Identification of Methylated Proteins in the Yeast Small Ribosomal Subunit: A Role for SPOUT Methyltransferases in Protein Arginine Methylation

Abstract

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation on Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…can be targets for ubiquitin-E3 linked degradation (21), can facilitate or block protein-protein interactions or enzyme-substrate interactions (6,22), and can modulate interactions with RNA (7,23,24). An important recent discovery is that the three hydrogen atoms on methyl groups bound to positively charged nitrogen or sulfur atoms may themselves be able to serve as hydrogen bond donors, greatly expanding the possibilities for interactions (25,26; Figure 1).…”

“…In the process, we used high-capacity, high-resolution chromatographic assays to detect radiolabeled methylated amino acid residues based on sulfonated polystyrene cation exchange resins. Using in vivo and in vitro methyl labeling with high specific activity S-adenosyl-[ 3 H-methyl]methionine labeled with three tritium atoms per methyl group (giving a specific activity of about 100 cpm per femtomole of methyl groups), and our ability to detect less than 10 cpm by extended liquid scintillation counting, we can detect attomole levels of methyl groups in mg or larger amounts of protein (23). This extraordinary sensitivity allows us to detect modifications that would be missed by other commonly used approaches.…”

“…However, recent work has emphasized the importance of methylating non-histone substrates at these residues, particularly ribosomal proteins, translation factors, and transcription factors in a wide variety of organisms (7,8,15,18,19). Furthermore, the methylation of lysine and arginine residues represents just the tip of the iceberg in protein methylation -modifications have also been established at histidine, modified histidine (diphthamide), glutamate, glutamine, asparagine, Lisoaspartate, D-aspartate, cysteine, isoprenylcysteine, methionine, N-terminal, and C-terminal residues (1,4 RNA (7,23,24). An important recent discovery is that the three hydrogen atoms on methyl groups bound to positively charged nitrogen or sulfur atoms may themselves be able to serve as hydrogen bond donors, greatly expanding the possibilities for interactions (25,26; Figure 1).…”

“…These proteins, and the enzymes that modify them, have been extensively studied from the intersection of two research directions. In the first place, the combination of bioinformatics and the biology of Saccharomyces cerevisiae have allowed an approach to identify methylated sites in yeast proteins (23,24,29,30). Bioinformatic analyses have allowed for the identification of open reading frames for candidate methyltransferases from genomic DNA sequences both of the major seven beta-strand family and of the SET domain, SPOUT, and other structural families (31-35).…”

“…Significantly, the availability of gene knockout strains of yeast allows for biochemical analyses of mutants to identify the methyltransferases responsible for each of the modifications. Secondly, mass spectrometric analyses, particularly top-down intact mass measurements, have allowed the location of methylated residues in the amino acid sequence of the modified proteins (23,24,37). The identification of the modifying enzymes can then allow for the study of the physiological effects of each methylation reaction by examining the biology of the mutant strains lacking the methyltransferase.…”