Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

Erce, Melissa A.; Abeygunawardena, Dhanushi; Low, Jason K. K.; Hart‐Smith, Gene; Wilkins, Marc R.

doi:10.1074/mcp.m113.031500

Cited by 35 publications

(53 citation statements)

References 66 publications

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“…The trimethylation may therefore enhance the tRNA-binding capacity of eEF1A. Alternatively, it may affect the proteinprotein interactions of eEF1A in a manner similar to that of lysine and arginine methylation of proteins (2,43,44). Given the conservation of this modification between yeast and human, it will be of interest to investigate the functional role of eEF1A N-terminal methylation in more detail.…”

Section: Methyltransferases That Act On Lysine 79 In Eef1a Arementioning

confidence: 99%

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Hamey

Winter

Yagoub

et al. 2016

Molecular & Cellular Proteomics

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102

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Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys 79 in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys 79 methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated. Molecular & Cellular Proteomics

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Section: Methyltransferases That Act On Lysine 79 In Eef1a Arementioning

confidence: 99%

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Hamey

Winter

Yagoub

et al. 2016

Molecular & Cellular Proteomics

Self Cite

102

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“…For experiments in which the abovementioned strategies are not feasible, we recommend, at minimum, the following: (i) separate methyl-PSM FDR estimates should be employed when filtering datasets using the target-decoy approach; (ii) sources of false positive methyl-PSMs likely to be present in the samples of interest should be identified, and the peptides giving rise to these false positive methyl-PSMs should be characterized and removed from datasets; and (iii) tryptic methyl-PSMs with C-terminal di-or tri-methylation should also be removed from datasets. When interpreting datasets derived from these filtering criteria alone, we suggest that methylation sites of particular interest should be independently validated; for example by comparing native peptide-and synthetic peptidederived MS/MS spectra; through radiolabeling experiments using purified methyltransferases and substrates (11,17,34); or through in vitro or ex vivo methylation experiments employing putative methyltransferases, followed by in-depth LC-MS/MS analyses of purified putative methyltransferase substrates (17,41).…”

Section: Methylpeptide False Discovery Rates Can Be Expected To Remaimentioning

confidence: 99%

“…1B. The Uniprot/literature-derived methylpeptides unable to be uncovered in this study were previously described from either enriched or overexpressed methylprotein samples (14,16,17,41,42), including samples of overexpressed poly(A)-binding protein, which featured methylated glutamic acid residues following Coomassie staining (42), or from sequence database search outputs derived from atypically broad search parameters (i.e. methyl-PSMs derived from Ϯ50 ppm precursor ion mass tolerances for LTQ Orbitrap XL-derived data (10)); it is therefore unsurprising that these methylpeptides were not identified from the data described here.…”

Section: Fig 1 Workflows Employed In the Investigation Of Methyl-psmentioning

confidence: 99%

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Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Hart‐Smith

Yagoub

Tay

et al. 2016

Molecular & Cellular Proteomics

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All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13 CD 3 -methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methylPSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-␤-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl- Post-translational methylation is a widespread protein modification, which predominantly occurs on lysine and arginine residues (1). Protein-lysine methyltransferases catalyze the methylation of lysine residues; these enzymes facilitate the incorporation of methyl groups into the N atoms of lysine residues to produce either mono-, di-, or tri-methyllysine (MML, 1 DML, and TML, respectively). Protein-arginine methyltransferases catalyze the methylation of arginine residues; these enzymes primarily act upon N G atoms to produce mono, asymmetric di-, or symmetric di-methylarginine, although the enzyme-mediated modification of N ␦ atoms to produce ␦-MMA has also been reported in Saccharomyces cerevisiae (2).Traditionally, lysine and arginine methylation have been closely associated with histone proteins, and their crucial roles in modifying chromatin structure have been extensively studied (3). In recent years, however, a growing number of large scale methylation site discovery experiments have indiFrom the ‡New South

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“…These results suggest that enzymes other than Hsl7p may be producing sDMA in yeast. This study adds to an increasing body of evidence that additional protein methyltransferases remain to be discovered in yeast (Niewmierzycka and Clarke 1999;Lipson et al 2010;Low and Wilkins 2012;Young et al 2012;Erce et al 2013).…”

Section: Discussionmentioning

confidence: 72%

Arginine methylation in yeast proteins during stationary-phase growth and heat shock

et al. 2015

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Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS(3)), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS(3) and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS(3) and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.

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Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

Cited by 35 publications

References 66 publications

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Arginine methylation in yeast proteins during stationary-phase growth and heat shock

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