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Kessler, Félix

Nom
Kessler, Félix
Affiliation principale
Fonction
Professeur.e ordinaire
Email
felix.kessler@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/320
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0000-0001-6409-5043

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  • Publication
    Accès libre
    The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery
    (2017)
    Zufferey-Arias, Mónica Alexandra
    ;
    Kessler, Félix 
    Le chloroplaste est un organite essentiel de la cellule végétale, il est le siège de la photosynthèse. Un événement d’endosymbiose est à l’origine du chloroplaste : une cellule eucaryote primitive a ingéré une cyanobactérie photosynthétique. Pendant l’évolution, la majorité des gènes du chloroplaste primitif ont été transférés vers le noyau. Les protéines issues des gènes transférés avec succès, sont maintenant synthétisées par des ribosomes dans le cytosol et importées dans les chloroplastes. Les protéines destinées au chloroplaste (pré-protéines) acquièrent une séquence additionnelle clivable codant pour un peptide à l’extrémité N-terminal (séquence d’adressage). La séquence d’adressage est reconnue par la machinerie d’importation du chloroplaste qui initie le transport des pré-protéines. La machinerie d’importation consiste en un translocon situé dans la membrane externe/interne du chloroplaste (TOC/TIC) (Translocon at the Outer/Inner membrane of Chloroplast). L’importation de centaines de différentes protéines dépend des complexes TOC et TIC. Le noyau du complexe TOC est composé de trois protéines, les récepteurs GTPase Toc159 et Toc34 ainsi que le canal Toc75. Ensemble ils reconnaissent et transfèrent les pré-protéines à travers la membrane externe du chloroplaste. Toc34 et Toc159 qui sont exposés à la surface du chloroplaste, fonctionnent en tant que récepteurs et ont des domaines G (GTP-binding) homologues. En plus du domaine G, Toc159 possède le domaine A (acide) à l’extrémité N-terminal qui s’étend dans le cytosol et contrôle la spécificité du récepteur, et le domaine M à l’extrémité C-terminal qui ancre la protéine à la membrane. Toc75 appartient à la famille OMP85, protéines de la membrane externe des bactéries gram négatives. Dans les chloroplastes elles ont évolué pour fournir un canal de translocation de protéines à travers la membrane externe.
    Toc159 joue un rôle essentiel dans la biogenèse du chloroplaste. Les bases de données de phosphoprotéomique montrent que le domaine A de Toc159 est fortement phosphorylé. La protéine cytosolique caséine kinase II phosphoryle le domaine A in vitro. Toutefois d’autres kinases ayant la même fonction ont aussi été prédites. Tandis que la phosphorylation contrôle l’assemblage et l’activité des complexes d’importation de protéines dans les chloroplastes et les mitochondries, aucune kinase organite-spécifique n’a été identifiée jusqu’à présent. Par co-purification avec Toc159, nous avons découvert une protéine kinase dans la membrane externe du chloroplaste (KOC1 « Kinase at the Outer Chloroplast membrane 1 »). KOC1 est une protéine intégrale de membrane orientée vers le cytosol et associée de manière stable avec le complexe TOC. KOC1 phosphoryle le domaine A chez les membres de la famille Toc159 in vitro. Dans les chloroplastes des mutants koc1, l’efficience de l’importation des protéines a été réduite. Par ailleurs, les plantules koc1 ont un taux de survie réduit quand elles sont déplacées de l’obscurité à la lumière, quand une importation rapide des pré-protéines est nécessaire pour une biogenèse de chloroplastes complète. Nos résultats indiquent que KOC1 est un composant de la machinerie d’importation TOC en phosphorylant les récepteurs, en soutenant l’importation de pré-protéines et en contribuant à une biogenèse de chloroplastes efficiente., The chloroplast constitutes the site of photosynthesis and is an essential organelle in plant cells. An endosymbiotic event was at the origin of the chloroplast, an ancestral eukaryotic cell engulfing a photosynthetic cyanobacterium. During evolution, the majority of ancestral chloroplast genes were lost or transferred to the nucleus. The protein products of the successfully transferred genes are now synthesized by cytosolic ribosomes and imported into the chloroplast. The chloroplast destined proteins (preproteins) acquired an additional sequence that encodes a cleavable N-terminal targeting peptide (transit peptides). The transit peptide is recognized by the chloroplast import machinery, which initiates import. The import machinery consists of translocon complexes at the outer (TOC) and inner membrane of the chloroplast (TIC). The import of hundreds of different chloroplast proteins depends on TOC and TIC complexes. The TOC complex core contains three proteins, the GTPase receptors: Toc159, Toc34 and the channel Toc75, together they recognize and transfer the pre-proteins across the outer membrane of the chloroplast. Both Toc34 and Toc159 are exposed at the surface of the chloroplast, consistent with a receptor function, and have homologous GTP-binding domains (G-domain). In addition to the G-domain, Toc159 has a N-terminal A- (acidic) domain that extends into the cytosol and controls receptor specificity and a C-terminal membrane anchoring M-domain. Toc75 belongs to the OMP85 family that serves to integrate proteins into the outer membrane of gram negative bacteria, in chloroplasts it has evolved to provide a protein translocation channel across the outer membrane.
    Toc159 plays an essential role in chloroplast biogenesis. Phosphoproteomics databases show that Toc159 is highly phosphorylated at the A domain. Cytosolic casein kinase II phosphorylates the A-domain in vitro, however other A-domain kinases have been predicted.
    While phosphorylation controls assembly and activity of protein import complexes in both mitochondria and chloroplasts no organelle-specific kinases have been identified so far. By co-purification with Toc159, we discovered "Kinase at the Outer Chloroplast membrane 1" (KOC1). KOC1 is an integral membrane protein facing the cytosol and stably associating with TOC. KOC1 phosphorylated the A-domain of Toc159 family members in vitro. In mutant koc1 chloroplasts preprotein import efficiency was diminished. Moreover, koc1 seedlings had reduced survival rates when moved from the dark to the light when protein import is required to rapidly complete chloroplast biogenesis. Our data indicate that KOC1 is a functional component of the TOC machinery phosphorylating import receptors, supporting preprotein import and contributing to efficient chloroplast biogenesis.
  • Publication
    Accès libre
    Identification and characterization of putative Toc159 interacting partners
    (2015)
    Montandon, Cyrille
    ;
    Kessler, Félix 
    Le chloroplaste est l’organelle qui caractérise les plantes terrestres ainsi que les autres eucaryotes photosynthétiques. Il remplit diverses fonctions métaboliques, mais la photosynthèse est son activité principale, sa structure et sa composition protéique en témoignent. Le chloroplaste est le résultat d’une endosymbiose entre un eucaryote ancestral et une cyanobactérie photosynthétique. Le génome du chloroplaste, vestige de son ancienne autonomie, encode environ une centaine de protéines. Les 2000-3000 autres protéines présentes dans le chloroplaste sont codées dans le noyau et traduites dans le cytosol et doivent donc être importées dans le chloroplaste. Le « transit peptide » (peptide de transit), une courte séquence à la terminaison aminée des protéines destinées au chloroplaste, est suffisant et nécessaire pour l’import spécifique dans le chloroplaste. La voie Toc/Tic (Translocon at the Outer/Inner membrane of the chloroplast envelope: « Translocon à la membrane externe/interne de l’enveloppe du chloroplaste ») reconnait et import ces protéines en présence d’ATP et de GTP. Le noyau du complexe Toc est composé de Toc159 et Toc34/33, 2 récepteurs possédant un domaine GTPase, et de Toc75 qui constitue le pore du complexe. Toc159 est formé de 3 domaines, un domaine ancrant la protéine dans la membrane à l’extrémité carboxyle (domaine M), un domaine GTPase (domaine G) et un domaine acide (domaine A). Le taux d’import des protéines et sa spécificité envers les protéines clientes varient en fonction du développement de la plante, du tissu ou du type de plaste. Les différents homologues de Toc159 et de Toc34/33 chez A. thaliana forment des complexes distincts. Ils montrent une spécificité d’import différente et leur expression varie en fonction du stade de développement ou de la partie de la plante. Le domaine A de Toc159 et de ses homologues détermine partiellement la spécificité envers les différents types de protéines clientes. Le domaine A est hyper-phosphorylé et existe sous une forme soluble, certainement le résultat d’un clivage spécifique. Ces modifications post-traductionnelles pourraient influencer la spécificité de Toc159 envers les protéines clientes. Le contrôle de l’activité GTPase de Toc159 ou de Toc34/33 pourrait également influencer l’activité d’import. Le but de ce travail de thèse était la caractérisation de protéines co-précipitées avec une version taguée de Toc159 et identifiées par spectrométrie de masse, qui pourraient être potentiellement responsable des modifications mentionnées ci-dessus. L’effort principal a été fait sur Emb2004/AT1G10510, une protéine LRR (Leucine Rich Repeat) et des contributions ont été faites à la caractérisation d’une protéine kinase potentielle/AT4G32250 et de Tic56/AT5G01590., The chloroplast is the distinctive organelle of land plants and other photosynthetic eukaryotes. It carries out a variety of metabolic process, but photosynthesis is its main task and this is reflected in its structures and protein composition. Chloroplasts evolved from an endosymbiosis between a photosynthetic cyanobacterium and an ancestral eukaryote. Reminiscent of this autonomous origin, the chloroplast genome encodes approximately 100 proteins. The majority of the remaining 2000-3000 proteins identified in the chloroplast are encoded in the nucleus and translated in the cytosol and must be imported into the chloroplast. The transit peptide, a small sequence at the N-terminus of the proteins destined for the chloroplast, is necessary and sufficient for specific import into the chloroplast. The Toc/Tic (Translocon at the Outer/Inner membrane of the chloroplast envelope) pathway mediates the recognition and the translocation of these proteins in an ATP- and GTP-dependent way. The Toc core complex is constituted of two receptors with a GTPase domain, Toc159 and Toc34/33, and a channel, Toc75. Toc159 is formed of three domains, a C-terminal membrane anchoring domain (M domain), a GTPase domain (G domain) and an acidic domain (A-domain). The protein import rate and specificity toward client proteins vary depending on the developmental stage, tissue or plastid type. The different A. thaliana homologues of Toc159 and Toc34/33 form distinct complexes. They have different import specificities and their expression level depends on the developmental stage and the anatomical part of the plant. The A-domain of Toc159 and its homologues partially determine the specificity toward the different types of client proteins. The A-domain is hyper-phosphorylated and exists as a soluble form, likely the result of a specific cleavage. These post-translational modifications of the A-domain might play a role in determining the affinity of Toc159 toward client proteins. The control of the GTPase activity of Toc159 or Toc34/33 might also have an influence on the import. The aim of this thesis was the characterization of proteins potentially responsible for these modifications and that were co-purified with a tagged Toc159 and identified by mass-spectrometry. The main effort was made on Emb2004/AT1G10510, a LRR (Leucine Rich Repeat) protein and contributions were made for the characterization of a predicted protein kinase (AT4G32250) and Tic56 (AT5G01590).
  • Publication
    Accès libre
    A plant split-ubiquitin system and TAP-tagging to study in vivo protein interactions in the chloroplast protein import machinery
    (2008)
    Rahim, Gwendoline
    ;
    Kessler, Félix 
    Most chloroplast proteins are synthesized as precursor proteins in the cytosol. The import of these precursor proteins is mediated by molecular complexes located at the outer and inner membrane of the chloroplast. These complexes are called Toc (translocon at the outer envelope membrane) and Tic (translocon at the inner envelope membrane) respectively. In Arabidopsis, the Toc complex consists of three principle components: two homologous receptor GTPases, atToc159 and atToc33 and a protein-import channel: atToc75. During import, the two GTPases undergo complex interactions with precursor proteins and amongst themselves although precise mechanisms remain unknown. In vitro studies revealed that Toc159 and Toc33 interact with each other via the dimerization of their GTP-binding domain (G-domain). Moreover, the crystal structure of the pea Toc33 ortholog, psToc34 indicates that it can stably homodimerize via its G-domain. However, neither Toc159/Toc33 heterodimers nor Toc33 homodimerization have been demonstrated in planta. To get new insight into the in vivo interactions of Toc GTPases, we have developed a plant split-ubiquitin system. This method, originally developed for yeast, was adapted to study interactions between the Toc GTPases atToc159 and atToc33 in Arabidopsis and tobacco protoplasts. We also demonstrated that the peroxisomal membrane protein atPex11e, used initially as a model membrane protein in our system, self-interacts as does its yeast homolog. The plant split-ubiquitin system proves to be widely usable. Another approach of this thesis was to get more information on the import mechanism via the identification of interaction partners of the Toc GTPase atToc33. atToc33 and proteins associated were isolated from Arabidopsis plants, using the tandem affinity purification (TAP) tag. We proved that this technique is suitable to purify Toc33, which encourages us to purify Toc proteins and complexes at a larger scale.
  • Publication
    Accès libre
    AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery
    (2004)
    Hiltbrunner, Andreas
    ;
    Grünig, Kathrin
    ;
    Alvarez-Huerta, Mayte
    ;
    Infanger, Sibylle
    ;
    Bauer, Jörg
    ;
    Kessler, Félix 
    AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.
  • Publication
    Accès libre
    The Novel Chloroplast Outer Membrane Kinase KOC1 Is a Required Component of the Plastid Protein Import Machinery
    Zufferey, Mónica
    ;
    Montandon, Cyrille
    ;
    Douet, Véronique 
    ;
    Demarsy, Emilie
    ;
    Agne, Birgit
    ;
    Baginsky, Sacha
    ;
    Kessler, Félix 
    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis.
  • Publication
    Accès libre
    Border control: selectivity of chloroplast protein import and regulation at the TOC-complex
    Demarsy, Emilie
    ;
    Lakshmanan, Ashok M
    ;
    Kessler, Félix 
    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC–TIC (translocon of outer membrane complex–translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.
  • Publication
    Accès libre
    The Chloroplast Import Receptor Toc90 Partially Restores the Accumulation of Toc159 Client Proteins in the Arabidopsis thaliana ppi2 Mutant
    Infanger, Sibylle
    ;
    Bischof, Sylvain
    ;
    Hiltbrunner, Andreas
    ;
    Agne, Birgit
    ;
    Baginsky, Sacha
    ;
    Kessler, Félix 
    Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- (translocon at the outer chloroplast membrane) and Tic- (translocon at the inner chloroplast membrane) complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families: Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.