Yeast ribosomal/cytochrome c SET domain methyltransferase subfamily: identification of Rpl23ab methylation sites and recognition motifs.
Ribosomal protein L23ab is specifically dimethylated at two distinct sites by the SET domain-containing enzyme Rkm1 in the yeast Saccharomyces cerevisiae. Using liquid column chromatography with electrospray-ionization mass spectrometry, we determined that Rpl23ab purified from the Deltarkm1 deletion strain demonstrated a loss in mass of approximately 56 Da when compared with Rpl23ab purified from the wild type strain. When Rpl23ab was proteolyzed, using proteinase ArgC or CNBr, and the peptides derived were analyzed by tandem mass spectrometry, no sites of methylation were found in Rpl23ab purified from the Deltarkm1 deletion strain, whereas two sites of dimethylation were observed in the wild type strain at lysine residues 105 and 109. We show that both Rpl23a and Rpl23b are expressed and methylated in vivo in yeast. Using polysomal fractionation, we demonstrate that the deletion of RKM1 has no effect on ribosomal complex formation. Comparison of SET domain methyltransferase substrates in yeast reveal sequence similarities around the lysine methylation sites and suggest that an (Asn/Pro)-Pro-Lys consensus sequence may be a target for methylation by subfamily 2 SET domain methyltransferases. Finally, we show the presence of Rkm1 homologs in fungi, plants, and mammals including humans.
Porras-Yakushi
Tanya R
TR
Department of Chemistry and Biochemistry, the Molecular Biology Institute, UCLA, Los Angeles, CA 90095-1569, USA.
Whitelegge
Julian P
JP
Clarke
Steven
S
D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D010944:Plants / Q000201:enzymology / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
C506085:RPL23A protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011495:Histone-Lysine N-Methyltransferase; C506086:Rkm1 protein, S cerevisiae