The Yeast Translation Release Factors Mrf1p and Sup45p (eRF1) Are Methylated, Respectively, by the Methyltransferases Mtq1p and Mtq2p

Abstract

The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N 5 -glutamine of the GGQ motif by PrmC methyltransferase. This motif is conserved in organisms from bacteria to higher eukaryotes. The Saccharomyces cerevisiae RFs, mitochondrial Mrf1p and cytoplasmic Sup45p (eRF1), have sequence similarities to the bacterial RFs, including the potential site of glutamine methylation in the GGQ motif. A computational analysis revealed two yeast proteins, Mtq1p and Mtq2p, that have strong sequence similarity to PrmC. Mass spectrometric analysis demonstrated that Mtq1p and Mtq2p methylate Mrf1p and Sup45p, respectively, in vivo. A tryptic peptide of Mrf1p, GGQHVNTTD-SAVR, containing the GGQ motif was found to be ϳ50% methylated at the glutamine residue in the normal strain but completely unmodified in the peptide from mtq1-⌬. Moreover, Mtq1p methyltransferase activity was observed in an in vitro assay. In similar experiments, it was determined that Mtq2p methylates Sup45p Chem. 280, 2439 -2445). Analysis of the deletion mutants showed that although mtq1-⌬ had only moderate growth defects on nonfermentable carbon sources, the mtq2-⌬ had multiple phenotypes, including cold sensitivity and sensitivity to translation fidelity antibiotics paromomycin and geneticin, to high salt and calcium concentrations, to polymyxin B, and to caffeine. Also, the mitochondrial mit ؊ mutation, cox2-V25, containing a premature stop mutation, was suppressed by mtq1-⌬. Most interestingly, the mtq2-⌬ was significantly more resistant to the anti-microtubule drugs thiabendazole and benomyl, suggesting that Mtq2p may also methylate certain microtubule-related proteins.Post-translational modification of proteins extends molecular structures beyond the limits imposed by the 20 encoded amino acids and, if reversible, allows a means of control and signaling. A wide range of prokaryotic and eukaryotic proteins are methylated post-translationally, including, for example, cytochrome c, ribosomal proteins, translation factors, and histones (1). The modifications occur by either N-methylation or carboxymethylation reactions, with the former reactions usually involving N-methylation of lysine, arginine, histidine, alanine, proline, glutamine, phenylalanine, asparagine, and methionine, whereas the latter reactions usually involving O-methylesterification of glutamic and aspartic acid. The enzymes catalyzing these methylation reactions generally use S-adenosylmethionine (AdoMet) 4 as the methyl donor to transfer the methyl group to the free amino group on the side chain of an amino acid residue (2). The extent of methylation can be complete or almost complete, as in the case of cytochrome c, or can be partial, as in case of ribosomal proteins. Once incorporated, the methyl groups do not appear to be removed from most proteins. However, reversible methylation of glutamic acid residues is involved in the chemotactic response of bacteria (3); also reversible methylation of the C subunit of the phosphoprotein phosphatase 2A (PP2A) at...

Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

“…ICM and trophoblastic giant cells (TGC) in outgrowth at day 9 are pointed out. Scale bars, 100 m. (22). The observed phenotypes in these two lower organisms are mild and can be only loosely related to inefficient termination of protein translation.…”

“…Methylation of the release factors ensures efficient translation termination and release of newly synthesized peptide from the ribosome (16). Similarly, the yeast HemK homologue, YDR140w (Mtq2p), was confirmed to methylate the eukaryotic release factor eRF1 on a corresponding glutamine residue (9,22). More recently, the human homologue N6amt1 (HemK2) was reported to methylate release factor 1 (eRF1) in vitro (5).…”

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

“…In one study, it was reported that the alteration of the translation release factor eRF1/Sup45 also leads to zymocin resistance (13). eRF1 possesses the universally conserved tripeptide GGQ, the Gln of which is methylated by the complex Mtq2-Trm112p (7,15,16). More specifically, a substitution of GGQ by the nonmethylatable sequences GGE or GGN induces strong zymocin resistance (13).…”

Two-subunit enzymes involved in eukaryotic post-transcriptional tRNA modification

“…4,114 In S. cerevisiae, Sc Mtq2-Trm112 is the enzyme responsible for N 5 methylation of the glutamine residue of the GGQ tripeptide motif of eukaryotic release factor 1 (eRF1). 4,115,116 The crystal structure of Mtq2-Trm112 from Encephalitozoon cuniculi consists of a single heterodimer, and suggests that E. cuniculi Trm112 may function to solubilize E. cuniculi Mtq2 by masking a hydrophobic patch involved in the dimer interface. 114 Indeed, the presence of Sc Trm112 increases the solubility of Sc Mtq2 when the proteins are expressed in E. coli.…”