Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

Editor's Choice article: Free access to Full TextInternational audienceMethylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function

Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0095512International audiencePost-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology

Structural basis for oligomerization of auxin transcriptional regulators.

Supplementary Information Supplementary Figures 1-3 and Supplementary Tables 1-2International audienceThe plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin

Main features of the identified chloroplastic methylproteins and methylation sites.

<p>(a) Curated subcellular/subplastidial location of methylproteins (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone-0095512-t002" target="_blank">Table 2</a>). (b) Functional categories of chloroplastic methylproteins. Annotated proteins from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone-0095512-t002" target="_blank">Table 2</a> were grouped to create categories ‘metabolism (other than photosynthesis)’ and ‘protein synthesis and targeting’. (c) Amino acid motif surrounding Lys methylation sites was created using WebLogo (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>). (d) Positioning of the Lys395 methylation site on the 3D-structure model of fructose bisphosphate aldolase (FBA1, At2g21330). The model was generated with the Phyre² server using the 3D structure of aldolase from rabbit muscle (PDB entry 1ZAI) and imaged with the PyMOL software.</p

Biochemical validation of methylproteins through <i>in vitro</i> methylation assays using stroma from Arabidopsis chloroplasts.

<p>Methylation assays were done in the presence of 20 µg purified recombinant targets (FBA2, GAPA1, PRPL11, or ATP-B), 80 µg stroma from Arabidopsis Col-0 chloroplasts, 20 µM [methyl-³H]-AdoMet and 100 nM <i>S</i>-adenosylhomocysteine hydrolase. After incubation at 30°C for 1 to 2 hours, assays were split into two equals parts and radioactivity incorporated into proteins was counted by liquid scintillation (panel a) and analyzed by phosphorimaging (panels b–c). In panels (a–c), the symbol Ø means that no recombinant protein was added to the stromal extract. Purified recombinant substrates are indicated by asterisks: ATP-B, 54 kDa; FBA2, 40 kDa; GAPA1, 38 kDa; PRPL11, 18 kDa (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512.s003" target="_blank">Fig. S3</a>). Activities with the FBA2, PRPL11 and ATP-B substrates were strictly dependent on the addition of stroma. Values are mean ± SD of two to six independent determinations.</p

Inventory of Arabidopsis PRMT and PKMTs known or predicted to be targeted to chloroplasts.

<p>Potential protein Lys/Arg methyltransferases found in the Arabidopsis genome were analyzed for their predicted subcellular localization by using SUBA3, AtSubP, and ChloroP 1.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Tanz1" target="_blank">[44]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Emanuelsson1" target="_blank">[46]</a>. Proteins with a confident prediction for plastid targeting are shown. Data extracted from the predictors are: for SUBA3, consensus localization and number of predictors (out of 18) indicating a plastid targeting; for AtSubP, prediction using the best hybrid-based classifier (AA+PSSM+N-Center-C+PSI-BLAST); for ChloroP 1.1, presence of a predictable chloroplast transit peptide (cTP). Experimental localizations using GFP-tagging or mass spectrometry (MS) have been reported in the indicated references.</p

Filtering pipeline for the identification of methylpeptides in the AT_CHLORO datasets.

<p>(a) Following the initial database search and parsing of results, the workflow is composed of six steps combining automatic filtering and expert examination of spectra. 1/selection of methyl peptide-spectrum matches (PSMs) with score ≥ 50; 2/manual inspection to assess spectral quality; 3/selection of methyl-PSMs (score <50) with identical methylation sites; 4/removal of sites with only one PSM; 5/removal of ambiguities due to amino acid substitutions; 6/removal of ambiguities due to trimethylation vs. acetylation. The final AT_CHLORO_Me list consisted in methylpeptides validated from the mass spectrometry aspect. Peptide counts represent the number of distinct peptide sequences compiled from a larger number of PSMs. (b) Score distribution of methyl-PSMs matching a reverse or true (forward) Arabidopsis protein library. The threshold score of 50 in step 1 was selected from these distributions. (c) Illustration of the diversity among PSMs that identify a methylation site (step 3). In this example (FBA1 protein, At2g21330), three PSMs point out a trimethyl-Lys with two distinct overlapping peptide sequences and various modification patterns of a Met residue (mono or dioxidized).</p

Immunodetection of Lys- and Arg-methylated proteins in chloroplast stroma and membranes subfractions.

<p>Chloroplasts from Arabidopsis, spinach and pea leaves were purified using Percoll gradients and fractionated into soluble (stroma) and membrane (thylakoids and envelope) fractions. Fifty µg of proteins were analyzed by SDS-PAGE (Coomassie blue staining) and immunoblotting with antibodies against trimethyl-Lys (anti-K_me3), mono- and dimethyl-Lys (anti-K_me1/2), or mono- and dimethyl-Arg (anti-R_me1/2). The major polypeptides detected by the anti-K_me3 antibodies are RbcL (*) and fructose bisphosphate aldolases (**) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Mininno1" target="_blank">[30]</a>.</p

Main properties of Lys and Arg methylproteins and methylation sites from the AT_CHLORO_Me inventory.

<p>Protein accession, symbol and description were from TAIR, Uniprot and PPDB databases. Functional annotation was done according to MapMan bins and sub-bins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Thimm1" target="_blank">[67]</a>. Subcellular and subplastidial location was curated using data from AT_CHLORO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Ferro1" target="_blank">[36]</a>, PPDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Sun1" target="_blank">[62]</a> and dedicated studies. The trimethylated vs. acetylated status of Lys residues was confirmed (c) or remained ambiguous (a). Modeling of 3D-structures was done using the Phyre² server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Kelley1" target="_blank">[52]</a>. NA, not available (the protein structure could not be modeled or the model does not cover the methylation site).</p