Regulation by glutathionylation of isocitrate lyase from Chlamydomonas reinhardtii.
Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys(178), whereas a second residue, Cys(247), becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.
Bedhomme
Mariette
M
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France.
Zaffagnini
Mirko
M
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France.
Marchand
Christophe H
CH
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS/Université Paris-Sud, Bâtiment 430, 91405 Orsay, Cedex, France.
Gao
Xing-Huang
XH
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France.
Moslonka-Lefebvre
Mathieu
M
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France.
Michelet
Laure
L
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France.
Decottignies
Paulette
P
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS/Université Paris-Sud, Bâtiment 430, 91405 Orsay, Cedex, France.
Lemaire
Stéphane D
SD
Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Bâtiment 630, 91405 Orsay, Cedex, France. Electronic address: stephane.lemaire@u-psud.fr.
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D007522:Isocitrate Lyase / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
D020418:Algal Proteins; D054477:Glutaredoxins; D015800:Protozoan Proteins; D007522:Isocitrate Lyase; D005978:Glutathione