Identification of Two SET Domain Proteins Required for Methylation of Lysine Residues in Yeast Ribosomal Protein Rpl42ab
ABSTRACT We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.
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ABSTRACT: Here we describe the discovery of S. cerevisiae protein YJR129Cp as a new eukaryotic seven-beta-strand lysine methyltransferase. An immunoblotting screen of 21 putative methyltransferases showed a loss in the methylation of elongation factor 2 (EF2) on knockout of YJR129C. Mass spectrometric analysis of EF2 tryptic peptides localized this loss of methylation to lysine 509, in peptide LVEGLKR. In vitro methylation, using recombinant methyltransferases and purified EF2, validated YJR129Cp as responsible for methylation of lysine 509 and Efm2p as responsible for methylation at lysine 613. Contextualised on previously described protein structures, both sites of methylation were found at the interaction interface between EF2 and the 40S ribosomal subunit. In line with the recently discovered Efm1 and Efm2 we propose that YJR129C be named elongation factor methyltransferase 3 (Efm3). The human homolog of Efm3 is likely to be the putative methyltransferase FAM86A, according to sequence homology and multiple lines of literature evidence.Biochemical and Biophysical Research Communications 07/2014; 451(2). DOI:10.1016/j.bbrc.2014.07.110 · 2.28 Impact Factor
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ABSTRACT: Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine-243, a residue that contacts the 25S rRNA near the P-site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven beta strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nuclei-containing organelle fraction, but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated, but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal media. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in orchestrating the early assembly of the large ribosomal subunit and in faithful protein production.Molecular and Cellular Biology 05/2014; 34(15). DOI:10.1128/MCB.01634-13 · 5.04 Impact Factor
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ABSTRACT: Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in many important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 43 methylated proteins, containing 68 methylation events associated with 64 methylation sites. More than 90% of these methylation events were previously unannotated in Uniprot database. Our results indicated, 1) over 2.6% of identified S. cerevisiae proteins are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys >Arg>Asp>Asn≈Gln≈His>Glu>Cys, 3) the methylation state on nitrogen center is largely exclusive. As our dataset covers various types of methylation centers, it provides rich information about yeast methylproteome and should significantly contribute to the field of protein methylation.Journal of Proteomics 08/2014; 114. DOI:10.1016/j.jprot.2014.07.032 · 3.93 Impact Factor