Saccharomyces cerevisiae eukaryotic elongation factor 1A (eEF1A) is methylated at Lys-390 by a METTL21-like methyltransferase

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)

A System for Enzymatic Lysine Methylation in a Desired Sequence Context

A number of lysine-specific methyltransferases (KMTs) are responsible for the post-translational modification of cellular proteins on lysine residues. Most KMTs typically recognize specific motifs in unstructured, short peptide sequences. However, we have recently discovered a novel KMT that appeared to have a more relaxed sequence specificity, namely, valosin-containing protein (VCP)-KMT, which trimethylates Lys-315 in the molecular chaperone VCP. On the basis of this, here, we explored the possibility of using the VCP-KMT/VCP system to obtain specific lysine methylation of desired sequences grafted onto a VCP-derived scaffold. We generated VCP-derived proteins in which three amino acid residues on each side of Lys-315 had been replaced by various sequences representing lysine methylation sites in histone H3. We found that all of these chimeric proteins were subject to efficient VCP-KMT-mediated methylation in vitro, and methylation was also observed in mammalian cells. Thus, we here describe a versatile system for introducing lysine methylation into a desired peptide sequence, and the approach should be readily expandable for generating combinatorial libraries of methylated sequences

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1–8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins

Methylation status of eEF1A in mammalian cells.

<p>(A) Alignment of various eEF1A sequences from <i>Homo sapiens</i> (<i>Hs</i>), <i>Oryctolagus cuniculus</i> (<i>Oc</i>; rabbit) and <i>Saccharomyces cerevisiae</i> (<i>Sc</i>), showing the region surrounding the lysine targeted by Ynl024c (arrow). Shown proteins are <i>Hs</i> eEF1A1 (NP_001393.1), <i>Oc</i> eEF1A1 (NP_001075808.1) and <i>Sc</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131426#pone.0131426.s004" target="_blank">S4 Fig</a>.</p

<i>In vitro</i> MTase activity of Ynl024c.

<p>Protein extract from the <i>ynl024cΔ</i> yeast strain (denoted “<i>Sc ynl024c</i>Δ”) was treated with a protein extract from <i>E</i>. <i>coli</i> either expressing 6xHis-tagged Ynl024c from the pET28a-<i>YNL024C</i> plasmid (denoted “<i>Ec</i>+ Ynl024c”) or devoid of an expression plasmid (denoted “<i>Ec</i>”), 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).</p

Ynl024c-mediated methylation of eEF1A <i>in vivo</i>.

<p>(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 <i>S</i>. <i>cerevisiae</i> eEF1A from wild-type and Ynl024c deficient (<i>ynl024cΔ</i>) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::<i>YNL024C</i>”or “<i>ynl024cΔ</i>::<i>YNL024C</i>“, 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131426#pone.0131426.s002" target="_blank">S2 Fig</a>.</p

Closest homologs of Ynl024c in <i>S</i>. <i>cerevisiae</i> and <i>H</i>. <i>sapiens</i>.

<p>*Identified by BLAST search versus non-redundant proteins sequences from all organisms using Ynl024c (NP_014374.1) as query.</p><p>Hits with e-value < 1e-5 were considered to be homologs.</p><p>Closest homologs of Ynl024c in <i>S</i>. <i>cerevisiae</i> and <i>H</i>. <i>sapiens</i>.</p

Sequence analysis and structural modelling of Ynl024c.

<p>(A) Topology diagram of the canonical 7BS MTase fold. Arrows and rectangles indicate β-strands and α-helices, respectively. The seven β-strands are designated “1–7”, the α-helices connecting them “A-F”, and the secondary structure elements preceding the 7BS-fold denoted “Z”. B, Protein sequence alignment of Ynl024c and human METTL21 proteins. Motifs “I”, “Post I” and “II”, which are shared by all 7BS MTases, as well as the DXXY motif, a hallmark of MTF16, are indicated by boxes. Above the alignment are indicated the secondary structure elements from the solved crystal structures of VCP-KMT (red; pdb 4LG1), METTL21A (green; pdb 4LEC), METTL21B (blue; pdb 4QPN) and METTL21C (orange; pdb 4MTL), a predicted Ynl024c structure (black; see also (C)), as well as a secondary structure prediction for Ynl024c, performed with Jpred 3 (dashed, black). β-strands and α-helices are indicated by arrows and thick lines, respectively, and the numbering/lettering of these are as outlined in A). Asterisks indicate conserved active site residues represented in (D). (C) Predicted structural model of Ynl024c. The model was generated by one-to-one threading with Phyre2, using METTL21A as a template. (D) localization of putatively important catalytic residues (green) in the active site of the Ynl024c structural model. AdoMet is shown in purple. The shown residues are indicated by asterisk in the sequence alignment in (B). (E) Structural alignment of human VCP-KMT, METTL21A, METTL21B and METTL21C (Color code as in (B)).</p

Lysine methylated peptides in eEF1A in wild type and Ynl024c deficient yeast.

<p>*Methylated residue indicated in bold.</p><p>**Exclusively detected in wild type cells.</p><p>Lysine methylated peptides in eEF1A in wild type and Ynl024c deficient yeast.</p