residues is increasingly found to be both prevalent throughout the proteome, and also relevant to human disease. Hpm1p mono-methylates H243 in the ribosomal protein Rpl3 and represents the only 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 aimed to understand how methylation of one histidine in one ribosomal protein could result in such 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 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 use in budding yeast, which resulted in the identification of 1,267 unique in vivo lysine-lysine pairs. By reproducibly monitoring over 350, we detected changes to membrane protein structure, chromatin compaction, and mitochondrial protein-protein interactions, independently of changes in protein abundance themselves. 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Tara Bartolec

LAB HEAD: Marc R. Wilkins

PROVIDER: PXD027953 | Pride | 2022-08-12

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	01022021_Hpm1Proteome_FWD_L_WT_H_Hpm1_F1_5uL_1.raw	Raw
	01022021_Hpm1Proteome_FWD_L_WT_H_Hpm1_F1_5uL_2.raw	Raw
	01022021_Hpm1Proteome_FWD_L_WT_H_Hpm1_F2_5uL_1.raw	Raw
	01022021_Hpm1Proteome_FWD_L_WT_H_Hpm1_F2_5uL_2.raw	Raw
	01022021_Hpm1Proteome_FWD_L_WT_H_Hpm1_F3_5uL_1.raw	Raw

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Publications

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

Bartolec Tara K TK Hamey Joshua J JJ Keller Andrew A Chavez Juan D JD Bruce James E JE Wilkins Marc R MR

Molecular & cellular proteomics : MCP 20220521 7

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 ...[more]

PMID: 35609787

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Project description:Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution. Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.