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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).

No MeSH data available.


Related in: MedlinePlus

Methylation status of eEF1A in mammalian cells.(A) Alignment of various eEF1A sequences from Homo sapiens (Hs), Oryctolagus cuniculus (Oc; rabbit) and Saccharomyces cerevisiae (Sc), showing the region surrounding the lysine targeted by Ynl024c (arrow). Shown proteins are Hs eEF1A1 (NP_001393.1), Oc eEF1A1 (NP_001075808.1) and Sc eEF1A (NP_009676.1). (B) Methylation status of eEF1A1 in HeLa cells and in rabbit reticulocytes. Chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 389–397 in human and rabbit eEF1A1. Tandem mass spectra supporting the identity of analyzed peptides are shown in S4 Fig.
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pone.0131426.g005: Methylation status of eEF1A in mammalian cells.(A) Alignment of various eEF1A sequences from Homo sapiens (Hs), Oryctolagus cuniculus (Oc; rabbit) and Saccharomyces cerevisiae (Sc), showing the region surrounding the lysine targeted by Ynl024c (arrow). Shown proteins are Hs eEF1A1 (NP_001393.1), Oc eEF1A1 (NP_001075808.1) and Sc eEF1A (NP_009676.1). (B) Methylation status of eEF1A1 in HeLa cells and in rabbit reticulocytes. Chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 389–397 in human and rabbit eEF1A1. Tandem mass spectra supporting the identity of analyzed peptides are shown in S4 Fig.

Mentions: Having firmly established that Lys390 in eEF1A is methylated by Ynl024c in S. cerevisiae, we considered the possibility that one of the human METTL21 proteins (or another MTase) could perform the same methylation on mammalian eEF1A. eEF1A is extremely well conserved among eukaryotes, and a sequence alignment shows that the mammalian protein indeed contains a lysine residue at the position corresponding to Lys390 in the yeast sequence (Fig 5A). We analyzed the methylation status of this residue (Lys392) in protein extracts from rabbit (O. cuniculus) reticulocytes, a commonly used system for in vitro translation, as well as in human HeLa cells (Fig 5 and S4 Fig). Lys392 was exclusively detected as unmethylated in these experiments, indicating that enzymatic methylation of this residue is not conserved within eukaryotes, and that mammalian METTL21 enzymes do not target this site.


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)

Methylation status of eEF1A in mammalian cells.(A) Alignment of various eEF1A sequences from Homo sapiens (Hs), Oryctolagus cuniculus (Oc; rabbit) and Saccharomyces cerevisiae (Sc), showing the region surrounding the lysine targeted by Ynl024c (arrow). Shown proteins are Hs eEF1A1 (NP_001393.1), Oc eEF1A1 (NP_001075808.1) and Sc eEF1A (NP_009676.1). (B) Methylation status of eEF1A1 in HeLa cells and in rabbit reticulocytes. Chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 389–397 in human and rabbit eEF1A1. Tandem mass spectra supporting the identity of analyzed peptides are shown in S4 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482628&req=5

pone.0131426.g005: Methylation status of eEF1A in mammalian cells.(A) Alignment of various eEF1A sequences from Homo sapiens (Hs), Oryctolagus cuniculus (Oc; rabbit) and Saccharomyces cerevisiae (Sc), showing the region surrounding the lysine targeted by Ynl024c (arrow). Shown proteins are Hs eEF1A1 (NP_001393.1), Oc eEF1A1 (NP_001075808.1) and Sc eEF1A (NP_009676.1). (B) Methylation status of eEF1A1 in HeLa cells and in rabbit reticulocytes. Chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 389–397 in human and rabbit eEF1A1. Tandem mass spectra supporting the identity of analyzed peptides are shown in S4 Fig.
Mentions: Having firmly established that Lys390 in eEF1A is methylated by Ynl024c in S. cerevisiae, we considered the possibility that one of the human METTL21 proteins (or another MTase) could perform the same methylation on mammalian eEF1A. eEF1A is extremely well conserved among eukaryotes, and a sequence alignment shows that the mammalian protein indeed contains a lysine residue at the position corresponding to Lys390 in the yeast sequence (Fig 5A). We analyzed the methylation status of this residue (Lys392) in protein extracts from rabbit (O. cuniculus) reticulocytes, a commonly used system for in vitro translation, as well as in human HeLa cells (Fig 5 and S4 Fig). Lys392 was exclusively detected as unmethylated in these experiments, indicating that enzymatic methylation of this residue is not conserved within eukaryotes, and that mammalian METTL21 enzymes do not target this site.

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