title: The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation.
abstract: In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post-translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A(4)-glyceraldehyde-3-phosphate dehydrogenase (A(4)-GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H(2)O(2). The formation of a mixed disulfide between glutathione and A(4)-GAPDH resulted in the inhibition of enzyme activity. A(4)-GAPDH was also inhibited by oxidants such as H(2)O(2). However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the A(n)B(n)-GAPDH isozyme in either A(2)B(2) or A(8)B(8) conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3-bisphosphoglycerate. In the presence of 1,3-bisphosphoglycerate, A(4)-GAPDH was fully protected from either oxidation or glutathionylation. Site-directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active-site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A(4)-GAPDH from irreversible oxidation under stress.
authors.affiliation: Laboratory of Molecular Plant Physiology, University of Bologna, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.forename: Laure
authors.initials: L
authors.lastname: Michelet
authors.forename: Christophe
authors.initials: C
authors.lastname: Marchand
authors.forename: Francesca
authors.initials: F
authors.lastname: Sparla
authors.forename: Paulette
authors.initials: P
authors.lastname: Decottignies
authors.forename: Pierre
authors.initials: P
authors.lastname: Le Maréchal
authors.forename: Myroslawa
authors.initials: M
authors.lastname: Miginiac-Maslow
authors.forename: Graham
authors.initials: G
authors.lastname: Noctor
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000378:metabolism / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D020033:Protein Isoforms / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018724:Spinacia oleracea / Q000201:enzymology / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D016877:Oxidants; D020033:Protein Isoforms; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide