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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

In vitro MTase activity of Ynl024c.Protein extract from the ynl024cΔ yeast strain (denoted “Sc ynl024cΔ”) was treated with a protein extract from E. coli either expressing 6xHis-tagged Ynl024c from the pET28a-YNL024C plasmid (denoted “Ec+ Ynl024c”) or devoid of an expression plasmid (denoted “Ec”), at 37°C for 60 min in the presence of [3H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).
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pone.0131426.g004: In vitro MTase activity of Ynl024c.Protein extract from the ynl024cΔ yeast strain (denoted “Sc ynl024cΔ”) was treated with a protein extract from E. coli either expressing 6xHis-tagged Ynl024c from the pET28a-YNL024C plasmid (denoted “Ec+ Ynl024c”) or devoid of an expression plasmid (denoted “Ec”), at 37°C for 60 min in the presence of [3H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).

Mentions: As our efforts to purify soluble recombinant Ynl024c from E. coli had proven unsuccessful, we investigated whether a crude protein extract from an E. coli strain overexpressing Ynl024c would manifest the anticipated eEF1A-methylating activity in vitro. To this end, such an extract was incubated with a ynl024cΔ yeast extract in the presence of [3H]AdoMet. Proteins were then separated by SDS-PAGE, and incorporation of [3H]methyl groups was detected by fluorography. eEF1A is a GTP-binding protein, and since we previously observed that the addition of ATP enhances the methylation of HSPA8 by human METTL21A [21], we also investigated the effect of GTP addition on Ynl024c-mediated methylation of eEF1A in vitro. In the absence of GTP, we observed only very weak labelling of the ~50 kDa region corresponding to the molecular weight of eEF1A (Fig 4). This weak activity was not influenced by the presence of the Ynl024c E. coli extract, suggesting that it may reflect eEF1A methylation by one of the other eEF1A-specific yeast MTases present in the yeast extract. In contrast, when GTP was present, a markedly stronger, Ynl024c-specific labelling was observed in the 50 kDa region of the ynl024cΔ yeast extract (Fig 4). Taken together, these results suggest that the Ynl024c protein is capable of directly methylating eEF1A, and that the observed effects of YNL024C deletion or over-expression on eEF1A methylation are not indirect, but reflect KMT activity of Ynl024c on eEF1A.


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)

In vitro MTase activity of Ynl024c.Protein extract from the ynl024cΔ yeast strain (denoted “Sc ynl024cΔ”) was treated with a protein extract from E. coli either expressing 6xHis-tagged Ynl024c from the pET28a-YNL024C plasmid (denoted “Ec+ Ynl024c”) or devoid of an expression plasmid (denoted “Ec”), at 37°C for 60 min in the presence of [3H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131426.g004: In vitro MTase activity of Ynl024c.Protein extract from the ynl024cΔ yeast strain (denoted “Sc ynl024cΔ”) was treated with a protein extract from E. coli either expressing 6xHis-tagged Ynl024c from the pET28a-YNL024C plasmid (denoted “Ec+ Ynl024c”) or devoid of an expression plasmid (denoted “Ec”), at 37°C for 60 min in the presence of [3H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).
Mentions: As our efforts to purify soluble recombinant Ynl024c from E. coli had proven unsuccessful, we investigated whether a crude protein extract from an E. coli strain overexpressing Ynl024c would manifest the anticipated eEF1A-methylating activity in vitro. To this end, such an extract was incubated with a ynl024cΔ yeast extract in the presence of [3H]AdoMet. Proteins were then separated by SDS-PAGE, and incorporation of [3H]methyl groups was detected by fluorography. eEF1A is a GTP-binding protein, and since we previously observed that the addition of ATP enhances the methylation of HSPA8 by human METTL21A [21], we also investigated the effect of GTP addition on Ynl024c-mediated methylation of eEF1A in vitro. In the absence of GTP, we observed only very weak labelling of the ~50 kDa region corresponding to the molecular weight of eEF1A (Fig 4). This weak activity was not influenced by the presence of the Ynl024c E. coli extract, suggesting that it may reflect eEF1A methylation by one of the other eEF1A-specific yeast MTases present in the yeast extract. In contrast, when GTP was present, a markedly stronger, Ynl024c-specific labelling was observed in the 50 kDa region of the ynl024cΔ yeast extract (Fig 4). Taken together, these results suggest that the Ynl024c protein is capable of directly methylating eEF1A, and that the observed effects of YNL024C deletion or over-expression on eEF1A methylation are not indirect, but reflect KMT activity of Ynl024c on eEF1A.

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