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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
_
0000-0001-6409-5043

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  • Publication
    Accès libre
    Pheophytin Pheophorbide Hydrolase (Pheophytinase) Is Involved in Chlorophyll Breakdown during Leaf Senescence in Arabidopsis[W],[OA]
    (2009)
    Schelbert, Silvia
    ;
    Aubry, Sylvain
    ;
    Burla, Bo
    ;
    Agne, Birgit
    ;
    Kessler, Félix 
    ;
    Krupinska, Karin
    ;
    Hörtensteiner, Stefan
    During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.
  • Publication
    Accès libre
    Protein transport in organelles: The Toc complex way of preprotein import
    (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.
  • Publication
    Accès libre
    A Toc159 Import Receptor Mutant, Defective in Hydrolysis of GTP, Supports Preprotein Import into Chloroplasts
    (2009)
    Agne, Birgit
    ;
    Infanger, Sibylle
    ;
    Wang, Fei
    ;
    Hofstetter, Valère
    ;
    Rahim, Gwendoline
    ;
    Martin, Meryll
    ;
    Lee, Dong Wook
    ;
    Hwang, Inhwan
    ;
    Schnell, Danny
    ;
    Kessler, Félix 
    The heterotrimeric Toc core complex of the chloroplast protein import apparatus contains two GTPases, Toc159 and Toc34, together with the protein-conducting channel Toc75. Toc159 and Toc34 are exposed at the chloroplast surface and function in preprotein recognition. Together, they have been shown to facilitate the import of photosynthetic proteins into chloroplasts in Arabidopsis. Consequently, the ppi2 mutant lacking at Toc159 has a non-photosynthetic albino phenotype. Previous mutations in the conserved G1 and G3 GTPase motifs abolished the function of Toc159 in vivo by disrupting targeting of the receptor to chloroplasts. Here, we demonstrate that a mutant in a conserved G1 lysine (atToc159 K868R) defective in GTP binding and hydrolysis can target and assemble into Toc complexes. We show that atToc159 K868R can support protein import into isolated chloroplasts, albeit at lower preprotein binding and import efficiencies compared with the wild-type receptor. Considering the absence of measurable GTPase activity in the K868R mutant, we conclude that GTP hydrolysis at atToc159 is not strictly required for preprotein translocation. The data also indicate that preprotein import requires at least one additional GTPase other than Toc159.
  • Publication
    Accès libre
    In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system
    (2008)
    Rahim, Gwendoline
    ;
    Bischof, Sylvain
    ;
    Kessler, Félix 
    ;
    Agne, Birgit
    The GTPases atToc33 and atToc159 are pre-protein receptor components of the translocon complex at the outer chloroplast membrane in Arabidopsis. Despite their participation in the same complex in vivo;, evidence for their interaction is still lacking. Here, a split-ubiquitin system is engineered for use in plants, and the in vivo; interaction of the Toc GTPases in Arabidopsis and tobacco protoplasts is shown. Using the same method, the self-interaction of the peroxisomal membrane protein atPex11e is demonstrated. The finding suggests a more general suitability of the split-ubiquitin system as a plant in vivo; interaction assay.
  • Publication
    Accès libre
    The role of GTP binding and hydrolysis at the atToc159 preprotein receptor during protein import into chloroplasts
    (2008)
    Wang, Fei
    ;
    Agne, Birgit
    ;
    Kessler, Félix 
    ;
    Schnell, Danny J.
    The majority of nucleus-encoded chloroplast proteins are targeted to the organelle by direct binding to two membrane-bound GTPase receptors, Toc34 and Toc159. The GTPase activities of the receptors are implicated in two key import activities, preprotein binding and driving membrane translocation, but their precise functions have not been defined. We use a combination of in vivo and in vitro approaches to study the role of the Toc159 receptor in the import reaction. We show that atToc159-A864R, a receptor with reduced GTPase activity, can fully complement a lethal insertion mutation in the ATTOC159 gene. Surprisingly, the atToc159-A864R receptor increases the rate of protein import relative to wild-type receptor in isolated chloroplasts by stabilizing the formation of a GTP-dependent preprotein binding intermediate. These data favor a model in which the atToc159 receptor acts as part of a GTP-regulated switch for preprotein recognition at the TOC translocon.
  • 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
    Characterization of Chloroplast Protein Import without Tic56, a Component of the 1-Megadalton Translocon at the Inner Envelope Membrane of Chloroplasts
    Köhler, Daniel
    ;
    Montandon, Cyril
    ;
    Hause, Gerd
    ;
    Majovsky, Petra
    ;
    Kessler, Félix 
    ;
    Baginsky, Sacha
    ;
    Agne, Birgit
    We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. In both mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane.
  • Publication
    Accès libre
    The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein
    Agne, Birgit
    ;
    Andrès, Charles
    ;
    Montandon, Cyril
    ;
    Christ, Bastien
    ;
    Ertan, Anouk
    ;
    Jung, Friederike
    ;
    Infanger, Sibylle
    ;
    Bischof, Sylvain
    ;
    Baginsky, Sacha
    ;
    Kessler, Félix 
    The translocon at the outer membrane of the chloroplast assists the import of a large class of preproteins with amino-terminal transit sequences. The preprotein receptors Toc159 and Toc33 in Arabidopsis (Arabidopsis thaliana) are specific for the accumulation of abundant photosynthetic proteins. The receptors are homologous GTPases known to be regulated by phosphorylation within their GTP-binding domains. In addition to the central GTP-binding domain, Toc159 has an acidic N-terminal domain (A-domain) and a C-terminal membrane-anchoring domain (M-domain). The A-domain of Toc159 is dispensable for its in vivo activity in Arabidopsis and prone to degradation in pea (Pisum sativum). Therefore, it has been suggested to have a regulatory function. Here, we show that in Arabidopsis, the A-domain is not simply degraded but that it accumulates as a soluble, phosphorylated protein separated from Toc159. However, the physiological relevance of this process is unclear. The data show that the A-domain of Toc159 as well as those of its homologs Toc132 and Toc120 are targets of a casein kinase 2-like activity.
  • 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.
  • Publication
    Accès libre
    Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency
    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.