Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae

Abstract

Eukaryotic elongation factor 1A (eEF1A1) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

“…In addition to screening the SET-domain methyltransferase gene knock-out strains, we also screened the seven ␤-strand methyltransferase gene family for loss of methylation in gene deletions. We found one seven ␤-strand methyltransferase involved in modifying eEF1a, presumably at a lysine residue (13), and one involved in methylating the small ribosomal protein Rps2 at one or more arginine residues (18). Finally, a seven ␤-strand methyltransferase has been shown to modify Rpl12ab and Rps25a/Rps25b at their N-terminal proline residues (6).…”

“…We have used mass spectrometry to screen for loss of methylation in the intact ribosomal proteins of yeast strains lacking a single known or putative methyltransferase. Using this approach, SET-domain methyltransferases responsible for modifying Rpl12ab, Rpl23ab, Rpl42ab, and eEF1a at specific lysine residues have been identified (13)(14)(15)(16)(17). In addition to screening the SET-domain methyltransferase gene knock-out strains, we also screened the seven ␤-strand methyltransferase gene family for loss of methylation in gene deletions.…”

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

“…Most characterized lysine-specific MTases are SET domain proteins acting primarily on histones, but a few lysine-specific protein MTases have also been found in Class I. In humans, these are the histone-specific MTase DOT1L 3 and the calmodulin-specific MTase CaM-KMT 4 ; in addition, human METTL10 is a likely ortholog of yeast See1, which methylates elongation factor 1A 5 . The MTases responsible for many lysine methylations remain elusive 6 , and some are likely to be found among the numerous uncharacterized Class I MTases.…”

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

“…To date, most protein lysine methyltransferases have been identified as members of the SET domain methyltransferase family. However, the YLR137W gene product, now designated Rkm5, now joins four other protein lysine methyltransferases of the seven-␤-strand superfamily: the Dot1 methyltransferase acting on histone H3 (25,26), the CaM KMT acting on calmodulin (27), the See1 methyltransferase acting on elongation factor 1A (17), and the PrmA methyltransferase acting on prokaryotic ribosomal protein L11 (28).…”

“…One of the major types of these reactions is the transfer of methyl groups from S-adenosylmethionine to a variety of residues including arginine (2-5), lysine (6 -11), glutamine (12), histidine (13), and N-terminal (14,15) residues. Ribosomal proteins (1-5, 7-11, 13, 14, 16), elongation factor 1A (1,6,17), and translational release factors (12) are common substrates of protein methyltransferases. These modifications are known to enhance resistance to ribosome-targeting antibiotics, facilitate ribosomal protein transport into and out of the nucleus, allow efficient ribosomal assembly, and increase translational accuracy (1,5).…”