Jason K. K. Low

Protein arginine methylation in Saccharomyces cerevisiae

Recent research has implicated arginine methylation as a major regulator of cellular processes, including transcription, translation, nucleocytoplasmic transport, signalling, DNA repair, RNA processing and splicing. Arginine methylation is evolutionarily conserved, and it is now thought that it may rival other post‐translational modifications such as phosphorylation in terms of its occurrence in the proteome. In addition, multiple recent examples demonstrate an exciting new theme: the interplay between methylation and other post‐translational modifications such as phosphorylation. In this review, we summarize our current understanding of arginine methylation and the recent advances made, with a focus on the lower eukaryote Saccharomyces cerevisiae. We cover the types of methylated proteins, their responsible methyltransferases, where and how the effects of arginine methylation are seen in the cell, and, finally, discuss the conservation of the biological function of methylarginines between S. cerevisiae and mammals.

Analysis of the Proteome of Saccharomyces cerevisiae for Methylarginine

Arginine methylation is a post-translational modification that has been implicated in a plethora of cellular processes. In the present manuscript, using two antimethylarginine antibodies and combinatorial deletion mutants of arginine methyltransferases, we found evidence of widespread arginine methylation in the Saccharomyces cerevisiae proteome. Immunoprecipitation was used for enrichment of methylarginine-containing proteins, which were identified via tandem mass spectrometry. From this, we identified a total of 90 proteins, of which 5 were previously known to be methylated. The proteins identified were involved in known methylarginine-associated biological functions such as RNA processing, nuclear transport, carbohydrate metabolic process, GMP biosynthetic process and protein folding. Through in vivo methylation by the incorporation of [3H]-methyl groups, we validated the methylation of 7 proteins (Ded1, Imd4, Lhp1, Nop1, Cdc11, Gus1, Pob3). By LC-MS/MS, we then confirmed a total of 15 novel methylarginine sites on 5 proteins (Ded1, Lhp1, Nop1, Pab1, and Ugp1). By examination of methylation on proteins from the triple knockout of methyltransferases Hmt1, Hsl7, Rmt2, we present evidence for the existence of additional unidentified arginine methyltransferases in the Saccharomyces cerevisiae proteome.

Interactions affected by arginine methylation in the yeast protein-protein interaction network

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.

Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners

RNase E is an essential enzyme that catalyses RNA processing. Microdomains which mediate interactions between RNase E and other members of the degradosome have been defined. To further elucidate the role of these microdomains in molecular interactions, we studied RNase E from Vibrio angustum S14. Protein sequence analysis revealed that its C-terminal half is less conserved and structured than its N-terminal half. Within this structural disorder, however, exist five small regions of predicted structural propensity. Four are similar to interaction-mediating microdomains identified in other RNase E proteins; the fifth did not correspond to any known functional motif. The function of the V. angustum S14 enolase-binding microdomain was confirmed using bacterial two-hybrid analysis, demonstrating the conserved function of this microdomain for the first time in a species other than Escherichia coli. Further, PNPase in V. angustum S14 was shown to interact with the last 80 amino acids of the C-terminal region of RNase E. This raises the possibility that PNPase interacts with the small ordered region at residues 1026-1041. The role of RNase E as a hub protein and the implications of microdomain-mediated interactions in relation to specificity and function are discussed.

The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose).

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.

Analysis of the RNA degradosome complex in Vibrio angustum S14.

The RNA degradosome is built on the C‐terminal half of ribonuclease E (RNase E) which shows high sequence variation, even amongst closely related species. This is intriguing given its central role in RNA processing and mRNA decay. Previously, we have identified RhlB (ATP‐dependent DEAD‐box RNA helicase)‐binding, PNPase (polynucleotide phosphorylase)‐binding and enolase‐binding microdomains in the C‐terminal half of Vibrio angustum S14 RNase E, and have shown through two‐hybrid analysis that the PNPase and enolase‐binding microdomains have protein‐binding function. We suggest that the RhlB‐binding, enolase‐binding and PNPase‐binding microdomains may be interchangeable between Escherichia coli and V. angustum S14 RNase E. In this study, we used two‐hybrid techniques to show that the putative RhlB‐binding microdomain can bind RhlB. We then used Blue Native‐PAGE, a technique commonly employed in the separation of membrane protein complexes, in a study of the first of its kind to purify and analyse the RNA degradosome. We showed that the V. angustum S14 RNA degradosome comprises at least RNase E, RhlB, enolase and PNPase. Based on the results obtained from sequence analyses, two‐hybrid assays, immunoprecipitation experiments and Blue Native‐PAGE separation, we present a model for the V. angustum S14 RNA degradosome. We discuss the benefits of using Blue Native‐PAGE as a tool to analyse the RNA degradosome, and the implications of microdomain‐mediated RNase E interaction specificity.

Stoichiometry of Saccharomyces cerevisiae Lysine Methylation: Insights into Non-histone Protein Lysine Methyltransferase Activity

Post-translational lysine methylation is well established as a regulator of histone activity; however, it is emerging that these modifications are also likely to play extensive roles outside of the histone code. Here we obtain new insights into non-histone lysine methylation and protein lysine methyltransferase (PKMT) activity by elucidating absolute stoichiometries of lysine methylation, using mass spectrometry and absolute quantification (AQUA), in wild-type and 5 PKMT gene deletion strains of Saccharomyces cerevisiae. By analyzing 8 sites of methylation in 3 non-histone proteins, elongation factor 1-α (EF1α), elongation factor 2 (EF2), and 60S ribosomal protein L42-A/B (Rpl42ab), we find that production of preferred methylation states on individual lysine residues is commonplace and likely occurs through processive PKMT activity, Class I PKMTs can be associated with processive methylation, lysine residues are selectively methylated by specific PKMTs, and lysine methylation exists over a broad range of stoichiometries. Together these findings suggest that specific sites and forms of lysine methylation may play specialized roles in the regulation of non-histone protein activity. We also uncover new relationships between two proteins previously characterized as PKMTs, SEE1 and EFM1, in EF1α methylation and show that past characterizations of EFM1 as having direct PKMT activity may require reinterpretation.

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex.

Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.

A conditional two-hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post-translational modification

The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two‐hybrid (C2H) system, in which bait and prey proteins can be co‐expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co‐expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S‐tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).