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.

2009, Agne, Birgit, Kessler, Félix

Most of the estimated 1000 or so chloroplast proteins are synthesized as cytosolic preproteins with N-terminal cleavable targeting sequences (transit peptide). Translocon complexes at the outer (Toc) and inner chloroplast envelope membrane (Tic) concertedly facilitate post-translational import of preproteins into the chloroplast. Three components, the Toc34 and Toc159 GTPases together with the Toc75 channel, form the core of the Toc complex. The two GTPases act as GTP-dependent receptors at the chloroplast surface and promote insertion of the preprotein across the Toc75 channel. Additional factors guide preproteins to the Toc complex or support their stable ATP-dependent binding to the chloroplast. This minireview describes the components of the Toc complex and their function during the initial steps of preprotein translocation across the chloroplast envelope.

2006, Kessler, Félix, Schnell, Danny

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus-encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non-photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc-Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.

2003, Kessler, Félix, Neuhaus, Jean-Marc

Eucaryotic cells (plants, animals, fungi, etc.) are subdivided in membrane-bound compartments (organelles), such as the nucleus, mitochondria, chloroplasts, vacuoles, etc. Most organellar proteins are encoded in the nucleus and synthesized in the cytoplasm. Proper sorting of proteins is required to establish and maintain organellar identity. Molecular machineries at the organelle surfaces specifically recognize targeting sequences of their cognate proteins and mediate their translocation across membranes. Proteins destined for the vacuoles are first translocated across the endoplasmic reticulum membrane, packaged into vesicles, transported to the Golgi, where they are sorted into specific vesicles and subsequently carried to the different types of vacuoles. Though plant cells share many features with animal and yeast cells, chloroplasts and distinct lytic and storage vacuoles are unique to plants. Here, we discuss import of proteins into the chloroplast as well as selective sorting of proteins to either the lytic or the storage vacuole.

2012, Venkatasalam, Shanmugabalaji, Kessler, Félix

Plastoglobules (PG) are lipid droplets existing in most types of plastids. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the surface of PG, but to various extents also at the thylakoid membrane. PGLs are thought to have a structural function in PG, and it is known that almost the complete protein is required for PGLs to assemble on PG. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. These findings suggest that PGL35-HIVp24 folds correctly after its synthesis inside the chloroplast and is then assembled not only on plastoglobules but also on thylakoid membranes. The fusion protein accumulated up to a 1% of the total protein and could be purified by biotin affinity chromatography of a total membrane extract. Targeting of PGL35 fusion proteins to plastoglobules has the potential to become an interesting expression system but it will be necessary to master the parameters that govern the partioning between plastoglobules and thylakoid membranes for this to become an effective technique. This study represents a step forward in this direction.
In addition to lipids and PGLs, the PG harbours other proteins: The PG proteome in Arabidopsis consist of about two dozen proteins including uncharacterized plastoglobule protein AtSOUL4, which has a predicted heme-binding motif. The Arabidopsis genome contains six proteins with a SOUL motif; two of them AtSOUL4 and AtSOUL5 are predicted to be plastid localized and AtSOUL5 was found in the thylakoid proteome. A SOUL homolog, AtSOUL1, is highly expressed and the soul1 mutant is hypersensitive to red light. In addition, C. reinhardtii eyespot phosphoproteins contained SOUL3. However, due to the presence of SOUL-heme binding proteins in light perception systems and their implication in plant photomorphogenesis, the SOUL heme binding proteins may be involved in light signalling. The heterologously expressed recombinant AtSOUL4 protein binds to heme in vitro as well as in vivo. Moreover, AtPGL35 and AtSOUL4 fluorescent fusion proteins colocalized in chloroplasts. Furthermore, immunoblot analysis of chloroplast membrane fractions shows that AtSOUL4 localized at the plastoglobule. AtSOUL4 has a highly conserved CKII phosphorylation site in C-terminal. Moreover, In vitro phosphorylation studies revealed that AtSOUL4 is phosphorylated most likely by a stromal CKII like kinase. The double knock out of AtSOUL4 and AtSOUL5 revealed a conditional shade avoidance behavior, indicating that AtSOUL4 heme-binding protein might also be involved in the light signalling.

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.

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.

2010, Zbierzak, Anna Maria, Kanwischer, Marion, Wille, Christina, Vidi, Pierre-Alexandre, Giavalisco, Patrick, Lohmann, Antje, Briesen, Isabel, Porfirova, Svetlana, Bréhélin, Claire, Kessler, Félix, Dörmann, Peter

Plastoglobules, lipid–protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to γ-tocopherol and γ-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

2006, Kessler, Félix, Schnell, Danny J.

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus-encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non-photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc–Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.

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.