Limits...
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

Evaluating Ynl024c as a functional homolog of the Hsp70-specific METTL21A.(A) Protein sequence alignment of various Hsp70 proteins from H. sapiens (Hs) and S. cerevisiae (Sc) showing the region surrounding the lysine targeted by METTL21A (arrow). Shown proteins are: HSPA1(P08107), HSPA5(P11021), HSPA8(P11021), Ssa1(P10591), Ssa3(P09435), Ssb1(P11484) and Ssb2(P40150). (B) Activity of METTL21A on recombinant yeast Hsp70 proteins. Recombinant METTL21A was incubated with the indicated recombinant E. coli-expressed yeast Hsp70 proteins, as well as with human HSPA8, in the presence of [3H]AdoMet, and the amount of TCA-insoluble radioactivity measured by scintillation counting. Error bars indicate standard deviation (n = 3). (C) Lys556 in Ssa1 is not methylated in vivo. Yeast whole cell extract (WCE) (20 μg) was incubated in the absence (upper) or presence (lower) of METTL21A (100 pmol). MS chromatograms of Asp-N generated peptides encompassing residues D555-A560 of Ssa1 (as shown in (E) and (F)), gated for m/z corresponding to peptides with un-, mono-, di- and trimethylated Lys556. Peaks corresponding to an unrelated peptide with a m/z-ratio matching the unmethylated species of the peptide are indicated by asterisks. (D) Lys565 in Ssb1/Ssb2 is not methylated in vivo. Same as in (C), except that the analysed peptide corresponds to L546-R568 from Ssb1/Ssb2 and was generated by cleavage with Arg-C. Tandem mass spectra of relevant peptides are shown in S1 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482628&req=5

pone.0131426.g002: Evaluating Ynl024c as a functional homolog of the Hsp70-specific METTL21A.(A) Protein sequence alignment of various Hsp70 proteins from H. sapiens (Hs) and S. cerevisiae (Sc) showing the region surrounding the lysine targeted by METTL21A (arrow). Shown proteins are: HSPA1(P08107), HSPA5(P11021), HSPA8(P11021), Ssa1(P10591), Ssa3(P09435), Ssb1(P11484) and Ssb2(P40150). (B) Activity of METTL21A on recombinant yeast Hsp70 proteins. Recombinant METTL21A was incubated with the indicated recombinant E. coli-expressed yeast Hsp70 proteins, as well as with human HSPA8, in the presence of [3H]AdoMet, and the amount of TCA-insoluble radioactivity measured by scintillation counting. Error bars indicate standard deviation (n = 3). (C) Lys556 in Ssa1 is not methylated in vivo. Yeast whole cell extract (WCE) (20 μg) was incubated in the absence (upper) or presence (lower) of METTL21A (100 pmol). MS chromatograms of Asp-N generated peptides encompassing residues D555-A560 of Ssa1 (as shown in (E) and (F)), gated for m/z corresponding to peptides with un-, mono-, di- and trimethylated Lys556. Peaks corresponding to an unrelated peptide with a m/z-ratio matching the unmethylated species of the peptide are indicated by asterisks. (D) Lys565 in Ssb1/Ssb2 is not methylated in vivo. Same as in (C), except that the analysed peptide corresponds to L546-R568 from Ssb1/Ssb2 and was generated by cleavage with Arg-C. Tandem mass spectra of relevant peptides are shown in S1 Fig.

Mentions: We have previously reported that Ynl024c is not a functional homolog of VCP-KMT/METTL21D, which trimethylates Lys315 in VCP, as the corresponding lysine residue is unmethylated in Cdc48, the yeast orthologue of VCP [22]. However, as Ynl024c is more similar to the Hsp70-specific MTase METTL21A (HSPA-KMT), we set out to investigate whether Ynl024c targets Hsp70 proteins. Human METTL21A has been reported to trimethylate Lys561 in the constitutively expressed Hsp70 protein HSC70/HSPA8 as well as the corresponding residue in several other human Hsp70/HSPA proteins [20,21]. A sequence alignment of various Hsp70 proteins from H. sapiens and S. cerevisiae demonstrates that a lysine residue is present at the corresponding position in several of the main Hsp70s in S. cerevisiae, e.g. SSA1, SSA3, SSB1, and SSB2 (the latter two are highly similar, showing >99% sequence identity) (Fig 2A). Initially, we therefore set out to test whether recombinant yeast Hsp70 proteins could be methylated by recombinant Ynl024c, but we were unfortunately unable to express and purify soluble, recombinant Ynl024c protein. However, we found that the yeast Hsp70 proteins SSA1 and SSA3, but not SSB2, could be methylated by human METTL21A in vitro, thus suggesting that yeast HSP70 proteins have the potential to become methylated by a METTL21A-like enzyme (Fig 2B). To further investigate the possibility that Ynl024c catalyses methylation of Hsp70 proteins, we analysed the methylation state of S. cerevisiae Hsp70s in vivo. To this end, an endoprotease digest of yeast proteins excised from the Hsp70-containing region of a SDS-PAGE gel was analysed by mass spectrometry. In this analysis, peptides covering the relevant Lys residue in SSA1 (Lys556) and SSB1/SSB2 (Lys565) were exclusively detected as unmethylated in wild-type cells (Fig 2C, 2D and S1 Fig). To exclude the possibility that our inability to detect methylated SSA1 was due to technical problems, we also investigated the methylation status of SSA1 in a yeast extract incubated with human METTL21A, and in this case SSA1 was found almost exclusively in the trimethylated state (Fig 2C). These results demonstrate that yeast Hsp70 proteins are unmethylated at the Lys residue corresponding to Lys561 in human HSPA8, and, consequently, that Ynl024c is likely not a functional orthologue of human METTL21A.


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)

Evaluating Ynl024c as a functional homolog of the Hsp70-specific METTL21A.(A) Protein sequence alignment of various Hsp70 proteins from H. sapiens (Hs) and S. cerevisiae (Sc) showing the region surrounding the lysine targeted by METTL21A (arrow). Shown proteins are: HSPA1(P08107), HSPA5(P11021), HSPA8(P11021), Ssa1(P10591), Ssa3(P09435), Ssb1(P11484) and Ssb2(P40150). (B) Activity of METTL21A on recombinant yeast Hsp70 proteins. Recombinant METTL21A was incubated with the indicated recombinant E. coli-expressed yeast Hsp70 proteins, as well as with human HSPA8, in the presence of [3H]AdoMet, and the amount of TCA-insoluble radioactivity measured by scintillation counting. Error bars indicate standard deviation (n = 3). (C) Lys556 in Ssa1 is not methylated in vivo. Yeast whole cell extract (WCE) (20 μg) was incubated in the absence (upper) or presence (lower) of METTL21A (100 pmol). MS chromatograms of Asp-N generated peptides encompassing residues D555-A560 of Ssa1 (as shown in (E) and (F)), gated for m/z corresponding to peptides with un-, mono-, di- and trimethylated Lys556. Peaks corresponding to an unrelated peptide with a m/z-ratio matching the unmethylated species of the peptide are indicated by asterisks. (D) Lys565 in Ssb1/Ssb2 is not methylated in vivo. Same as in (C), except that the analysed peptide corresponds to L546-R568 from Ssb1/Ssb2 and was generated by cleavage with Arg-C. Tandem mass spectra of relevant peptides are shown in S1 Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131426.g002: Evaluating Ynl024c as a functional homolog of the Hsp70-specific METTL21A.(A) Protein sequence alignment of various Hsp70 proteins from H. sapiens (Hs) and S. cerevisiae (Sc) showing the region surrounding the lysine targeted by METTL21A (arrow). Shown proteins are: HSPA1(P08107), HSPA5(P11021), HSPA8(P11021), Ssa1(P10591), Ssa3(P09435), Ssb1(P11484) and Ssb2(P40150). (B) Activity of METTL21A on recombinant yeast Hsp70 proteins. Recombinant METTL21A was incubated with the indicated recombinant E. coli-expressed yeast Hsp70 proteins, as well as with human HSPA8, in the presence of [3H]AdoMet, and the amount of TCA-insoluble radioactivity measured by scintillation counting. Error bars indicate standard deviation (n = 3). (C) Lys556 in Ssa1 is not methylated in vivo. Yeast whole cell extract (WCE) (20 μg) was incubated in the absence (upper) or presence (lower) of METTL21A (100 pmol). MS chromatograms of Asp-N generated peptides encompassing residues D555-A560 of Ssa1 (as shown in (E) and (F)), gated for m/z corresponding to peptides with un-, mono-, di- and trimethylated Lys556. Peaks corresponding to an unrelated peptide with a m/z-ratio matching the unmethylated species of the peptide are indicated by asterisks. (D) Lys565 in Ssb1/Ssb2 is not methylated in vivo. Same as in (C), except that the analysed peptide corresponds to L546-R568 from Ssb1/Ssb2 and was generated by cleavage with Arg-C. Tandem mass spectra of relevant peptides are shown in S1 Fig.
Mentions: We have previously reported that Ynl024c is not a functional homolog of VCP-KMT/METTL21D, which trimethylates Lys315 in VCP, as the corresponding lysine residue is unmethylated in Cdc48, the yeast orthologue of VCP [22]. However, as Ynl024c is more similar to the Hsp70-specific MTase METTL21A (HSPA-KMT), we set out to investigate whether Ynl024c targets Hsp70 proteins. Human METTL21A has been reported to trimethylate Lys561 in the constitutively expressed Hsp70 protein HSC70/HSPA8 as well as the corresponding residue in several other human Hsp70/HSPA proteins [20,21]. A sequence alignment of various Hsp70 proteins from H. sapiens and S. cerevisiae demonstrates that a lysine residue is present at the corresponding position in several of the main Hsp70s in S. cerevisiae, e.g. SSA1, SSA3, SSB1, and SSB2 (the latter two are highly similar, showing >99% sequence identity) (Fig 2A). Initially, we therefore set out to test whether recombinant yeast Hsp70 proteins could be methylated by recombinant Ynl024c, but we were unfortunately unable to express and purify soluble, recombinant Ynl024c protein. However, we found that the yeast Hsp70 proteins SSA1 and SSA3, but not SSB2, could be methylated by human METTL21A in vitro, thus suggesting that yeast HSP70 proteins have the potential to become methylated by a METTL21A-like enzyme (Fig 2B). To further investigate the possibility that Ynl024c catalyses methylation of Hsp70 proteins, we analysed the methylation state of S. cerevisiae Hsp70s in vivo. To this end, an endoprotease digest of yeast proteins excised from the Hsp70-containing region of a SDS-PAGE gel was analysed by mass spectrometry. In this analysis, peptides covering the relevant Lys residue in SSA1 (Lys556) and SSB1/SSB2 (Lys565) were exclusively detected as unmethylated in wild-type cells (Fig 2C, 2D and S1 Fig). To exclude the possibility that our inability to detect methylated SSA1 was due to technical problems, we also investigated the methylation status of SSA1 in a yeast extract incubated with human METTL21A, and in this case SSA1 was found almost exclusively in the trimethylated state (Fig 2C). These results demonstrate that yeast Hsp70 proteins are unmethylated at the Lys residue corresponding to Lys561 in human HSPA8, and, consequently, that Ynl024c is likely not a functional orthologue of human METTL21A.

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