title: Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.
abstract: Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.
authors.affiliation: Department of Plant Physiology, University of Osnabrueck, D-49069 Osnabrueck, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Holtgrefe
authors.forename: Jochen
authors.initials: J
authors.lastname: Gohlke
authors.forename: Julia
authors.initials: J
authors.lastname: Starmann
authors.forename: Samantha
authors.initials: S
authors.lastname: Druce
authors.forename: Susanne
authors.initials: S
authors.lastname: Klocke
authors.forename: Bianca
authors.initials: B
authors.lastname: Altmann
authors.forename: Joanna
authors.initials: J
authors.lastname: Wojtera
authors.forename: Christian
authors.initials: C
authors.lastname: Lindermayr
authors.forename: Renate
authors.initials: R
authors.lastname: Scheibe
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects* / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011523:Protoplasts / Q000187:drug effects / Q000201:enzymology; D011817:Rabbits; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D050505:Mutant Proteins; D013438:Sulfhydryl Compounds; D026422:S-Nitrosoglutathione; C042431:S-ethyl glutathione; D004247:DNA; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C585192:GAPC1 protein, Arabidopsis; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D005978:Glutathione; D003545:Cysteine