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Recherche de nouveaux systèmes de transport à travers l'enveloppe du chloroplaste. Caractérisation de nouvelles protéines hydrophobes.

Abstract : The chloroplast is an organelle totally integrated in the metabolism of the plant cell. It contains its own metabolic pathways like photosynthesis, aminoacid synthesis. The chloroplast is limited by the envelope composed of two membranes and an intermembrane space. Envelope membranes are the site of transport of metabolites, ions, proteins and information between the plastid and the cytosol. Then, they contain many transport systems, but only some of them have been characterised. Hydrophobicity and low representation are the main limitations for the study of these proteins. In order to characterised new transport systems, we have developed an approach that allowed us to identify new proteins.
This approach is based of the hydrophobicity of translocators that allows solubilisation of these proteins in organic solvents. Hydrophobic proteins were differentially extracted in various mixture of chloroform/methanol according to their hydrophobicity. Then, proteins were separated by SDS-PAGE and analysed by microsequencing. Several new proteins were identified including IE16 and IE18, localized in the inner membrane of the chloroplast envelope.
Functional characterisation of these proteins was continued by analysing sequences homologies with proteins of known function. We have also obtained mutants of cyanobacteria and Arabidopsis thaliana in which genes coding for IE16 and IE18 are disrupted. The analysis of their phenotypes provide informations on the function of these proteins. In particular, IE18 could be involved in the transport of K+ and/or H+. In addition, these proteins have been expressed in heterologous systems. They are produced in the system baculovirus/insect cells. Their biochemical and electrophysiological characterisation are currently in progress.
One of the proteins extracted in organic solvent was demonstrated to correspond to a 35 kDa annexin. The binding of annexin to chloroplasts and its function were studied. This annexin copurifies with chloroplasts and envelope membranes in the presence of calcium. The sulfolipid, a chloroplast specific lipid, is a high affinity site for interaction with annexin. This protein does not form an ionic channel when integrated in lipidic planar bilayers. This annexin does not possess a peroxydase activity. The signification of the interaction of annexin with the chloroplast envelope remains to be determined.
We have developed an approach that can be used systematically for studying transport in various membranes systems.
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  • HAL Id : tel-00181078, version 1
  • PRODINRA : 251170

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Daphné Seigneurin-Berny. Recherche de nouveaux systèmes de transport à travers l'enveloppe du chloroplaste. Caractérisation de nouvelles protéines hydrophobes.. Sciences du Vivant [q-bio]. Université Joseph-Fourier - Grenoble I, 2000. Français. ⟨tel-00181078⟩

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