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Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase.

Jakobsson ME, Davydova E, Małecki J, Moen A, Falnes PØ - PLoS ONE (2015)

Bottom Line: Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue.Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified.Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, 0316, Norway.

ABSTRACT
The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).

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Related in: MedlinePlus

Ynl024c-mediated methylation of eEF1A in vivo.(A) Qualitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. MS chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 387–395 of S. cerevisiae eEF1A from wild-type and Ynl024c deficient (ynl024cΔ) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::YNL024C”or “ynl024cΔ::YNL024C“, respectively. The expected elution time for the relevant peptide is indicated by an arrow above the upper trace. The peak corresponding to an unrelated peptide with a m/z-ratio matching the trimethylated peptide species is indicated by an asterisk. (B) Quantitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. Quantitative representation of the data from (A). The fractional occupancy of the various lysine methylation states was determined as the relative signal for corresponding peptides in (A), determined by integration. C-E, MS/MS fragmentation pattern supporting the identity of analysed peptides. Representative annotated spectra for un- (C), mono- (D) and trimethylated (E) peptides corresponding to peaks in (A) are shown. Spectra for the dimethylated peptide is shown in S2 Fig.
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pone.0131426.g003: Ynl024c-mediated methylation of eEF1A in vivo.(A) Qualitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. MS chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 387–395 of S. cerevisiae eEF1A from wild-type and Ynl024c deficient (ynl024cΔ) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::YNL024C”or “ynl024cΔ::YNL024C“, respectively. The expected elution time for the relevant peptide is indicated by an arrow above the upper trace. The peak corresponding to an unrelated peptide with a m/z-ratio matching the trimethylated peptide species is indicated by an asterisk. (B) Quantitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. Quantitative representation of the data from (A). The fractional occupancy of the various lysine methylation states was determined as the relative signal for corresponding peptides in (A), determined by integration. C-E, MS/MS fragmentation pattern supporting the identity of analysed peptides. Representative annotated spectra for un- (C), mono- (D) and trimethylated (E) peptides corresponding to peaks in (A) are shown. Spectra for the dimethylated peptide is shown in S2 Fig.

Mentions: Our inability to detect of methylation of eEF1A at Lys390 in the ynl024cΔ yeast suggested Ynl024c as the MTase responsible for this modification. To further investigate this, we set out to analyse the relative abundance of the various methylated forms of Lys390, and to address the potential effect of Ynl024c over-expression. We used the endoprotease Asp-N to generate a Lys390-encompassing peptide for relative quantification of the different methylated forms by MS, since trypsin-mediated cleavage is inhibited by lysine methylation, and therefore introduces a methylation-dependent bias in peptide generation [35,36]. These experiments revealed that, although Lys390 in eEF1A is primarily found in the unmethylated state in wild-type cells, a considerable portion (~20%) is monomethylated at this residue (Fig 3A–3D). In contrast, Lys390 in eEF1A from ynl024cΔ cells was exclusively detected in the unmethylated state (Fig 3A–3C).


Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase.

Jakobsson ME, Davydova E, Małecki J, Moen A, Falnes PØ - PLoS ONE (2015)

Ynl024c-mediated methylation of eEF1A in vivo.(A) Qualitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. MS chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 387–395 of S. cerevisiae eEF1A from wild-type and Ynl024c deficient (ynl024cΔ) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::YNL024C”or “ynl024cΔ::YNL024C“, respectively. The expected elution time for the relevant peptide is indicated by an arrow above the upper trace. The peak corresponding to an unrelated peptide with a m/z-ratio matching the trimethylated peptide species is indicated by an asterisk. (B) Quantitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. Quantitative representation of the data from (A). The fractional occupancy of the various lysine methylation states was determined as the relative signal for corresponding peptides in (A), determined by integration. C-E, MS/MS fragmentation pattern supporting the identity of analysed peptides. Representative annotated spectra for un- (C), mono- (D) and trimethylated (E) peptides corresponding to peaks in (A) are shown. Spectra for the dimethylated peptide is shown in S2 Fig.
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pone.0131426.g003: Ynl024c-mediated methylation of eEF1A in vivo.(A) Qualitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. MS chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 387–395 of S. cerevisiae eEF1A from wild-type and Ynl024c deficient (ynl024cΔ) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::YNL024C”or “ynl024cΔ::YNL024C“, respectively. The expected elution time for the relevant peptide is indicated by an arrow above the upper trace. The peak corresponding to an unrelated peptide with a m/z-ratio matching the trimethylated peptide species is indicated by an asterisk. (B) Quantitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. Quantitative representation of the data from (A). The fractional occupancy of the various lysine methylation states was determined as the relative signal for corresponding peptides in (A), determined by integration. C-E, MS/MS fragmentation pattern supporting the identity of analysed peptides. Representative annotated spectra for un- (C), mono- (D) and trimethylated (E) peptides corresponding to peaks in (A) are shown. Spectra for the dimethylated peptide is shown in S2 Fig.
Mentions: Our inability to detect of methylation of eEF1A at Lys390 in the ynl024cΔ yeast suggested Ynl024c as the MTase responsible for this modification. To further investigate this, we set out to analyse the relative abundance of the various methylated forms of Lys390, and to address the potential effect of Ynl024c over-expression. We used the endoprotease Asp-N to generate a Lys390-encompassing peptide for relative quantification of the different methylated forms by MS, since trypsin-mediated cleavage is inhibited by lysine methylation, and therefore introduces a methylation-dependent bias in peptide generation [35,36]. These experiments revealed that, although Lys390 in eEF1A is primarily found in the unmethylated state in wild-type cells, a considerable portion (~20%) is monomethylated at this residue (Fig 3A–3D). In contrast, Lys390 in eEF1A from ynl024cΔ cells was exclusively detected in the unmethylated state (Fig 3A–3C).

Bottom Line: Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue.Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified.Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, 0316, Norway.

ABSTRACT
The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).

No MeSH data available.


Related in: MedlinePlus