Kessler, Félix

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.

2001, Hiltbrunner, Andreas, Bauer, Jörg, Alvarez-Huerta, Mayte, Kessler, Félix

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.

, Eugeni Piller, Lucia, Glauser, Gaétan, Kessler, Félix, Besagni, Céline

Plants are exposed to ever changing light environments and continuously forced to adapt. Excessive light intensity leads to the production of reactive oxygen species that can have deleterious effects on photosystems and thylakoid membranes. To limit damage, plants increase the production of membrane soluble antioxidants such as tocopherols. Here, untargeted lipidomics after high light treatment showed that among hundreds of lipid compounds alpha-tocopherol is the most strongly induced, underscoring its importance as an antioxidant. As part of the antioxidant mechanism, a-tocopherol undergoes a redox cycle involving oxidative opening of the chromanol ring. The only enzyme currently known to participate in the cycle is tocopherol cyclase (VTE1, At4g32770), that re-introduces the chromanol ring of a-tocopherol. By mutant analysis, we identified the NAD(P)H-dependent quinone oxidoreductase (NDC1, At5g08740) as a second enzyme implicated in this cycle. NDC1 presumably acts through the reduction of quinone intermediates preceding cyclization by VTE1. Exposure to high light also triggered far-ranging changes in prenylquinone composition that we dissect herein using null mutants and lines overexpressing the VTE1 and NDC1 enzymes.

2004, Hiltbrunner, Andreas, Grunig, 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.

, 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.

2002, Bauer, Jörg, Hiltbrunner, Andreas, Weibel, Petra, Vidi, Pierre-Alexandre, Alvarez-Huerta, Mayte, Smith, Matthew, Schnell, Danny, Kessler, Félix

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

, Aronsson, Henrik, Combe, Jonathan, Patel, Ramesh, Agne, Birgit, Martin, Meryll, Kessler, Félix, Jarvis, Paul

The atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.