S20-006 Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii A Goyer 1 , P Decottignies 2 , Camilla Haslekås 3 , U Klein 3 , P Le Marechal 2 , JP Jacquot 4 , M Miginiac-Maslow 1 1 IBP, Université Paris-Sud 91405 Orsay Cedex, France. goyer@ibp.u-psud.fr 2 IBBMC, Université Paris-Sud 91405 Orsay Cedex, France 3 Department of Biology, University of Oslo, Norway 4 Interaction Arbres-Microorganismes Université Nancy I, France Keywords: peroxiredoxin, thioredoxin, redox signaling, oxidative stress Introduction Peroxiredoxins (Prx) form a ubiquitous group of peroxidases that were found in almost all living organisms. Prx featuring 2 conserved cysteine residues (2Cys-Prx) are reduced by the AhpF protein in bacteria, and by the thioredoxin/thioredoxin reductase system in yeast, animals and plants. 2Cys-Prxs catalyze in vitro the reduction of alkyl hydroperoxides and hydrogen peroxide. They exist as homodimers, both subunits are linked by an intermolecular disulfide (Chae et al., 1994). In animals, yeast, and plants, the disulfide is reduced via a thiol/disulfide redox interchange with reduced thioredoxin (Trx), thus regenerating an active peroxidase. The present study was aimed at setting up a purification system for trapping proteins that react with thioredoxin, based on their ability to form mixed disulfide-linked adducts with a single cysteine mutant thioredoxin. The system allowed the purification and identification of a 2Cys-Prx protein (Ch-Prx1) from the green alga Chlamydomonas reinhardtii. The purified protein was characterized by its peroxidase activity, and its ability to use different thioredoxin isoforms as hydrogen donors. The regulation of the Ch-Prx1 gene expression suggests that Ch-Prx1 is involved in detoxification of ROS in the Chlamydomonas chloroplast. Materials and methods Algal strains and culture conditions-Chlamydomonas reinhardtii cell-wall-less strain CW15 and strain CC 125 were grown in a photoautotrophic minimal medium (HSM) at 25°C or 32°C under continuous stirring and bubbling with 2 or 5 % CO 2 enriched air. Light intensity was 150 or 300 μmol/m 2 sec. Cultures were kept in a continuous light regime or in a 12 h light/dark regime. Purification of the Ch-Prx1 native protein from Chlamydomonas cells-Chlamydomonas CW15 cells were pelleted, resuspended in 30 mM Tris-HCl pH 7.9 (Tris buffer), and broken by 2 cycles of freeze-thawing. Broken cells were centrifuged, the supernatant was adjusted to 2 % (w/v) streptomycin sulfate and precipitated nucleic acids were pelleted. The supernatant was adjusted to 95 % (w/v) ammonium sulfate. Precipitated proteins were pelleted, resuspended in Tris buffer, and dialyzed against the same buffer. The protein solution was loaded onto a column made of a mutated Chlamydomonas h-type thioredoxin (C39S mutant) grafted on a CNBr activated sepharose support according to recommandations of the supplier (Pharmacia-Amersham) and equilibrated with Tris buffer. After loading on the column, the