Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.014142, 1-15, 2012.Protein post-translational modifications (PTMs) 1 play a pivotal role in cellular signaling (1). Recently, redox PTMs have emerged as important mechanisms of signaling and regulation in all organisms. Our increasing understanding of the molecular mechanism of cell signaling has revealed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) act as signaling molecules to transfer extracellular or intracellular information and elicit specific responses. ROS and RNS have generally been considered to be toxic molecules that have to be continuously scavenged and efficiently detoxified. Plant cells exhibit a remarkable ability to cope with high rates of ROS/RNS production as a result of a complex scavenging system that includes either antioxidant molecules or enzymes (2). In photosynthetic organisms, ROS and RNS are continuously produced during normal aerobic metabolism but are also produced transiently in response to various types of endogenous or exogenous signals, such as biotic and abiotic stresses. This production activates specific signaling pathways, resulting in transcriptional, post-transcriptional and post-translational responses that will, in fine, allow adaptation to new environmental conditions. These past decades, redox modifications have emerged as central mechanisms in these processes, at the interface between ROS/RNS and the adaptative responses to environmental changes. ROS/RNS signaling operates mainly through a set of PTMs of thiol residues on proteins (3). Indeed, cysteine residues can undergo different states of oxidation such as sulfenic, sulfinic, and sulfonic acids in addition to protein disulfide bridges (intra-or intermolec...

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