In vivo targets of S-thiolation in Chlamydomonas reinhardtii.
Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.
Michelet
Laure
L
Institut de Biotechnologie des Plantes, UMR 8618, Bâtiment 630, Orsay cedex, France.
Zaffagnini
Mirko
M
Vanacker
Hélène
H
Le Maréchal
Pierre
P
Marchand
Christophe
C
Schroda
Michael
M
Lemaire
Stéphane D
SD
Decottignies
Paulette
P
D019747:Aldose-Ketose Isomerases / Q000737:chemistry; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000737:chemistry; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D010788:Photosynthesis; D010940:Plant Proteins; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D015800:Protozoan Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds
D004220:Disulfides; D018840:HSP70 Heat-Shock Proteins; C102156:HSP70B protein, Chlamydomonas; D010940:Plant Proteins; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; D054464:Peroxiredoxins; D010735:Phosphoglycerate Kinase; D019747:Aldose-Ketose Isomerases; C022548:ribosephosphate isomerase; D003545:Cysteine