Stoichiometry of <i>Saccharomyces cerevisiae</i> Lysine Methylation: Insights into Non-histone Protein Lysine Methyltransferase Activity

Hart‐Smith, Gene; Chia, Samantha Z.; Low, Jason K. K.; McKay, Matthew J.; Molloy, Mark P.; Wilkins, Marc R.

doi:10.1021/pr401251k

Cited by 23 publications

(20 citation statements)

References 74 publications

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“…The differences in stoichiometry of the methylation sites, which ranges from ϳ85% for the N-terminal methylation to between 2.5 and 13% for other sites (50), is also suggestive of this. However, the stoichiometry observed here for the N-terminal methylation should be confirmed by a more accurate measurement, such as absolute quantification, to allow a direct comparison with previous studies (50). It is likely that all methylation sites can co-occur, as previous whole protein mass spectrometric analysis found that eEF1A exists at an average mass roughly equal to what is expected for all lysine methylation sites plus N-terminal acetylation (17), now known to be N-terminal trimethylation.…”

Section: Methyltransferases That Act On Lysine 79 In Eef1a Arementioning

confidence: 88%

“…While the exact function of their resulting methylation sites is unknown, the fact that they act upon one protein suggests they may modulate various functions of eEF1A (16). The differences in stoichiometry of the methylation sites, which ranges from ϳ85% for the N-terminal methylation to between 2.5 and 13% for other sites (50), is also suggestive of this. However, the stoichiometry observed here for the N-terminal methylation should be confirmed by a more accurate measurement, such as absolute quantification, to allow a direct comparison with previous studies (50).…”

Section: Methyltransferases That Act On Lysine 79 In Eef1a Arementioning

confidence: 99%

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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: 88%

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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“…Gel lanes were excised into 28 slices according to protein mass, which were destained (when required), reduced, and alkylated following standard procedures (29). In-gel tryptic digestions and peptide extractions were performed following procedures described previously (30). Peptide extraction solutions were dried in a SpeedVac TM and reconstituted in 20 l of 0.1% (v/v) formic acid.…”

Section: Methodsmentioning

confidence: 99%

“…This methylpeptide identifies arginine methylation on a known protein-arginine methyltransferase substrate motif, RGG (6,8,18,34), further supporting the designation of this match as a true positive. In addition, three methylpeptides designated as true positives identified two alternative forms of lysine methylation on known elongation factor 1-␣ methylation sites: di-methylation on Lys-390 (previously only characterized as MML) and tri-methylation on Lys-316 (previously only characterized as MML and DML) (30,37). XIC and synthetic peptide-derived MS/MS data used to validate these methyl-PSMs are presented in supplemental Figs.…”

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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The activity of a yeast Family 16 methyltransferase, Efm2, is affected by a conserved tryptophan and its N‐terminal region

et al. 2016

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The Family 16 methyltransferases are a group of eukaryotic nonhistone protein methyltransferases. Sixteen of these have recently been described in yeast and human, but little is known about their sequence and structural features. Here we investigate one of these methyltransferases, Saccharomyces cerevisiae elongation factor methyltransferase 2 (Efm2), by site‐directed mutagenesis and truncation. We show that an active site‐associated tryptophan, invariant in Family 16 methyltransferases and at position 222 in Efm2, is important for methyltransferase activity. A second highly conserved tryptophan, at position 318 in Efm2, is likely involved in S‐adenosyl methionine binding but is of lesser consequence for catalysis. By truncation analysis, we show that the N‐terminal 50–200 amino acids of Efm2 are critical for its methyltransferase activity. As N‐terminal regions are variable among Family 16 methyltransferases, this suggests a possible role in determining substrate specificity. This is consistent with recently solved structures that show the core of Family 16 methyltransferases to be near‐identical but the N termini to be structurally quite different. Finally, we show that Efm2 can exist as an oligomer but that its N terminus is not necessary for oligomerisation to occur.

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Stoichiometry of Saccharomyces cerevisiae Lysine Methylation: Insights into Non-histone Protein Lysine Methyltransferase Activity

Cited by 23 publications

References 74 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

The activity of a yeast Family 16 methyltransferase, Efm2, is affected by a conserved tryptophan and its N‐terminal region

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