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

Author

Claude Alban, Marianne Tardif, Morgane Mininno, Sabine Brugière, Annabelle Gilgen, Sheng Ma, Meryl Mazzoleni, Océane Gigarel, Jacqueline Martin-Laffon, Myriam Ferro, and Stéphane Ravanel

Subjects
Medicine, Science
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.

Published
2014
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2. Supplementary Table from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Author

Nicolas Reynoird, Nima Mosammaparast, Pawel K. Mazur, Pierre Hainaut, Michiel Vermeulen, Yohann Couté, Pascal W.T.C. Jansen, Marianne Tardif, Rebecca Rodell, Jessica Vayr, Ana Morales Benitez, Florent Chuffart, Alexandre G. Casanova, Joshua R. Brickner, Ning Tsao, Tanveer Ahmad, Clement Oyeniran, Gael S. Roth, Simone Hausmann, and Valentina Lukinović

Abstract

Supplementary Table from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Published
2023

3. Data from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Author

Nicolas Reynoird, Nima Mosammaparast, Pawel K. Mazur, Pierre Hainaut, Michiel Vermeulen, Yohann Couté, Pascal W.T.C. Jansen, Marianne Tardif, Rebecca Rodell, Jessica Vayr, Ana Morales Benitez, Florent Chuffart, Alexandre G. Casanova, Joshua R. Brickner, Ning Tsao, Tanveer Ahmad, Clement Oyeniran, Gael S. Roth, Simone Hausmann, and Valentina Lukinović

Abstract

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy.Significance:SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy.This article is highlighted in the In This Issue feature, p. 2007

Published
2023

4. Supplementary Data from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Author

Nicolas Reynoird, Nima Mosammaparast, Pawel K. Mazur, Pierre Hainaut, Michiel Vermeulen, Yohann Couté, Pascal W.T.C. Jansen, Marianne Tardif, Rebecca Rodell, Jessica Vayr, Ana Morales Benitez, Florent Chuffart, Alexandre G. Casanova, Joshua R. Brickner, Ning Tsao, Tanveer Ahmad, Clement Oyeniran, Gael S. Roth, Simone Hausmann, and Valentina Lukinović

Abstract

Supplementary Data from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Published
2023

5. Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism

Author

Andreas P.M. Weber, Phillip Westhoff, Marianne Tardif, Denis Falconet, Erika Guglielmino, Dagmar Lyska, Benoit Gallet, Giovanni Finazzi, Gilles Curien, Clément Hallopeau, Michele Carone, Davide Dal Bo, Claire Remacle, Johan Decelle, Sabine Brugière, Myriam Ferro, Janina Janetzko, Light Photosynthesis & Metabolism (Photosynthesis), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ)-Universität zu Köln = University of Cologne, Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Photosymbiose, Institut de biologie structurale (IBS - UMR 5075), LIPID, Université de Liège, ARC grant (DARKMET proposal) for Concerted Research Actions (17/21-08), financed by the French Community of Belgium (Wallonia-Brussels Federation)., Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111, ANR-17-CE05-0029,MoMix,Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria(2017), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), European Project: 833184, ChloroMito, Martin-Laffon, Jacqueline, Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria - - MoMix2017 - ANR-17-CE05-0029 - AAPG2017 - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, Chloroplast and Mitochondria interactions for microalgal acclimation - ChloroMito - 833184 - INCOMING, Universität zu Köln-Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ), and InBios/Phytosystems Research Unit, University of Liege

Subjects
Proteomics, 0106 biological sciences, 0301 basic medicine, photorespiration, Physiology, Heterotroph, Plant Science, Photosynthesis, 7. Clean energy, 01 natural sciences, Galdieria sulphuraria, Extremophiles, 03 medical and health sciences, mixotrophy, Total inorganic carbon, Botany, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, red algae, photosynthesis, biology, Chemistry, Research, RuBisCO, Heterotrophic Processes, Carbon Dioxide, Full Papers, 15. Life on land, Carbon, 030104 developmental biology, Rhodophyta, biology.protein, Photorespiration, Energy source, Mixotroph, 010606 plant biology & botany
Abstract

Summary Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon.Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2.Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (Rubisco) and decreasing photorespiration.We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

Published
2021

6. SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Author

Valentina Lukinović, Simone Hausmann, Gael S. Roth, Clement Oyeniran, Tanveer Ahmad, Ning Tsao, Joshua R. Brickner, Alexandre G. Casanova, Florent Chuffart, Ana Morales Benitez, Jessica Vayr, Rebecca Rodell, Marianne Tardif, Pascal W.T.C. Jansen, Yohann Couté, Michiel Vermeulen, Pierre Hainaut, Pawel K. Mazur, Nima Mosammaparast, Nicolas Reynoird, Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-16-CE11-0018,S3S,Signalisation physiologique et pathologique de la lysine méthyltransférase SMYD3(2016), and Reynoird, Nicolas

Subjects
MESH: Cell Nucleus, MESH: RNA Processing, Post-Transcriptional, Lung Neoplasms, MESH: DNA Helicases, MESH: AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase, [SDV.CAN]Life Sciences [q-bio]/Cancer, Methylation, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, MESH: Methylation, MESH: DNA Methylation, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, methylation signaling, MESH: Neoplasms, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphorylation, Molecular Biology, alkylation, E3 ligase, SMYD3, MESH: Humans, MESH: R-Loop Structures, MESH: Transcription, Genetic, SCLC, Histone-Lysine N-Methyltransferase, ASCC, MESH: RNA, Neoplasm, Small Cell Lung Carcinoma, DNA-Binding Proteins, Oncology, RNF113A, MESH: HEK293 Cells, MESH: HeLa Cells, RNA methylation, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, MESH: Ubiquitination, transcription, Protein Processing, Post-Translational, MESH: Nuclear Proteins, genome stability, MESH: Spliceosomes, MESH: DNA-Binding Proteins
Abstract

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. Significance: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007

Published
2022

7. Quantitative proteomic analyses reveal the impact of nitrogen starvation on the proteome of the model diatom Phaeodactylum tricornutum

Author

Josselin Lupette, Marianne Tardif, Sabine Brugière, Yohann Couté, Juliette Salvaing, Eric Maréchal, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Laboratoire de biogenèse membranaire (LBM), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), Salvaing, Juliette, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, and IDEX UGA - - UGA2015 - ANR-15-IDEX-0002 - IDEX - VALID

Subjects
Diatoms, Proteomics, Proteome, Nitrogen, Nitrogen starvation, Phaeodactylum, Fatty Acids, Diatom, Biochemistry, Carbon, Proteome remodelling, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Quantitative proteomics, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Molecular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Triglycerides
Abstract

Data Paper; International audience; Diatoms are one of the largest groups in phytoplankton biodiversity. Understanding their response to nitrogen variations, present from micromolar to near-zero levels in oceans and fresh waters, is essential to comprehend their ecological success. Nitrogen starvation is used in biotechnological processes, to trigger the remodeling of carbon metabolism in the direction of fatty acids and triacylglycerol synthesis. We evaluated whole proteome changes in Phaeodactylum tricornutum after 7 days of cultivation with 5.5-mM nitrate (+N) or without any nitrogen source (-N). On a total of 3768 proteins detected in biological replicates, our analysis pointed to 384 differentially abundant proteins (DAP). Analysis of proteins of lower abundance in -N revealed an arrest of amino acid and protein syntheses, a remodeling of nitrogen metabolism, and a decrease of the proteasome abundance suggesting a decline in unselective whole-proteome decay. Analysis of proteins of higher abundance revealed the setting up of a general nitrogen scavenging system dependent on deaminases. The increase of a plastid palmitoyl-ACP desaturase appeared as a hallmark of carbon metabolism rewiring in the direction of fatty acid and triacylglycerol synthesis. This dataset is also valuable to select gene candidates for improved biotechnological properties.

Published
2022

8. Unravelling hidden components of the chloroplast envelope proteome: opportunities and limits of better MS sensitivity

Author

Daniel Salvi, Norbert Rolland, Lucas Moyet, Imen Bouchnak, Marianne Tardif, Sabine Brugière, Marcel Kuntz, Sophie Le Gall, Dynamique du protéome et biogenèse du chloroplaste (ChloroGenesis), Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), French National Research Agency (ANR) ANR-15-IDEX-02 ANR-10-LABX-49-01 ANR-10-INBS-08, Institut National de la Recherche Agronomique (INRA), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-15-IDEX-02,DATA@UGA,Grenoble Alpes Data Institute(2016), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)

Subjects
Cell Extracts, Chloroplasts, Proteome, Arabidopsis thaliana, Arabidopsis, Plant Biology, Cellular organelles, Biochemistry, Chloroplast membrane, Subcellular Separation, Chloroplast, Cell fractionation, Analytical Chemistry, 03 medical and health sciences, Chloroplast Proteins, Organelle, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Plastid, Databases, Protein, Molecular Biology, 030304 developmental biology, 0303 health sciences, Subcellular analysis, Mass spectrometry, Chemistry, Arabidopsis Proteins, Subcellular localization, Research, 030302 biochemistry & molecular biology, Membrane Proteins, food and beverages, Proteins, Intracellular Membranes, Chloroplast envelope, Biogenesis, Subcellular Fractions
Abstract

By quantitatively comparing the proteomes of total leaf (crude cell extract) from Arabidopsis and purified chloroplast envelope fractions, this study makes available a novel parameter (calculated Enrichment Factor) for each putative envelope protein. This parameter provides important information to enable the more confident identification of genuine envelope components, distinguishing them from contaminants from other cellular/chloroplast compartments., Graphical Abstract Highlights Identification of previously undetected chloroplast envelope proteins. Up to date manual annotation of genuine (or shared) envelope components. New hypotheses for localizations, functions, interactions among cell compartments. A new resource of significant value to the broader plant science community., The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

Published
2019

9. The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

Author

Khawla Seddiki, Juliette Salvaing, Fabrice Rébeillé, Juliette Jouhet, Denis Falconet, Sabine Brugière, Yohann Couté, Eric Maréchal, Marcel Kuntz, Pierre-Henri Jouneau, Hubert Schaller, Antoine Jaussaud, Josselin Lupette, Marianne Tardif, Christian Morabito, Jean-Luc Putaux, LIPID, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de biologie moléculaire des plantes (IBMP), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Dynamique du protéome et biogenèse du chloroplaste (ChloroGenesis), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Flagship program from the CEA High Commissioner, French National Research Agency (ANR) ANR-11-BTBR-0008 ANR-15-IDEX-02 ANR-10-INBS-08, ANR-11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-BTBR-0008/11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-02,DATA@UGA,Grenoble Alpes Data Institute(2016), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Jouneau, Pierre-Henri, Biotech - Bioressources - Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques - - OCEANOMICS2011 - ANR-11-BTBR-0008 - BTBR - VALID, IDEX UGA - - UGA2015 - ANR-15-IDEX-0002 - IDEX - VALID, and Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID

Subjects
0106 biological sciences, 0301 basic medicine, Betaine lipid, Phaeodactylum, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], 01 natural sciences, Ribosome, Triacylglycerol, [SDV.BV.BOT] Life Sciences [q-bio]/Vegetal Biology/Botanics, Histones, 03 medical and health sciences, Lipid droplet, Protein biosynthesis, ERAD pathway, Phaeodactylum tricornutum, Plastid, Secondary plastid, ComputingMilieux_MISCELLANEOUS, Diatoms, biology, Chemistry, Endoplasmic reticulum, fungi, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Lipid droplets, Carotenoids, 030104 developmental biology, Biochemistry, Proteome, Agronomy and Crop Science, 010606 plant biology & botany
Abstract

International audience; Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previously validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl-beta-alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of beta-carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function includes protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.

Published
2019

10. Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii

Author

Yuanxue Liang, Sabine Brugière, Gilles Peltier, Bertrand Legeret, Marianne Tardif, Yonghua Li-Beisson, Brian Jones, Pascaline Auroy, Fred Beisson, Stéphan Cuiné, Hugh D. Goold, Hélène Javot, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Bioénergie et Microalgues (EBM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Signalisation de l'Adaptation des Végétaux à l'Environnement (SAVE), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Plant Environmental Physiology and Stress Signaling (PEPSS), Synthèse et étude de systèmes à intêret biologique (SEESIB), and Centre National de la Recherche Scientifique (CNRS)-Université Blaise Pascal - Clermont-Ferrand 2 (UBP)

Subjects
2. Zero hunger, 0301 basic medicine, biology, Physiology, [SDV]Life Sciences [q-bio], Endoplasmic reticulum, Photobioreactor, Chlamydomonas reinhardtii, Biomass, Lipid metabolism, Plant Science, biology.organism_classification, Photosynthesis, 7. Clean energy, Polar membrane, 03 medical and health sciences, 030104 developmental biology, Biochemistry, Lipid droplet, Genetics, Biophysics, ComputingMilieux_MISCELLANEOUS
Abstract

Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

Published
2016

11. Optical tweezing using tunable optical lattices along a few-mode silicon waveguide

Author

F. de Fornel, Emmanuel Picard, Jean-Baptiste Jager, Benoit Cluzel, Marianne Tardif, Christophe Pin, Emmanuel Hadji, Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Bourgogne (UB)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Silicon Nanoelectronics Photonics and Structures (SiNaps), PHotonique, ELectronique et Ingénierie QuantiqueS (PHELIQS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service de Physique des Matériaux et Microstructures (SP2M - UMR 9002), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ANR-11-LABX-0001,ACTION,Systèmes intelligents intégrés au cœur de la matière(2011), Laboratoire Interdisciplinaire Carnot de Bourgogne [Dijon] (LICB), and Université de Bourgogne (UB)-Université de Technologie de Belfort-Montbeliard (UTBM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Silicon, Materials science, Optical Tweezers, Biomedical Engineering, Nanophotonics, Holography, chemistry.chemical_element, Physics::Optics, Bioengineering, 02 engineering and technology, Trapping, Models, Biological, 01 natural sciences, Biochemistry, Waveguide (optics), law.invention, 010309 optics, law, Lab-On-A-Chip Devices, 0103 physical sciences, Tweezers, Light beam, Particle Size, business.industry, General Chemistry, 021001 nanoscience & nanotechnology, Microspheres, [SPI.ELEC]Engineering Sciences [physics]/Electromagnetism, chemistry, Optical tweezers, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, Nanoparticles, Optoelectronics, 0210 nano-technology, business
Abstract

International audience; Fourteen years ago, optical lattices and holographic tweezers were considered as a revolution, allowing for trapping andmanipulating multiple particles at the same time using laser light. Since then, near-field optical forces have arousedtremendous interest as they enable efficient trapping of a wide range of objects, from living cells to atoms, in integrateddevices. Yet, handling at will multiple objects using a guided light beam remains a challenging task for current on-chipoptical trapping techniques. We demonstrate here on-chip optical trapping of dielectric microbeads and bacteria usingone-dimensional optical lattices created by near-field mode beating along a few-mode silicon nanophotonic waveguide.This approach allows not only for trapping a large number of particles in periodic trap arrays with various geometries, butalso for manipulating them via diverse transport and repositioning techniques. Near-field mode-beating optical latticesmay be readily implemented in lab-on-a-chip devices, addressing numerous scientific fields ranging from bio-analysis tonanoparticle processing.

Published
2018

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

Author

Morgane Mininno, Jacqueline Martin-Laffon, Sabine Brugière, Claude Alban, Mélanie Leroux, Marianne Tardif, Sylvie Figuet, Meryl Mazzoleni, Stéphane Ravanel, Annabelle Gilgen, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), INRA (Department of Plant Biology ), ANR (Proteomics French Infrastructure) [ANR-10-INBS-08-01/10-INBS-0008], Université Grenoble Alpes, ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)

Subjects
Ribosomal Proteins, Chloroplasts, Methyltransferase, Arabidopsis thaliana, Physiology, Molecular Sequence Data, Mutant, Arabidopsis, Germination, Plant Science, Dual targeting, Chloroplast, Methylation, Ribosome, Mitochondrial Proteins, Ribosomal protein, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Photosynthesis, Mitochondrion, Binding site, Phylogeny, biology, Arabidopsis Proteins, Genetic Complementation Test, Methyltransferases, Plant, Cell Biology, General Medicine, biology.organism_classification, Molecular biology, Mitochondria, Protein Transport, Biochemistry, Protein Biosynthesis, Acetyltransferase, Mutation, Peptides, Ribosomes, Subcellular Fractions, Post-translational modifications
Abstract

Editor's Choice article: Free access to Full Text; International audience; Methylation 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.

Published
2015

13. ChloroKB:a web-application for the integration of knowledge related to chloroplast metabolic network

Author

Daphné Seigneurin-Berny, Sylvain Bournais, Christophe Bruley, Claude Alban, Gilles Curien, Marianne Tardif, Marcel Kuntz, Stéphane Ravanel, Pauline Gloaguen, Michel Matringe, Myriam Ferro, Yves Vandenbrouck, Norbert Rolland, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Biologie, Informatique et Mathématiques, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR (GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology) [ANR-10-LABEX-04], ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and ANR–10–LABEX–04 ,GRAL,Labex

Subjects
0301 basic medicine, Chloroplasts, Traceability, Arabidopsis thaliana, Physiology, Knowledge Bases, [SDV]Life Sciences [q-bio], Arabidopsis, Metabolic network, Context (language use), Plant Science, Computational biology, Bioinformatics, Chloroplast, Metabolic engineering, Knowledge base, 03 medical and health sciences, Software, Genetics, Web application, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Internet, biology, business.industry, Plant, Breakthrough Technologies, biology.organism_classification, Biocuration, Biological network visualization, ComputingMethodologies_PATTERNRECOGNITION, 030104 developmental biology, Metabolism, Metabolic pathways, business, Metabolic Networks and Pathways, Subcellular Fractions
Abstract

International audience; Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers.

Published
2017

14. Single-cell bacterium identification with a SOI optical microcavity

Author

Emmanuel Picard, Emmanuel Hadji, Marianne Tardif, D. Peyrade, K. Uchiyamada, Pierre Marcoux, Jean-Baptiste Jager, Laboratoire des technologies de la microélectronique (LTM ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Silicon Nanoelectronics Photonics and Structures (SiNaps), PHotonique, ELectronique et Ingénierie QuantiqueS (PHELIQS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Service de Physique des Matériaux et Microstructures (SP2M - UMR 9002), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Département d'Architectures, Conception et Logiciels Embarqués-LETI (DACLE-LETI), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Graduate School of Pure and Applied Sciences, University of Tsukuba, Université de Tsukuba = University of Tsukuba, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Materials science, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Physics and Astronomy (miscellaneous), genetic structures, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Nanophotonics, Silicon on insulator, Physics::Optics, 02 engineering and technology, 01 natural sciences, law.invention, Quantitative Biology::Cell Behavior, 010309 optics, Quantitative Biology::Subcellular Processes, Optics, law, 0103 physical sciences, Tweezers, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Photonic crystal, Condensed Matter::Quantum Gases, Physics::Biological Physics, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], business.industry, 021001 nanoscience & nanotechnology, Optical microcavity, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, eye diseases, Wavelength, Optical tweezers, Optical cavity, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, Optoelectronics, sense organs, 0210 nano-technology, business
Abstract

International audience; Photonic crystals and microcavities can act as on-chip nano-optical tweezers for identification or manipulation of biological objects. So far, virus and bacteria optical trapping has been achieved, their presence in the vicinity of the optical resonator being deduced from a shift of the resonant wavelength. Here we show that bacteria can not only be trapped but identified as well if the trapping time is long enough. A silicon on insulator microcavity with a properly tuned quality factor allows to achieve enhanced trapping times. By combining spatial and temporal observation of bacteria-cavity interaction, the optical identification of three different kinds of bacteria is demonstrated.

Published
2016

15. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

Author

Océane Gigarel, Sabine Brugière, Jacqueline Martin-Laffon, Marianne Tardif, Morgane Mininno, Stéphane Ravanel, Claude Alban, Sheng Ma, Olivier Bastien, Laboratoire de Physiologie Cellulaire & Végétale [Grenoble] (UGA), Université Grenoble Alpes (UGA), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Department of Plant Biology and Breeding from the French National Institute for Agricultural Research (AAP BAP2013 INRA_Calvin Cycle-Me) and by the Proteomics French Infrastructure (ANR-10-INBS-08-01)., ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), French National Institute for Agricultural Research (AAP BAP2013 INRA_Calvin Cycle-Me), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Models, Molecular, Methyltransferase, Chloroplasts, Arabidopsis thaliana, Protein subunit, Ribulose-Bisphosphate Carboxylase, [SDV]Life Sciences [q-bio], Protein domain, Amino Acid Motifs, Plant Science, Biology, Substrate Specificity, Evolution, Molecular, 03 medical and health sciences, Protein Domains, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, SET-domain protein methyltransferase, green lineage, Site-directed mutagenesis, Rosaceae, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Pisum sativum, Aldehyde-Lyases, Genetics, molecular evolution, chimeric enzyme, Chlamydomonas rheinhardtii, RuBisCO, Carbon fixation, fungi, food and beverages, Fabaceae, Plant, Methylation, Chloroplast, Cucurbitaceae, 030104 developmental biology, Biochemistry, biology.protein, Mutagenesis, Site-Directed, methylation, site-directed mutagenesis, Site directed mutagenesis
Abstract

International audience; Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Published
2016

16. AtMic60 Is Involved in Plant Mitochondria Lipid Trafficking and Is Part of a Large Complex

Author

Denis Falconet, Michael R. Wozny, Juliette Jouhet, William A. Prinz, Maryse A. Block, Sabine Brugière, Morgane Michaud, Eric Maréchal, Marianne Tardif, Valérie Gros, Myriam Ferro, Jaideep Mathur, Alexandre Toulmay, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Department of Molecular and Cellular Biology, University of Guelph, EMBO (ASTF-638-2014), ANR-12-JSV2-0001,ChloroMitoLipid,Export de galactolipides des chloroplastes vers les mitochondries(2012), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR–10–LABEX–04 ,GRAL,Labex, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Arabidopsis, TIM/TOM complex, Mitochondrial Membrane Transport Proteins, Galactoglycerolipids, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Mitochondrial membrane transport protein, chemistry.chemical_compound, Organelle, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Digalactosyldiacylglycerol, Phosphate starvation, Membrane biogenesis, Phospholipids, Phosphatidylethanolamine, biology, Agricultural and Biological Sciences(all), Biochemistry, Genetics and Molecular Biology(all), Arabidopsis Proteins, Endoplasmic reticulum, Lipids transfer, Plant, Lipid Metabolism, Membrane contact site, Cell biology, Mitochondria, Protein Transport, 030104 developmental biology, Membrane, Biochemistry, chemistry, Lipoprotein complex, biology.protein, General Agricultural and Biological Sciences
Abstract

International audience; The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.

Published
2016

17. A Proteomic Survey of Chlamydomonas reinhardtii Mitochondria Sheds New Light on the Metabolic Plasticity of the Organelle and on the Nature of the -Proteobacterial Mitochondrial Ancestor

Author

Brigitte Gontero, Oliver Deusch, Jérôme Garin, Marianne Tardif, Norbert Rolland, Sabine Brugière, Tal Dagan, Jacques Joyard, William Martin, Annie Adrait, Robert van Lis, Lauriane Kuhn, Ariane Atteia, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institute of Botany, Heinrich-Heine-Universität Düsseldorf [Düsseldorf], Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
0106 biological sciences, carbon metabolism, Algae, proteome, Chlamydomonas reinhardtii, nucleotide metabolism, Mitochondrion, phylogeny, 01 natural sciences, Genome, Oxidative Phosphorylation, 03 medical and health sciences, evolution, Organelle, Genetics, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ecology, Evolution, Behavior and Systematics, Alphaproteobacteria, 030304 developmental biology, 0303 health sciences, biology, Chlamydomonas, metabolic pathway, sequence homology, bioinformatics, Vitamin biosynthesis, biology.organism_classification, Biological Evolution, Cell biology, mitochondria, enzyme, vitamin biosynthesis, targeting prediction, Proteome, Eukaryote, protein import, protein, metabolism, 010606 plant biology & botany
Abstract

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

Published
2009

18. Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

Author

Laurence Braun, François Boulay, Marianne Tardif, and Marie-Josèphe Rabiet

Subjects
Transcriptional Activation, Immunology, Guanosine, HL-60 Cells, GTPase, Biochemistry, chemistry.chemical_compound, Transactivation, Humans, Promoter Regions, Genetic, cdc42 GTP-Binding Protein, NADPH oxidase, biology, Superoxide, NADPH Oxidases, Cell Biology, Hematology, Tetracycline, Cell biology, Enzyme Activation, chemistry, biology.protein, Guanine nucleotide exchange factor, Signal transduction, Nicotinamide adenine dinucleotide phosphate, Signal Transduction
Abstract

A tetracycline-controlled expression system was adapted to the human promyelocytic HL-60 cell line by placement of the transactivator (tTA-off) sequence under the control of the human EF-1α promoter region. Constitutively active and dominant-inhibitory forms of Cdc42 (Cdc42V12 and Cdc42N17, respectively) were conditionally expressed in this system. The expression of Cdc42V12 had no marked effect on chemoattractant-mediated superoxide production, corroborating previous results indicating that the guanosine 5′-triphosphate (GTP)–bound form of Cdc42 is ineffective in directly activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in a cell-free system. However, the N17 mutant potently inhibited chemoattractant-induced superoxide production. The expression of Cdc42N17 interfered with the GTP-loading of Rac and Ras and with the activation of the MAP-kinase pathway. A drastic reduction of chemoattractant-induced inositol-1,4,5-trisphosphate formation and calcium mobilization was observed, corroborating previous in vitro study results identifying PLCβ2 as a Rac/Cdc42 effector. Cdc42N17 was also found to inhibit the translocation of Ras-GRF2, a guanine nucleotide exchange factor for Ras and Rac but not for Cdc42. Thus, the dominant-inhibitory mutant Cdc42N17 was found to interfere at multiple levels in the signaling pathways. The pleiotropic inhibitory effects of Cdc42N17 illustrate the potential pitfalls of using dominant-inhibitory proteins to study the function of Ras-family GTPases. In this regard, a number of conclusions drawn from the use of dominant-inhibitory mutants in myeloid cells might have to be reconsidered.

Published
2002

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

Author

Jacqueline Martin-Laffon, Océane Gigarel, Morgane Mininno, Sabine Brugière, Claude Alban, Myriam Ferro, Sheng Ma, Meryl Mazzoleni, Annabelle Gilgen, Marianne Tardif, Stéphane Ravanel, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Plant Biology INRA, Cluster 9 of the Region Rhone-Alpes, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Ravanel, Stéphane, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Martin-Laffon, Jacqueline

Subjects
Models, Molecular, 0106 biological sciences, Triose phosphate, Chloroplasts, Methyltransferase, ribulose bisphosphate carboxylase, Arabidopsis thaliana, Protein Conformation, Amino Acid Motifs, Intracellular Space, lcsh:Medicine, arginine, plant, Plant Science, Biochemistry, 01 natural sciences, Mass Spectrometry, Protein structure, Protein methylation, Databases, Protein, lcsh:Science, méthylation, 0303 health sciences, Multidisciplinary, chloroplaste, Plant Biochemistry, food and beverages, Methylation, Recombinant Proteins, Transport protein, Chloroplast, Protein Transport, rubisco, Glyceraldehyde-3-phosphate dehydrogenase, Protochlorophyllide reductase, Research Article, Biology, plastide, 03 medical and health sciences, Fructose-bisphosphate aldolase, Ribose 5-phosphate isomerase, Plastid ribosomal protein L11, chloroplast, Ribosomal protein, prpl11, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, métabolisme, 030304 developmental biology, lysine, Arabidopsis Proteins, lcsh:R, Biology and Life Sciences, Methyltransferases, Chloroplast stroma, arabidopsis, post-translational modification, méthyltransférase, Phosphoglycerate kinase, lcsh:Q, methyltransferase, protéine recombinante, methylation, protein, 010606 plant biology & botany
Abstract

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0095512; International audience; 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.

Published
2014

20. Human Complement 5a (C5a) Anaphylatoxin Receptor (CD88) Phosphorylation Sites and Their Specific Role in Receptor Phosphorylation and Attenuation of G Protein-mediated Responses

Author

Thierry Christophe, François Boulay, Marianne Tardif, Marie-Danielle Milcent, and Marie-Josèphe Rabiet

Subjects
Homologous desensitization, Enzyme-linked receptor, Phosphorylation, Protein phosphorylation, 5-HT5A receptor, Cell Biology, Biology, Receptor, Interleukin-13 receptor, Molecular Biology, Biochemistry, Molecular biology, C5a receptor
Abstract

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166–19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser334 was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser332 and Ser334 or Ser334 and Ser338, was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser334 and Ser338 is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8–2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

Published
2000

21. PredAlgo: a new subcellular localization prediction tool dedicated to green algae

Author

Laurent Cournac, Olivier Vallon, Sabine Brugière, Michael Specht, Norbert Rolland, Ariane Atteia, Myriam Ferro, Michael Hippler, Guillaume Cogne, Gilles Peltier, Christophe Bruley, Marianne Tardif, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institute for Plant Biochemistry and Biotechnology, Westfälische Wilhelms-Universität Münster (WWU), Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Physiologie membranaire et moléculaire du chloroplaste (PMMC), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), 'Bundesministerium für Bildung und Forschung' Grant 0315265C (GOFORSYS partner Golmer Forschungseinheit für Systembiologie)., Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de la Méditerranée - Aix-Marseille 2, Bioénergie et Microalgues (EBM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), Environnement, Bioénergie, Microalgues et Plantes (EBMP), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))

Subjects
0106 biological sciences, Chloroplasts, Algae, organellar import, Prasinophyceae, Chlamydomonas reinhardtii, transit peptide, Computational biology, Proteomics, 01 natural sciences, 03 medical and health sciences, proteomics, chloroplast, Transit Peptide, Botany, subcellular localization, Genetics, mitochondrion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, mass spectrometry, 0303 health sciences, biology, Trebouxiophyceae, Algal Proteins, Computational Biology, biology.organism_classification, Mitochondria, secretory pathway, Chloroplast, Proteome, Neural Networks, Computer, Chloroplast Proteins, protein, Software, 010606 plant biology & botany
Abstract

International audience; The unicellular green alga Chlamydomonas reinhardtii is a prime model for deciphering processes occurring in the intracellular compartments of the photosynthetic cell. Organelle-specific proteomic studies have started to delineate its various subproteomes, but sequence-based prediction software is necessary to assign proteins subcellular localizations at whole genome scale. Unfortunately, existing tools are oriented toward land plants and tend to mispredict the localization of nuclear-encoded algal proteins, predicting many chloroplast proteins as mitochondrion targeted. We thus developed a new tool called PredAlgo that predicts intracellular localization of those proteins to one of three intracellular compartments in green algae: the mitochondrion, the chloroplast, and the secretory pathway. At its core, a neural network, trained using carefully curated sets of C. reinhardtii proteins, divides the N-terminal sequence into overlapping 19-residue windows and scores the probability that they belong to a cleavable targeting sequence for one of the aforementioned organelles. A targeting prediction is then deduced for the protein, and a likely cleavage site is predicted based on the shape of the scoring function along the N-terminal sequence. When assessed on an independent benchmarking set of C. reinhardtii sequences, PredAlgo showed a highly improved discrimination capacity between chloroplast- and mitochondrion-localized proteins. Its predictions matched well the results of chloroplast proteomics studies. When tested on other green algae, it gave good results with Chlorophyceae and Trebouxiophyceae but tended to underpredict mitochondrial proteins in Prasinophyceae. Approximately 18% of the nuclear-encoded C. reinhardtii proteome was predicted to be targeted to the chloroplast and 15% to the mitochondrion.

Published
2012

22. Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants

Author

Stéphane Ravanel, Morgane Mininno, Sheng Ma, Myriam Ferro, Marianne Tardif, Claude Alban, Virginie Pautre, Sabine Brugière, Annabelle Gilgen, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Etude de la dynamique des protéomes (EDyP ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Department of Plant Biology INRA, Cluster 9 of the Region Rhone-Alpes, French National Research Agency [SVSE6], Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Martin-Laffon, Jacqueline, Laboratoire d'étude de la dynamique des protéomes (LEDyP), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0106 biological sciences, Chloroplasts, Fructose 1,6-bisphosphate, ribulose bisphosphate carboxylase, Arabidopsis thaliana, substrate specificity, Arabidopsis, Plant Biology, Fructose-bisphosphate aldolase, plant, 01 natural sciences, Biochemistry, protein lysine methyltransferase, chemistry.chemical_compound, kinetic properties, Fructose-Bisphosphate Aldolase, Protein methylation, 0303 health sciences, biology, food and beverages, chloroplast, enzyme kinetics, plant biochemistry, protein methylation, s adenosylmethionine, aldolase, rubisco, Chloroplast, protéine, plante, inorganic chemicals, LSMT, Ribulose-Bisphosphate Carboxylase, 6-bisphophate aldolase, complex mixtures, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Pisum sativum, 030304 developmental biology, Ribulose, RuBisCO, Aldolase A, fungi, Peas, Cell Biology, légume, biology.organism_classification, fructose 1, enzyme, chemistry, biology.protein, bacteria, methylation, Protein Processing, Post-Translational, metabolism, 010606 plant biology & botany
Abstract

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.

Published
2012

23. Actin polymerization induced by GTP gamma S in permeabilized neutrophils is induced and maintained by free barbed ends

Author

Marianne Tardif, Sherry Huang, Daniel Safer, Tim Redmond, Sally H. Zigmond, and M. Pring

Subjects
Cell Membrane Permeability, Time Factors, Cytochalasin B, Neutrophils, Phalloidine, Guanosine, macromolecular substances, In Vitro Techniques, Biochemistry, chemistry.chemical_compound, Bacterial Proteins, Animals, Deoxyribonuclease I, Cytochalasin, Molecular Biology, Actin, Fluorescent Dyes, biology, Depolymerization, Rhodamines, Cell Biology, Actins, Thymosin, Kinetics, chemistry, Profilin, Polymerization, Guanosine 5'-O-(3-Thiotriphosphate), Streptolysins, biology.protein, Biophysics, Streptolysin, Rabbits, Mathematics
Abstract

To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTP gamma S) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTP gamma S, while thymosin beta 4 or DNase I decreased this polymerization. These data suggested that GTP gamma S induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTP gamma S, demonstrating that GTP gamma S did not inhibit profilin's monomer sequestering ability. The F-actin induced by GTP gamma S was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTP gamma S, addition of more GTP gamma S induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTP gamma S maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.

Published
1995

24. INDUCTION OF ACTIN POLYMERIZATION IN PERMEABILIZED NEUTROPHILS - ROLE OF ATP

Author

Marianne Tardif, Sally H. Zigmond, and Tim Redmond

Subjects
Cell Membrane Permeability, GTP', Phalloidin, Neutrophils, Polymers, Guanosine, macromolecular substances, Pertussis toxin, Biochemistry, chemistry.chemical_compound, Adenosine Triphosphate, Bacterial Proteins, medicine, Animals, Ascitic Fluid, Molecular Biology, Actin, Guanylyl Imidodiphosphate, Chemotactic Factors, Dose-Response Relationship, Drug, Apyrase, Nucleotides, Cell Biology, Adenosine, Actins, chemistry, Guanosine 5'-O-(3-Thiotriphosphate), Streptolysins, Biophysics, Phosphorylation, Guanosine Triphosphate, Rabbits, Oligopeptides, medicine.drug, Signal Transduction
Abstract

We have used streptolysin-O (SO)-permeabilized neutrophils to investigate the signal transduction pathway through which chemoattractants induce actin polymerization. Chemoattractants stimulate phosphorylation of various proteins and lipids but whether these phosphorylations are required for actin polymerization is not known. Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to SO-permeabilized neutrophils induced a doubling of the F-actin. This induction of F-actin, assayed by TRITC-labeled phalloidin binding, did not require the addition of ATP. Neither addition of apyrase to deplete residual ATP nor addition of ADP or UDP to compete with residual endogenous ATP inhibited significantly the GTP gamma S-induced polymerization. Addition of ATP on its own caused no increase in F-actin and did not affect the time course or concentration dependence of GTP gamma S-induced F-actin. Addition of ATP did increase the maximal amount of F-actin induced by GTP gamma S by about 20%. N-Formylnorleucylleucylphenalanine (formyl-peptide) in the presence of GTP, but not in its absence, also stimulated an increase in F-actin in SO-permeabilized cells. The F-actin induced by formyl-peptide plus GTP was inhibited by pertussis toxin. The induction did not require addition of ATP and addition of ADP to compete with residual ATP only slightly decreased the level of actin. However, addition of UDP significantly reduced the response to formyl-peptide plus GTP. Addition of ATP enhanced the increase in F-actin induced by optimal concentrations of GTP with formyl-peptide. ATP also lowered the apparent Km for GTP, but not for N-formyl peptide. The non-hydrolyzable ATP analog, adenosine 5'-(beta, gamma-imino)triphosphate, did not enhance the actin polymerization. Rather its presence inhibited the response induced by formyl-peptide plus GTP. The data suggest that actin polymerization can be induced by GTP gamma S in an manner that is largely ATP-independent. A role for ATP cannot be ruled out in the induction of actin polymerization by formyl-peptide plus GTP.

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