Stress-induced protein S-glutathionylation in Arabidopsis

Abstract

S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [35S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.

Figure 1.

Protein-bound [³⁵S]GSH in Arabidopsis cell cultures following 0- to 4-h treatment with water (Control) or 1 mm BHP. Arabidopsis cell cultures were prelabeled with 25 μCi l-[³⁵S]Cys in the presence of cycloheximide for 4 h, then the protein-bound radioactive GSH content of each sample was determined by radio-HPLC at various times after treatment. Values are the average of duplicate experiments with the error bars showing the variation in the replicates.

Figure 2.

Silver-stained 2D-PAGE of in vivo thiolated (using GSSG-biotin) proteins from Arabidopsis cell culture extracts. Labeled arrows indicate locations of identified polypeptides, whereas spots that were picked but gave insufficient data for unambiguous identification are not labeled. Approximate mass (in kilodaltons) and pI range are shown.

Figure 3.

Deconvoluted mass spectra of fully reduced AtDHAR1 before (gray line) and after (black line) modification with GSSG-biotin, showing quantitative addition of a 758-D adduct corresponding to biotinylated glutathione.

Figure 4.

Two-dimensional PAGE of thiolated proteins from Arabidopsis cell culture extracts. Proteins and protein complexes labeled with GSSG-biotin were retained on a streptavidin matrix, with thiolated proteins then released from the column with a single treatment of DTT (A) or first washed with 6 m urea then released with DTT (B). Labeled arrows indicate locations of identified polypeptides, whereas spots that were picked but gave insufficient data for unambiguous identification are not labeled. Approximate mass (in kilodaltons) and pI range are shown. Gel analyses were performed in duplicate.

Figure 5.

Electrospray-MS analysis of AtGSTZ1 before (solid line) and after (dashed line) treatment with oxidized glutathione, showing efficient single glutathionylation of enzyme (E) and its dehydrated form (E − H₂O). Spectra are deconvoluted from multiply charged ion spectra. The expected mass for AtGSTZ1 (after cleavage of the initial Met) was 25,807.5 D.

Figure 6.

Western blots of recombinant proteins following exposure to GSSG-biotin. Recombinant proteins AtDHAR (blots A and B), AtADH (blots C and D), AtNit1 (blots E and F), and AtMetS (blots G and H) were exposed to GSSG-biotin under a variety of conditions. Gels A, C, E, and G were run under nonreducing conditions, whereas gels B, D, F, and H were run after reduction of the proteins. In each case, the polypeptide migrating as the expected monomeric species is arrowed, with any additional species being due either to intramolecular/intermolecular disulfide formation or to limited proteolysis. For each blot, lane 1 shows recombinant protein exposed to GSSG-biotin alone, lane 2 shows recombinant protein exposed to GSSG-biotin in the presence of crude Arabidopsis total protein extract, lane 3 shows recombinant protein exposed to GSSG-biotin in the presence of alkylated crude protein extract, lane 4 shows recombinant protein exposed to GSSG-biotin in the presence of crude protein extract and pCMB, and lane 5 shows recombinant protein exposed to crude protein extract in the absence of GSSG-biotin. Blots A, C, E, and G show GSSG-biotin-labeled proteins, whereas blots B, D, F, and H show His-tag detection of identical samples to the corresponding lanes in blots A, C, E, and G, showing equivalent recoveries of recombinant protein.