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.

FIGURE 1.

Three-dimensional modeling of C. reinhardtii ICL. Modeling was made using Swiss-Model workspace (available on the World Wide Web) based on the known structure of ICL from Mycobacterium tuberculosis (Protein Data Bank code 1F61). The structure was generated with the Swiss-PDB viewer software and rendered with POV-Ray (available on the World Wide Web). The catalytic cysteine 178 appears in orange, and the three other cysteines are shown in blue. Inset, black broken lines represent the distance between sulfur atoms (in Å). Two histidine residues potentially important for ICL reactivity (His¹⁶⁷ and His¹⁸⁰) and located near Cys¹⁶⁵ and Cys¹⁷⁸ are also represented.

FIGURE 2.

Effect of oxidative treatments on CrICL activity. Reduced CrICL (control) was incubated for 30 min in the presence of different oxidants as indicated. The reversibility of CrICL inactivation was assessed by incubation in the presence of 20 mm DTT (+DTT). Data are represented as mean percentage of maximal activity ± S.D. (n = 3–5). 100% corresponds to the initial activity of reduced ICL.

FIGURE 3.

Kinetics of inactivation of CrICL by GSSG. Prereduced CrICL (10 μm) was incubated in the presence (closed circles) or absence (open squares) of 5 mm GSSG. Aliquots were withdrawn at the indicated times to assay enzyme activity. Activities are represented as mean percentage ± S.D. (n = 3–5) of the initial activity measured before treatments.

FIGURE 4.

Kinetics of reactivation of glutathionylated CrICL. A, reactivation in the presence of DTTred. CrICL was inactivated by incubation with 5 mm GSSG until no residual activity was detected and dialyzed overnight against HEPES-NaOH (pH 7.2). This GSSG-inactivated ICL was incubated with no addition (black circles), with DTTred alone (0.2 mm (closed squares) or 20 mm (closed diamonds)) or with 0.2 mm DTTred in the presence of 20 μm TRXh1 (open circles) or 1 μm GRX1 (open squares). The enzyme was also treated with 20 μm TRXh1 in the presence of 0.3 mm NADPH and 0.22 μm A. thaliana NADPH-thioredoxin reductase b (gray circles). B, reactivation in the presence of GSH and GRX. GSSG inactivated CrICL was incubated with 5 mm GSH (closed triangles) or with 5 mm GSH in the presence of 1 μm GRX1 alone (open triangles) or in the presence of 0.2 mm NADPH and yeast glutathione reductase (GR) (6 μg/ml) (gray triangles). 100% corresponds to the mean maximal reactivation measured after treatment with 20 mm DTT.

FIGURE 5.

Analysis of CrICL glutathionylation with BioGSSG. Recombinant WT CrICL and the variants C178S, C165S/C247S, and C165S/C247S/C301S were incubated for 1 h in the presence of BioGSSG (2 mm) with or without prior incubation with 100 mm IAM. Proteins were resolved by non-reducing SDS-PAGE and transferred to nitrocellulose for Western blotting with anti-biotin antibodies. The Coomassie Brilliant Blue staining of the gel shows equal loading in each lane. The reversibility of the reaction was assessed by treatment with 20 mm DTTred for 30 min as indicated. CrICL activity was measured on aliquots after BioGSSG treatment. Activities are represented as mean percentage ± S.D. (n = 3–5) of the initial activity measured before inactivation treatments.

FIGURE 6.

Reactivation of GSSG inactivated WT CrICL and variants by TRXh1 and GRX1. Recombinant WT CrICL and variants were inactivated by incubation with 5 mm GSSG until no residual activity was detected and dialyzed overnight against HEPES-NaOH (pH 7.2). The inactivated proteins were treated with 20 mm DTTred or 0.2 mm DTTred alone or supplemented with either 20 μm TRXh1 or 1 μm GRX1. The protein activity was determined on WT CrICL (black bar), CrICL C165S (white bar), CrICL C247S (gray bar), CrICL C301S (white bar with diagonals), and CrICL C165S/C247S (gray bar with diagonals). Activities are represented as mean percentage ± S.D. (n = 3–5) of the initial activity measured for each protein before GSSG treatment.

FIGURE 7.

Peptide mass fingerprinting of CrICL reveals that Cys¹⁷⁸ and Cys²⁴⁷ are glutathionylated. WT CrICL was incubated with 5 mm GSSG for 24 h and dialyzed overnight against HEPES-NaOH (pH 7.2). Peptide mass fingerprints of Glu-C digestions after alkylation of cysteines with iodoacetamide (+57 Da) showed a glutathione adduct (+305 Da) on peptides Asp¹⁷⁰–Glu¹⁹¹ and Gly²⁴⁴–Glu²⁷² (A). For both peptides, the 305-Da mass increase was reversed by DTTred treatment (B). The peptides containing Cys¹⁶⁵ and Cys³⁰¹ were found to be exclusively carbamidomethylated.