Yeast Ribosomal/Cytochrome c SET Domain Methyltransferase Subfamily

Abstract

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 ⌬rkm1 deletion strain demonstrated a loss in mass of ϳ56 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 ⌬rkm1 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.SET domain methyltransferases are a new family of methyltransferases known to specifically methylate lysine residues in a variety of proteins (1, 2). The family was named after the Drosophila genes in which it was first discovered, Su(var), Enhancer of zeste, and Trithorax (3), all of which were later shown to encode histone lysine methyltransferases (4 -6). The SET domain methyltransferases differ from other methyltransferases with known structures in that its catalytic core forms a knot-like structure with a conserved tyrosine residue in the active site necessary for catalysis (6 -12). SET domain methyltransferases have been shown to modify a variety of proteins including Rubisco 3 (13, 14), cytochrome c (15), and most notably histones (16). Currently, no physiological effect has been observed for the lysine methylation of Rubisco or cytochrome c (14, 15). However, in histones lysine methylation has been shown to play an important role in the activation or repression of transcription through modulating the structure of chromatin (16).Histone tails are highly decorated by lysine methylation reactions and the majority of these modifications occur in close proximity and can exhibit opposite effects. The most well characterized biology involves the interplay between the methylation of lysine 4 and lysine 9 of mammalian histone H3, catalyzed by distinct SET domain methyltransferases (16). Methylation of lysine 4 is correlated with active transcription and euchromatin, whereas methylation of lysine 9 is associated with repressed transcription and heterochromatin (17)(18)(19)(20). In yeast, histone H3 methylation has also been actively studied and a similar interplay has been observed, although the distance between the methylated sites is greater. Histone H...

A Conserved SET Domain Methyltransferase, Set11, Modifies Ribosomal Protein Rpl12 in Fission Yeast

“…If this is the case, at least four S. pombe SET methyltransferases, Set8, Set10, Set11, and Set13, are involved in the modification of ribosomal proteins. In this respect, one hypothesis to explain this is that a subfamily of SET methyltransferases may have evolved to target the lysine residues of highly basic proteins, such as histones and ribosomal proteins, that stably associate with DNAs and RNAs, respectively (16).…”

“…In the budding yeast Saccharomyces cerevisiae, direct mass spectrometric analysis of the large ribosomal proteins revealed that six of them, Rpl1, Rpl3, Rpl12, Rpl23, Rpl42, and Rpl43, are post-translationally modified by the addition of methyl groups (14). Rpl23 is specifically ⑀-N-dimethylated at two residues, Lys 105 and Lys 109 , and these modifications are catalyzed by the SET domain-containing methyltransferase Rkm1 (15,16). S. cerevisiae Rpl12, the counterpart of bacterial L11, is also modified,: by ⑀-N-dimethylated at Lys 3 , ⑀-N-trimethylation at Lys 10 , and ␦-N-monomethylation at Arg 66 (17,18).…”

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

“…Interestingly, the majority of the yeast point mutations displaying phenotypes 3 H-methylated large ribosomal subunits were isolated from [ 3 H]AdoMetlabeled intact BY4742 wild-type cells and reverse-phase HPLC was used to isolate a fraction containing a mixture of Rpl3 and Rpl23ab as described in "Experimental Procedures." The latter protein is a known coeluting species known to contain two dimethyllysine residues (17). This mixture was acid hydrolyzed and 1 mol each of ⑀-dimethyllysine and 3-methyl-L-histidine (-methyl-L-histidine, Vega Biochemicals 12608) standards were added.…”

“…We have used mass spectrometry to screen for loss of methylation in the intact ribosomal proteins of yeast strains lacking a single known or putative methyltransferase. Using this approach, SET-domain methyltransferases responsible for modifying Rpl12ab, Rpl23ab, Rpl42ab, and eEF1a at specific lysine residues have been identified (13)(14)(15)(16)(17). In addition to screening the SET-domain methyltransferase gene knock-out strains, we also screened the seven ␤-strand methyltransferase gene family for loss of methylation in gene deletions.…”

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

“…Rpl3 is modified to form a 3-methylhistidine residue at position 243 (13), whereas Rpl12ab is modified at three positions: a dimethylproline residue is present at the N terminus (14), an ⑀-trimethyllysine residue is found at position 3 (9,19), and an unusual ␦-monomethylarginine residue is found at position 66 (2). Rpl23ab is modified by ⑀-dimethyllysine formation at positions 105 and 109 (7), and Rpl42ab is modified by ⑀-monomethyllysine formation at positions 39 and 55 (9). The proposed methylation at Rpl43ab has not been observed to date in our studies.…”