Abstract
Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / chemistry
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Amino Acid Sequence
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Arginine / chemistry*
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Arginine / metabolism*
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Blotting, Western
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Chromatography, Liquid
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DEAD-box RNA Helicases / chemistry
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DEAD-box RNA Helicases / genetics
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DEAD-box RNA Helicases / metabolism
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Methylation
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Molecular Sequence Data
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Protein Interaction Maps
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Protein Processing, Post-Translational
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Protein-Arginine N-Methyltransferases / chemistry
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Protein-Arginine N-Methyltransferases / genetics
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Protein-Arginine N-Methyltransferases / metabolism
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Proteome / chemistry
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Proteome / genetics
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Proteome / metabolism
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Proteomics
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Ribonucleoprotein, U1 Small Nuclear / chemistry
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Ribonucleoprotein, U1 Small Nuclear / genetics
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Ribonucleoprotein, U1 Small Nuclear / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Substrate Specificity
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Tandem Mass Spectrometry
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Two-Hybrid System Techniques
Substances
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Adaptor Proteins, Signal Transducing
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Air2 protein, S cerevisiae
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Gbp2 protein, S cerevisiae
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NPL3 protein, S cerevisiae
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Nuclear Proteins
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Proteome
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RNA-Binding Proteins
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Recombinant Proteins
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Repressor Proteins
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Ribonucleoprotein, U1 Small Nuclear
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SNP1 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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YRA1 protein, S cerevisiae
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Arginine
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HMT1 protein, S cerevisiae
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Protein-Arginine N-Methyltransferases
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DED1 protein, S cerevisiae
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DEAD-box RNA Helicases