Abstract
The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N5-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, GGQHVNTTDSAVR, containing the GGQ motif was found to be approximately 50% methylated at the glutamine residue in the normal strain but completely unmodified in the peptide from mtq1-Delta. Moreover, Mtq1p methyltransferase activity was observed in an in vitro assay. In similar experiments, it was determined that Mtq2p methylates Sup45p. The Sup45p methylation by Mtq2p was recently confirmed independently (Heurgue-Hamard, V., Champ, S., Mora, L., Merkulova-Rainon, T., Kisselev, L. L., and Buckingham, R. H. (2005) J. Biol. Chem. 280, 2439-2445). Analysis of the deletion mutants showed that although mtq1-Delta had only moderate growth defects on nonfermentable carbon sources, the mtq2-Delta 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-Delta. Most interestingly, the mtq2-Delta was significantly more resistant to the anti-microtubule drugs thiabendazole and benomyl, suggesting that Mtq2p may also methylate certain microtubule-related proteins.
Highlights
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
Our search for PrmC orthologs in S. cerevisiae proteome revealed the two yeast proteins, Mtq1p (YNL063w) and Mtq2p (YDR140w), that have strong sequence similarity to the E. coli PrmC methyltransferase (Fig. 1). Both Mtq1p and Mtq2p have conserved functional domains responsible for binding of the cofactor, AdoMet, that is generally characteristic for protein methyltransferases [40]. This high protein sequence similarity suggested that Mrf1p and Sup45p may have glutamine residues methylated at the positions corresponding to the E. coli release factors (RFs), and those reactions could be carried out by Mtq1p and Mtq2p, respectively
Mrf1p was considered as a potential Mtq1p substrate because Mrf1p is a mitochondrial protein, and Mtq1p was predicted to reside in mitochondria by PSORT II, Sup45p is known to be in the cytoplasm
Summary
Primers Oligo and Oligo (Table 2) and plasmid pAB2630 (pBS1539) were used to prepare the PCR fragment, which were used in the construction of the mtq1-⌬::URA3 disruption. The DNA fragment required for producing the mtq2-⌬::URA3 disruption was prepared with Oligo and Oligo and plasmid pAB2630. Mtq, and mrf deletion strains with the marker kanMX4 in the B-8114 background, yeast genomic DNAs, which were prepared from the corresponding ORF::kanMX4 deletion strain (Invitrogen), were used as template for PCR with primers Oligo and 4, Oligo and 8, and Oligo and 15, respectively. Construction of the SUP45::TAP Yeast Strain—The SUP45::TAP strain was made by a PCR-based technique with Oligo and Oligo and with plasmid pAB2630 (pBS1539) as a template (Table 2). B-8114 was transformed with the resulting PCR product containing the SUP45::TAP::URA3 DNA fragment, and the transformants were
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