title: Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro.
abstract: Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys(149).
authors.affiliation: Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
authors.forename: Mariette
authors.initials: M
authors.lastname: Bedhomme
authors.forename: Mattia
authors.initials: M
authors.lastname: Adamo
authors.forename: Christophe H
authors.initials: CH
authors.lastname: Marchand
authors.forename: Jérémy
authors.initials: J
authors.lastname: Couturier
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D054477:Glutaredoxins; D007527:Isoenzymes; D011994:Recombinant Proteins; D013879:Thioredoxins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; D003545:Cysteine