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. 2014 Oct 31;289(44):30499-30510.
doi: 10.1074/jbc.M114.601658. Epub 2014 Sep 17.

Identification and characterization of a novel evolutionarily conserved lysine-specific methyltransferase targeting eukaryotic translation elongation factor 2 (eEF2)

Erna Davydova  1 , Angela Y Y Ho  1 , Jedrzej Malecki  1 , Anders Moen  1 , Jorrit M Enserink  2 , Magnus E Jakobsson  1 , Christoph Loenarz  3 , Pål Ø Falnes  4
Affiliations

Identification and characterization of a novel evolutionarily conserved lysine-specific methyltransferase targeting eukaryotic translation elongation factor 2 (eEF2)

Erna Davydova et al. J Biol Chem. .

Abstract

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

Keywords: Enzyme; Enzyme Catalysis; Post-translational Modification (PTM); Protein Methylation; Translation Elongation Factor.

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Figures

FIGURE 1.
FIGURE 1.
FAM86 is a 7BS-MTase conserved across Eukarya. A, the canonical core folds of the 7BS-MTases, α-helices, and β-strands are represented as barrels and arrows, respectively. B, alignment of FAM86 protein sequences from Homo sapiens (Hs; NP_958802.1), Rattus norvegicus (Rn; NP_001100445.1), Caenorhabditis elegans (Ce; NP_498985.3), Drosophila melanogaster (Dm; NP_573368.2), Arabidopsis thaliana (At; NP_198092.1), and S. cerevisiae (Sc; NP_012663.2). Boxes indicate motifs characteristic for 7BS-MTases (Motif I, Post I, and Motif II) as well as the (D/E)XX(Y/F) motif typical of MTase family 16. The predicted secondary structure for human FAM86A is shown above the alignment. C, alignment of human FAM86A and FAM86B2 protein sequences (NP_958802.1 and NP_001131082.1) with the Gallus gallus FAM86 homologue (NP_001025798.1). The G. gallus sequence is included as a representative of a typical vertebrate FAM86 protein. The underlined segment indicates residues missing in the FAM86B1 protein. Residues that are conserved in vertebrates but differ between FAM86A and FAM86B2 are boxed.
FIGURE 2.
FIGURE 2.
FAM86A and its yeast homologue Yjr129c are eEF2 methyltransferases. A, eEF2 co-purifies with FAM86A in TAP. Shown is a Coomassie Blue-stained SDS-polyacrylamide gel of TAP eluates from a Flp-In T-REx HEK293-derived cell line treated with doxycycline to induce expression of SBP- and CBP-tagged FAM86A (TAP-FAM86A) or the CBP-SBP tag alone (TAP). Purification buffers were supplemented with AdoMet (SAM) or AdoHcy (SAH) where indicated. The identity of the bait and co-purifying proteins was determined by peptide mass fingerprinting; asterisks indicate β-tubulin (50 kDa), pyruvate carboxylase (125 kDa), and acetyl-CoA carboxylase 1 (265 kDa). Molecular weight markers (in kilodaltons) are shown on the right. B, eEF2 from yjr129cΔ yeast extracts is efficiently methylated by FAM86A and Yjr129c. Top, MTase reactions with recombinant FAM86A or Yjr129c, [3H]AdoMet, and WT or yjr129cΔ yeast extracts separated by SDS-PAGE and subjected to fluorography; bottom, Ponceau S stain of the 100-kDa region of the same membrane. C, trimethylation of Lys-509 in eEF2. Shown are MS/MS fragmentation patterns of representative AspN-generated trimethylated peptides from the WT strain (top) and unmethylated peptides from the yjr129cΔ strain (bottom). D, Lys-509 in eEF2 is the target of methylation by the MTases Yjr129c and FAM86A. Shown are MS chromatograms of AspN-generated Lys-509-containing peptides from partially purified eEF2 from WT or yjr129cΔ yeast extracts. When indicated, the extracts had been incubated with recombinant Yjr129c or FAM86A in the presence of AdoMet. *, an unrelated peptide. A.u., arbitrary units. E, mutagenesis of Lys-509 in eEF2 abolishes FAM86A and Yjr129c-mediated methylation. Recombinant FAM86A or Yjr129c was incubated in the presence of [3H]AdoMet with WT or K509A His6-eEF2, expressed, and purified from yjr129cΔ mutant yeast. The MTase reactions were separated by SDS-PAGE and subjected to fluorography (top) and Ponceau S staining (bottom).
FIGURE 3.
FIGURE 3.
Methylation of mammalian eEF2. A, human and rabbit eEF2 is trimethylated on Lys-525. Shown is MS/MS fragmentation of a trypsin-generated human (HEK293) (Hs, top) and rabbit (Oc, bottom) eEF2-derived peptide, supporting Lys-525 as the target of methylation. B, eEF2 is trimethylated on Lys-525 in the rat. Shown is MS/MS fragmentation of AspN-generated trimethylated (top) and unmethylated (bottom) eEF2-derived peptides from the rat brain. C, eEF2 methylation status in a panel of rat tissues. Chromatograms were gated for different methylation states of AspN-generated Lys-525-containing peptides (as in B) from partially purified eEF2 from rat brain, kidney, and spleen. Relative intensities of signals for the different methylation states are indicated in percentages. A.u., arbitrary units. D, eEF2 from a hypomethylated human embryonic kidney cell line is efficiently methylated by both FAM86A and Yjr129c. A protein extract from HEK293 cells that had either been left untreated (HEK293) or treated with 20 μm AdOx to induce hypomethylation (HEK293(AdOx)) was incubated with recombinant FAM86A or Yjr129c in the presence of [3H]AdoMet. The MTase reactions were separated by SDS-PAGE and subjected to fluorography (top) and Ponceau S staining (bottom). E, Lys-525 in human eEF2 is subject to FAM86A- and Yjr129c-mediated trimethylation in vitro. eEF2 was partially purified from extracts from untreated and AdOx-treated cells and then subjected to LC-MS/MS analysis. When indicated, eEF2 had been incubated with recombinant Yjr129c or FAM86A in the presence of AdoMet. Chromatograms were gated for different methylation states of trypsin-generated Lys-525-containing eEF2 peptides (as shown in A). Arrows, peptides of interest; *, unrelated peptides. Relative intensities of signals for the different methylation states are indicated in percentages for the hypomethylated cell line.
FIGURE 4.
FIGURE 4.
Phenotypic analysis of the yjr129cΔ yeast strain. A, comparison of overall protein synthesis between WT and yjr129cΔ yeast strains. Cultures were grown to the same A600 in SD −Met/−Cys medium; a mix of 35S-labeled methionine and cysteine was added; and total protein synthesis was measured at each time point by TCA precipitation of total protein, followed by scintillation counting. Error bars, S.D. (n = 4). B, hypersensitivity of the yjr129cΔ yeast strain to sordarin on solid medium at 37 °C. Dilutions of WT yeast cells and two independently derived yjr129cΔ mutants (JEY9581 (yjr129cΔ) and JEY10800 (yjr129cΔ*)) were grown on YPD plates containing 5 μg/ml sordarin at 30 or 37 °C for 3 days. C, hypersensitivity of the yjr129cΔ yeast strain to sordarin in liquid medium. The graph shows the growth of the WT and yjr129cΔ (JEY9581) strains in liquid YPD medium containing 0 μg/ml (wt0 and Δ0), 5 μg/ml (wt5 and Δ5), or 10 μg/ml (wt10 and Δ10) sordarin over a period of 24 h at 37 °C. Error bars, range of duplicates. D, dual luciferase assays investigating the effects of YJR129C deletion on ribosomal frameshifting. YJR129C deletion significantly alters programmed ribosomal frameshifting of the HIV-1 gag-pol but not the Ty1 +1 site, relative to the luciferase activity of control sequences, which was unaffected. n = 3; mean ± S.D.; **, p < 0.01; n.s., not significant.
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