Abstract
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.
Highlights
The importance of redox regulation and redox signaling linked to post-translational modifications of cysteine residues of proteins starts to be widely recognized
In C. reinhardtii, isocitrate lyase was previously identified both as a putative TRX target retained on a monocysteinic TRX affinity column [10] and as a protein undergoing S-thiolation in
The results clearly show that purified C. reinhardtii ICL (CrICL) can be inactivated by glutathionylation and reactivated by GRX, whereas TRX does not appear to regulate isocitrate lyase (ICL) activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested
Summary
The importance of redox regulation and redox signaling linked to post-translational modifications of cysteine residues of proteins starts to be widely recognized. Cysteine residues can undergo different states of oxidation, such as sulfenic, sulfinic, and sulfonic acid, and protein disulfide bridges (intra- or intermolecular), S-thiolation (mainly glutathionylation), or nitrosylation These post-translational redox modifications are mainly under the control of two types of ubiquitous disulfide oxidoreductases: thioredoxins (TRXs) and glutaredoxins (GRXs). Besides TRX-dependent regulation of the redox state of protein disulfide bonds, glutathionylation has recently emerged, among other thiol-based post-translational modifications, as an important redox-based signaling mechanism [3,4,5]. This modification consists in the formation of a mixed disulfide between an accessible free thiol on a protein and a molecule of glutathione. Isocitrate lyase (ICL), was identified both as a putative TRX target and as an S-thiolated protein in vivo
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