Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

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.

Role of Glyoxylate Shunt in Oxidative Stress Response

“…1C). It has been reported that Chlamydomonas reinhardtii regulates isocitrate lyase by glutathionylation (51) and that a transaminase (PA0132), which was highly up-regulated in PQtreated WT cells (supplemental Table S5), can transform glyoxylate to glycine, a component of glutathione. Further experiments are warranted to investigate the role of glyoxylate in the regulation and production of glutathione in P. aeruginosa (Fig.…”

The Synechocystis PCC6803 MerA-Like Enzyme Operates in the Reduction of Both Mercury and Uranium under the Control of the Glutaredoxin 1 Enzyme

“…By analogy with these data, the presently described MerA-Grx1 interaction led us to speculate that MerA might be glutathionylated upon Hg 2ϩ reduction and subsequently deglutathionylated by Grx1. Hence, using standard in vitro procedures to assay glutathionylation (27,28), MerA was incubated with biotinylated glutathione (BioGSSG). Subsequently, the presence of glutathione adducts on MerA was analyzed by Western blotting using an antibiotin antibody (Fig.…”

“…The water-soluble biotinylation reagent EZ Link Sulfo-NHS-Biotin (Perbio Science) was used to couple biotin to the primary amino group of GSSG, as described previously (28). The biotinylation reagent (40 l; 48 mM) was incubated with GSSG (40 l; 32 mM) in 50 mM potassium phosphate buffer (pH 7.2) for 1 h at room temperature, generating biotinylated GSSG (BioGSSG).…”

“…All treatments were performed at room temperature. Proteins were then loaded onto nonreducing SDS-PAGE gels and analyzed by Western blotting using antibiotin antibodies, as described previously (28).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Proteins specifically produced in n-hexane grown cells but not in succinate grown cells were an isocytrate lyase (spots B and C), a malate dehydrogenase (6D) and the subunit beta of a succinyl CoA synthetase (6D). The two different pI of isocytrate lyase are most likely caused by post-translation modifications of the protein [360]. The over-expression of the isocytrate lyase and malate dehydrogenase would suggest the key role of glyoxylate cycle in n-hexane-grown BCP1 cells as it was described in A. borkumensis, since the isocytrate lyase is the central enzyme of the cycle and malate dehydrogense has a role in restoring the isocitrate used in the cycle.…”