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PMID:22650761

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Citation

Young, BD, Weiss, DI, Zurita-Lopez, CI, Webb, KJ, Clarke, SG and McBride, AE (2012) Identification of methylated proteins in the yeast small ribosomal subunit: a role for SPOUT methyltransferases in protein arginine methylation. Biochemistry 51:5091-104

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 of 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.

Links

PubMed PMC3383884 Online version:10.1021/bi300186g

Keywords

Arginine/genetics; Arginine/metabolism; Cysteine/metabolism; Methylation; Multigene Family/physiology; Mutagenesis, Site-Directed; Protein Processing, Post-Translational/genetics; Protein-Arginine N-Methyltransferases/genetics; Protein-Arginine N-Methyltransferases/metabolism; Ribosomal Proteins/genetics; Ribosomal Proteins/metabolism; Ribosome Subunits, Small/genetics; Ribosome Subunits, Small/metabolism; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/growth & development; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Zinc/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:YG5X

involved_in

GO:0032259: methylation

ECO:0000305: curator inference used in manual assertion

GO:0008168

P

Seeded From UniProt

complete

YEAST:YG5X

enables

GO:0008168: methyltransferase activity

ECO:0000250: sequence similarity evidence used in manual assertion

UniProtKB:Q12314

F

Seeded From UniProt

complete

YEAST:YM91

involved_in

GO:0032259: methylation

ECO:0000305: curator inference used in manual assertion

GO:0008168

P

Seeded From UniProt

complete

YEAST:YM91

enables

GO:0008168: methyltransferase activity

ECO:0000250: sequence similarity evidence used in manual assertion

UniProtKB:Q12314

F

Seeded From UniProt

complete

YEAST:SFM1

enables

GO:0035241: protein-arginine omega-N monomethyltransferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:SFM1

involved_in

GO:0018216: peptidyl-arginine methylation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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