Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3) B. McDonagh a, 1 , R. Requejo a, 1 , C.A. Fuentes-Almagro b , S. Ogueta b , J.A. Bárcena a, ⁎ , C.A. Padilla a a Department of Biochemistry and Molecular Biology, University of Córdoba and Córdoba Maimónides Institute for Biomedical Research (IMIBIC), Córdoba, Spain b Proteomics Facility, SCAI, University of Córdoba, Spain ARTICLE INFO ABSTRACT Article history: Received 20 January 2011 Accepted 18 April 2011 Available online 30 April 2011 Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential “Thiolic Redox Proteome” of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms. © 2011 Elsevier B.V. All rights reserved. Keywords: Glutathione Glutaredoxin Mitochondria Thiol-disulfide Yeast Redox proteome 1. Introduction In the era of functional genomics, the sequenced genome of Saccharomyces cerevisiae is more than ever an invaluable tool for studying fundamental mechanisms characteristic of the eukaryotic cell. Within proteins, the thiol group (- SH) of cysteines (Cys) plays a crucial role in chemical reactions. The reactive thiol group may undergo a number of oxidative modifications, two thiol groups may form a disulfide bridge which occurs in some proteins (PSSP), in oxidized glutathione (GSSG) or as a mixed disulfide in S-glutathionylated proteins (PSSG). Apart from these reactions the Cys can be reversibly oxidized by ROS to sulfenic acids (\SOH) and nitrosothiols (\SNO) [1]. There is increasing evidence regarding the number of proteins whose function is regulated by the modification of a JOURNAL OF PROTEOMICS 74 (2011) 2487 – 2497 ⁎ Corresponding author. E-mail address: ja.barcena@uco.es (J.A. Bárcena). 1 These authors contributed equally. 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.04.018 available at www.sciencedirect.com www.elsevier.com/locate/jprot