Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.DOI: http://dx.doi.org/10.7554/eLife.19105.001

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 (⌬ypl208w) 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 ⑀-[ 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.

The ribosomal protein L12ab (Rpl12ab) in Saccharomyces cerevisiae is modified by methylation at both arginine and lysine residues. Although the enzyme responsible for the modification reaction at arginine 66 has been identified (Rmt2), the enzyme(s) responsible for the lysine modification(s) has not been found, and the site(s) of methylation has not been determined. Here we demonstrate, using a combination of mass spectrometry and labeling assays, that the yeast gene YDR198c encodes the enzyme responsible for the predominant ⑀-trimethylation at lysine 10 in Rpl12ab. An additional site of predominant ⑀-dimethylation is observed at lysine 3; the enzyme catalyzing this modification is not known. The YDR198c gene encodes a SET domain similar to that of the Rkm1 enzyme responsible for modifying Rpl23ab, and we have now designated the YDR198c gene product as Rkm2 (ribosomal lysine methyltransferase 2). The effect of the loss of the enzyme on ribosomal complex stability was studied by polysomal fractionation. However, no difference was observed between the ⌬rkm2 deletion strain and its parent wild type strain. With the identification of this enzyme, it appears that the 12 SET domain family members in yeast can now be divided into two subfamilies based on function and amino acid sequence identity. One branch includes enzymes that modify histones, including Set1 and Set2; the other branch includes Rkm1, Rkm2, and Ctm1, the cytochrome c methyltransferase. These studies suggest that the remaining seven SET domain proteins may also be lysine methyltransferases.Ribosomal proteins are highly modified by post-translational methylation reactions (1). The extent to which ribosomal proteins are modified and the enzymes responsible for the modifications have only begun to be understood. For example, in the yeast Saccharomyces cerevisiae, of the three most highly methylated large subunit proteins (Rpl1ab, Rpl12ab, and Rpl23ab) (1, 2), methyltransferases have only been recently identified for two of these species. The Rmt2 enzyme catalyzes the monomethylation of arginine 66 of Rpl12ab (3), and the Rkm1 enzyme catalyzes the dimethylation of two lysine residues in Rpl23ab (4). A mass spectral analysis identified other potentially methylated species within the large subunit of S. cerevisiae (5). This study confirmed the modifications of Rpl1ab, Rpl12ab, and Rpl23ab and also provided evidence for the modification of Rpl3, Rpl42ab, and Rpl43ab. Studies of the small subunit in yeast have been less extensive, with the identification of methylated Rps2, Rps3, Rps13, Rps21ab, Rps23a, and Rps25ab (1, 6, 7). The physiological role(s) of these modifications is not known.In the fission yeast Schizosaccharomyces pombe, Prmt3 has been shown to be a ribosomal protein methyltransferase (8). This protein-arginine methyltransferase 3 homolog is responsible for asymmetrically dimethylating an arginine residue in the small ribosomal subunit protein S2 (8). The direct role this modification plays is not known. However, when the gene is absent, an accumul...

Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca. 2-fold increase in ubiquitylation site identifications in K-ε-GG peptide-enriched samples over traditional collisional-induced dissociation (CID) or higher-energy collision dissociation (HCD) methods. Precursor ions were predominantly observed as 3+ charged species or higher and in a mass range 300–1200 m/z. N-ethylmaleimide was used as an alkylating agent to reduce false positive identifications resulting from overalkylation with halo-acetamides. These results demonstrate that the application of ETD fragmentation, in addition to narrowing the mass range and using N-ethylmaleimide yields more high-confidence ubiquitylation site identification than conventional CID and HCD analysis.

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...