Thiol-based redox regulation in Chlamydomonas: the emerging role of glutathionylation and glutaredoxins

M. Bedhomme, M. Zaffagnini, C.H. Marchand, X.H. Gao, L. Michelet, P. Decottignies, and S.D. Lemaire

Institut de Biologie des Plantes, UMR 8618, CNRS, Univ Paris-Sud, 91405 Orsay Cedex, France

Protein glutathionylation is a reversible post-translational modification promoted by oxidative and nitrosative stresses and consisting of the formation of a mixed disulfide between glutathione and a protein cysteine residue. This modification can protect specific cysteines from irreversible oxidation but can also modulate protein activities and thereby play a role in many cellular processes. Glutathionylation was mainly studied in mammals but emerging evidence suggests that it could constitute an important mechanism of regulation and signaling in photosynthetic organisms. While the mechanism of glutathionylation prevailing in vivo remains unclear, the reverse reaction, called deglutathionylation, is mainly catalyzed by small disulfide oxidoreductases of the thioredoxin family named glutaredoxins (GRXs). In order to get more insight into the importance and function of protein glutathionylation in photosynthetic organisms, we developed several proteomic approaches based on radiolabeling, biotinylation or affinity purification to identify proteins undergoing glutathionylation in the unicellular green alga Chlamydomonas reinhardtii. These methods allowed identification, in vivo or in vitro, of more than 100 glutathionylated proteins involved in numerous cell processes and pathways. Several targets, including A4-glyceraldehyde-3-phosphate dehydrogenase and isocitrate lyase, were confirmed biochemically and the underlying molecular mechanisms were investigated in details by site-directed mutagenesis, mass spectrometry and kinetic analyses. Glutathionylation was found to affect the activity of all the proteins analyzed each of them exhibiting distinct reactivities with glutathione and GRXs. All these data indicate that glutathionylation likely constitutes an important mechanism of regulation in Chlamydomonas. (References: Zaffagnini et al. 2007 FEBS J., 274, 212; Michelet et al. 2008 JBC 283, 21571. Zaffagnini et al. 2008 JBC, 283, 8868; Gao et al. 2009 Mol. Plant. 2, 218; Bedhomme et al. 2009 JBC 284, 36282).

e-mail address of presenting author: mariette.bedhomme@u-psud.fr