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      Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

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          Abstract

          Post-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.

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          Most cited references59

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          Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.

          Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
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            Histone methylation in higher plants.

            Histone methylation plays a fundamental role in regulating diverse developmental processes and is also involved in silencing repetitive sequences in order to maintain genome stability. The methylation marks are written on lysine or arginine by distinct enzymes, namely, histone lysine methyltransferases (HKMTs) or protein arginine methyltransferases (PRMTs). Once established, the methylation marks are specifically recognized by the proteins that act as readers and are interpreted into specific biological outcomes. Histone methylation status is dynamic; methylation marks can be removed by eraser enzymes, the histone demethylases (HDMs). The proteins responsible for writing, reading, and erasing the methylation marks are known mostly in animals. During the past several years, a growing body of literature has demonstrated the impact of histone methylation on genome management, transcriptional regulation, and development in plants. The aim of this review is to summarize the biochemical, genetic, and molecular action of histone methylation in two plants, the dicot Arabidopsis and the monocot rice.
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              Empirical analysis of transcriptional activity in the Arabidopsis genome.

              Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                18 April 2014
                : 9
                : 4
                : e95512
                Affiliations
                [1 ]Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, Grenoble, France
                [2 ]CNRS, UMR5168, Grenoble, France
                [3 ]CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, Grenoble, France
                [4 ]INRA, USC1359, Grenoble, France
                [5 ]Université Grenoble Alpes, Grenoble, France
                [6 ]CEA, iRTSV, Biologie à Grande Echelle, Grenoble, France
                [7 ]INSERM, U1038, Grenoble, France
                Universität Stuttgart, Germany
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CA MT MF SR. Performed the experiments: CA MT M. Mininno SB AG SM M. Mazzoleni OG JML MF SR. Analyzed the data: CA MT MF SR. Wrote the paper: CA MT MF SR.

                Article
                PONE-D-14-07634
                10.1371/journal.pone.0095512
                3991674
                24748391
                339d88e0-02a0-4e91-9eab-937301e15da9
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 February 2014
                : 27 March 2014
                Page count
                Pages: 13
                Funding
                This study received financial support from the Department of Plant Biology INRA, the Cluster 9 of the Région Rhône-Alpes, the French National Research Agency (ANR-2010-BLAN-1610-01 Chloro-Pro), and the Proteomics French Infrastructure (ProFI; ANR-10-INBS-08-01). This work was supported by a PhD fellowship (to M. Mininno) funded by the French Ministry of Higher Education and Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Plant Biochemistry
                Plant Science

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