In vivo studies have shown that the ribosomal large subunit protein L23a (Rpl23ab) in Saccharomyces cerevisiae is methylated at lysine residues. However, the gene encoding the methyltransferase responsible for the modification has not been identified. We show here that the yeast YPL208w gene product, a member of the SET domain family of methyltransferases, catalyzes the reaction, and we have now designated it Rkm1 (ribosomal lysine (K) methyltransferase 1). Yeast strains with deletion mutations in candidate SET domain-containing genes were in vivo labeled with S-adenosyl-l-[methyl-(3)H]methionine. [(3)H]Methyl radioactivity was determined after lysates were fractionated by SDS gel electrophoresis. When compared with the parent strain or other candidate deletion strains, a loss of a radiolabeled 15-kDa species was observed in the rkm1 (Deltaypl208w) knock-out strain. Treatment of wild-type cell extracts with RNase or proteinase K demonstrated that the methyl-accepting substrate is a protein. Cellular lysates from parent and knockout strains were fractionated using high salt sucrose gradients. Analysis of the gradient fractions by SDS gel electrophoresis demonstrated that the 15-kDa methyl-accepting substrate elutes with the large ribosomal subunit. In vitro methylation experiments using purified ribosomes confirmed that the methyl-accepting substrate is a ribosomal protein. Amino acid analysis of the in vivo labeled 15 kDa polypeptide showed that it contains epsilon-[(3)H]dimethyllysine residues. Mass spectrometry of tryptic peptides of the 15 kDa polypeptide identified it as Rpl23ab. Analysis of the intact masses of the large ribosomal subunit proteins by electrospray mass spectrometry confirmed that the substrate is Rpl23ab and that it is specifically dimethylated at two distinct sites by Rkm1. These results show that SET domain methyltransferases can be involved in translational roles as well as in the previously described transcriptional roles.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|