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First published online July 29, 2005; 10.1104/pp.104.058917 Plant Physiology 138:2233-2244 (2005) © 2005 American Society of Plant Biologists
Stress-Induced Protein S-Glutathionylation in Arabidopsis1School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom
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.
The tripeptide glutathione (GSH;  -Glu-Cys-Gly) serves important functions in plants as a reductant, transiently accumulating under stress conditions as its oxidized disulfide (GSSG). As well as forming disulfides with itself, GSH can also form mixed disulfides with proteinaceous Cys. This S-glutathionylation of proteins is commonly termed thiolation and has recently become established as a widespread reversible posttranslational modification of proteins that occurs in animal and fungal cells exposed to oxidative stress (Klatt and Lamas, 2000 ). Although originally considered to be a protective mechanism to prevent the irreversible oxidation of reactive protein-bound Cys to their sulfinic and sulfonic acid derivatives, it is now recognized that thiolation can act as a redox-driven regulator of signal transduction cascades and metabolic pathways (Fratelli et al., 2004). As such, thiolation has clear parallels with redox regulation of proteins mediated through intramolecular or intermolecular disulfide formation with other proteinaceous Cys (Lee et al., 2004). In plants, proteomic approaches have focused upon the redox regulation of proteins forming protein disulfides with thioredoxin in differing subcellular compartments (Motohashi et al., 2001; Balmer et al., 2003, 2004; Marchand et al., 2004; Yamazaki et al., 2004). The total redox-responsive disulfide proteome of Arabidopsis (Arabidopsis thaliana) has also recently been investigated using large-scale proteomic techniques (Lee et al., 2004). However, few studies have focused specifically on the ability of proteins to form mixed protein disulfides with GSH and how this modification may regulate their activity. Ito et al. (2003) fed a biotinylated glutathione ester to Arabidopsis cell cultures and reported that some 20 proteins underwent thiolation, though only two of these polypeptides were subsequently identified by sequencing, highlighting the difficulties of working in vivo. In view of the unique oxidative stresses placed on plants, we were interested in exploring the thiolation of the Arabidopsis proteome, in particular comparing and contrasting the results obtained with those studies directed at the redox regulation of Arabidopsis proteins either by thioredoxin or by the reduction of intramolecular disulfides. To this end, we now report on an efficient in vitro method to systematically identify Arabidopsis polypeptides that are capable of undergoing rapid S-glutathionylation and the effect of this modification on the activity of specific thiolated proteins.
In Vivo Thiolation of Arabidopsis Proteins
Arabidopsis suspension cultures treated with a chemical oxidant were used as a classic system to perturb GSH metabolism and induce oxidative stress in a large mass of plant cells (May and Leaver, 1993
Identification of in Vivo Thiolated Proteins
Biotinylated, oxidized glutathione (GSSG-biotin) was prepared such that each GSH molecule was linked via its free amino group to biotin. Arabidopsis suspension cultures were then treated with the GSSG-biotin for 1 h followed by a 30-min oxidizing treatment with BHP, and biotinylated proteins present were purified using streptavidin-agarose. Proteins attached to the matrix by association with the biotinylated glutathione tag were selectively released using DTT to cleave the disulfides and then analyzed by 2D-PAGE (Fig. 2). The most abundant polypeptides were then identified by peptide mass fingerprinting (Table I). Eight protein families could be identified, though most of the polypeptides examined were derived from either
Identification of in Vitro Thiolated Proteins
Proteins were thiolated in vitro with GSSG-biotin and then directly purified using streptavidin-agarose. To confirm that the derivatization with biotin did not affect the ability of GSH to form disulfides, the GSSG-biotin was incubated with freshly reduced Arabidopsis dehydroascorbate reductase 1 (AtDHAR1), a plant enzyme known to undergo S-glutathionylation through a single highly reactive Cys residue (Dixon et al., 2002
Naturally biotinylated proteins were not present in the affinity-purified sample, as they would not have been released from the streptavidin matrix under the gentle, reducing conditions used for elution of the disulfide-bound proteins. However, the method used could not discriminate between proteins that were thiolated directly from those proteins that were present in the final preparation due to their close interaction with GS-biotin-tagged proteins. To test this possibility, an additional purification step was carried out, in which the proteins bound to the streptavidin matrix were washed with 6 M urea to disrupt protein-protein interactions. The remaining disulfide-bound proteins were then eluted with DTT and analyzed by 2D-PAGE (Fig. 4B). This additional wash substantially reduced the yield of recovered protein and also considerably altered the pattern of proteins obtained. Peptide mass fingerprinting identified 22 proteins from this sample, of which six had not been picked from the previous gels (Table II).
Having identified Arabidopsis proteins that underwent thiolation in vitro, it was then of interest to determine the associated mechanism of this derivatization and its effect on protein function. Individual proteins were cloned and expressed in Escherichia coli as His-tagged polypeptides, and the purified recombinant proteins incubated with GSSG to promote thiolation were then analyzed by ESI-MS. One of the proteins identified as being thiolated in the in vitro screen was AtDHAR1 (spot ID 26) that had already been shown to undergo quantitative thiolation with a single molecule of GSH (Fig. 3), at the site of the catalytic Cys-20 (Dixon et al., 2002
Since the studies with the recombinant proteins relied on spontaneous disulfide exchange between the polypeptide and GSSG, one possibility was that the thiolation of these polypeptides observed in the crude protein extracts was due to the intervention of a catalyst. To test this possibility, the incubations with the recombinant proteins were repeated using GSSG-biotin in the presence or absence of crude Arabidopsis protein extract. Thiolation of the proteins was then monitored after resolving the repurified polypeptides by nonreducing SDS-PAGE and probing blots with streptavidin to detect GS-biotin-conjugated proteins (Fig. 6). In the presence of GSSG alone, this assay confirmed the spontaneous thiolation of AtDHAR1, with the respective GS-biotin-labeled monomer, dimer, trimer, and tetramer adducts identified in the absence of reducing agent (Fig. 6A). AtADH and AtMetS did not undergo thiolation in the presence of GSSG alone, though biotinylation of AtNit1 was detected even though this modification had not been determined previously by ESI-MS (Fig. 6E). Under nonreducing conditions, AtNit1 ran as a doublet of GSH-biotin-labeled protein. As determined with the other proteins, when AtNit1 was analyzed by SDS-PAGE under reducing conditions, an anti-His-tag serum only recognized a single polypeptide species in the preparation, suggesting that the multiple bands were due to AtNit1 species showing different degrees of thiolation. From this it was concluded that AtNit1 could undergo modest thiolation in the presence of GSSG alone, though this could only be detected with the very sensitive streptavidin-coupled blotting reagent. When the recombinant proteins were incubated with GSSG in the presence of the crude preparation from Arabidopsis, both AtADH and AtMetS formed GS-biotin-protein mixed disulfides (Fig. 6, B and G). In both cases, multiple polypeptide species were identified using streptavidin. In the case of AtMetS, the low molecular weight species were most likely due to proteolysis. Similarly, in the 2D gel electrophoresis experiments, several polypeptides with differing masses were identified by MALDI-TOF MS as AtMetS consistent with limited proteolysis of the parent protein. With AtADH, the single major polypeptide identified under nonreducing conditions appeared to correspond to the native enzyme. The other labeled protein species observed must have resulted from different charge/size variants of the unreduced polypeptide due to aberrant oxidation of sulfhydryl groups since the anti-His-tag serum only recognized a single species when AtADH was analyzed by SDS-PAGE under reducing conditions (Fig. 6D). Whereas AtMetS and AtADH only became thiolated in the presence of the Arabidopsis protein extract, the effect of adding the catalyst to the AtDHAR1 and AtNit1 preparations reduced their conjugation with GS-biotin (Fig. 6, A and E). Since the amount of target protein was unchanged (Fig. 6, B and F), we concluded that the reduction in thiolation was due to the competition for GSSG-biotin by the other proteins present in the crude extract. Having established that AtADH and AtMetS required other Arabidopsis proteins to become thiolated, it was of interest to determine how the associated reaction might be catalyzed. To test for the involvement of protein Cys mediating disulfide exchange reactions, the crude mixture was treated with iodoacetamide or p-chloromercuribenzoate (pCMB), two treatments known to differentially inhibit Cys-dependent enzymes. Protein-mediated thiolation of AtADH and AtMetS was lost on alkylation of sulfhydryl groups with iodoacetamide but was not inhibited by pCMB (Fig. 6, B and G).
Using a combination of radiolabeling studies and in vivo and in vitro proteomics, we have demonstrated that protein thiolation is a widespread response to oxidizing conditions in Arabidopsis suspension cultures. By combining the results of the in vivo and in vitro GS-biotin-labeling studies, 2D-PAGE followed by MALDI MS identified 79 distinct proteins that can either be thiolated or form very stable complexes with glutathionylated proteins. It is also clear from the 2D gels that there are many other less abundant thiolated proteins to be identified. Surprisingly, the range of polypeptides found to undergo thiolation with GSSG-biotin was very different in the in vivo and in vitro labeling experiments. Two proteins, Suc synthase and a subunit of acetyl-CoA carboxylase, were identified as being thiolated in both studies, confirming that, where available, proteins would undergo mixed disulfide formation both in and outside the cellular environment. However, whereas in the in vitro studies a diverse range of polypeptides were thiolated, a much smaller range of proteins underwent this modification in vivo, and this was associated with their proteolytic degradation. There are many reasons to explain the differences in vivo and in vitro thiolation, including the bioavailability and compartmentalization of GS-biotin in the two test systems, and the redox status of the protein targets. For example, many of the proteins identified as undergoing thiolation in the sensitive in vitro screen may be constitutively S-glutathionylated in plant cells prior to feeding with GSSG-biotin and BHP. The proteolysis associated with the in vivo feeding study also indicated that the chemical treatments had seriously perturbed the cellular environment. For these reasons, the in vitro experiments give us a good appreciation of the total range of proteins that can undergo thiolation, as has recently been demonstrated with the Arabidopsis proteins undergoing S-nitrosylation (Lindermayr et al., 2005 ). The next technical challenge with these protein modification studies is to develop protocols allowing the reliable monitoring of these reactions within the cell while avoiding nonspecific side effects.
While there have been several recent large-scale proteomic studies directed at identifying redox reactive protein thiols in animal cells (Baty et al., 2002
Thiolation of protein sulfhydryl groups protects cysteinyl residues from irreversible oxidation to their sulfinic and sulfonic acid derivatives. As a freely reversible posttranslational modification, thiolation is known to regulate the activity of enzymes and regulatory proteins (Klatt and Lamas, 2000 Based on the known roles for thiolation in animals, it is possible to functionally group the proteins identified by our proteomics approach in Arabidopsis into three sets: those whose activity is likely to be directly modulated by S-glutathionylation, those that undergo the modification without altering their immediate activity, and those proteins that have been identified because they are closely associated with thiolated proteins rather than undergoing this modification in their own right. To distinguish between glutathionylated proteins and any associated nonthiolated proteins, the streptavidin-bound proteins were washed with the denaturant 6 M urea to disrupt noncovalent interactions. Therefore, the urea wash should largely consist of polypeptides bound by protein-protein interactions rather than by thiolation. However, as denaturation exposed previously inaccessible Cys residues, there is a possibility that the revealing of the free thiols could promote intramolecular disulfide bond formation leading to the displacement of GS-biotin, thereby losing some thiolated proteins in this wash. Thus, while we can be confident that the proteins left on the streptavidin beads after urea treatment that were selectively eluted with DTT treatment were thiolated, it is also possible that some of the S-glutathionylated proteins were lost on denaturation.
We first considered those proteins that were thiolated due to their possession of reactive Cys in the active site. Enzymes that utilize active-site Cys as the respective reactive thiolate anions to drive catalysis are particularly susceptible to thiolation. The lambda GSTLs and DHARs had previously been characterized as glutathione S-transferases that could readily undergo thiolation in vitro (Dixon et al., 2002
The use of Cys to coordinate catalytic metal ions will also predispose proteins to thiolation. One of the most abundant proteins in the purified sample of thiolated proteins was AtMetS, as shown by its intense spot on 2D-PAGE and by the identification of numerous degradation products in the in vitro labeling study. The recent crystal structure of AtMetS (Ferrer et al., 2004
Another group of proteins undergoing changes in function following thiolation do so by less well defined mechanisms. One major group of polypeptides identified in the thiolation screen contained components of the 20S proteasome. The 20S proteasome complex is made up of four seven-subunit rings (two
There were then those proteins that were identified in the screen where thiolation probably did not directly affect activity. Because of our interest in glutathione-dependent enzymes, the presence of phi (GSTF)- and tau (GSTU)-class glutathione transferases was intriguing, as based on available structural models these proteins are unlikely to have Cys in their active sites (Prade et al., 1998
Finally, there were examples of polypeptides that could not undergo thiolation and must have therefore been identified due to their close association with other S-glutathionylated proteins. One example was 20S proteasome
Major differences were determined in the proteins identified by the in vitro thiolation screen as compared with other searches for redox-reactive proteins in Arabidopsis. Only one previous study has reported protein thiolation in Arabidopsis. Using a carboxyethyl ester of biotinylated glutathione to aid uptake into intact living cells, thiolation of some 20 polypeptides resolved on a nonreducing SDS-PAGE gel was reported, with two identified as Fru-bisphosphate aldolase and triose phosphate isomerase (Ito et al., 2003
Although relatively little attention has been paid to the regulatory roles of thiolation in plants, there has been very significant progress made in identifying those proteins targeted for thioredoxin-mediated disulfide exchange (Buchanan and Balmer, 2005
From our analysis of the nature of the in vitro thiolation of five recombinant proteins by GSSG-biotin treatment, it is clear that Arabidopsis proteins can be thiolated by at least two mechanisms. AtDHAR1 and, to a lesser extent, AtNit1 underwent spontaneous thiolation in the presence of GSSG-biotin. By contrast, AtADH and AtMetS both required the inclusion of crude protein, with the associated thiolating activity inhibited by S-alkylation, providing good evidence for the requirement of an enzyme with a free sulfhydryl group to catalyze glutathionylation. The crude protein extract could promote thiolation by two mechanisms. First, the enzyme catalyst could increase the reactivity of the cysteinyl residue of the protein such that it can drive disulfide formation, for example, by converting the thiol to a reactive sulfenic acid derivative. Such a mechanism has been demonstrated for H2O2-mediated thiolation of 20S proteasome polypeptides in yeast (Demasi et al., 2003
Chemicals GSSG-biotin was synthesized by incubating 2.5 mM GSSG and 7.5 mM EZ-link sulfo-NHS-LC-LC-biotin (Perbio Science, Cramlington, UK) in 0.1 M potassium phosphate buffer, pH 8.0, for 2 h at 20°C. Unreacted biotin reagent was then removed by reacting with 20 mM Tris-Cl buffer, pH 7.5. The derivatization of both amino groups of GSSG was confirmed by ESI-MS with the expected reaction product (m/z for M + H+ = 1,517.6) determined. GSSG-biotin was stored at –20°C and was used without further purification.
Arabidopsis (Arabidopsis thaliana) cell cultures (50 mL) were maintained, used, and harvested in mid-log growth as described (Loutre et al., 2003
All thiol analyses were carried out in triplicate on cells treated with L-[35S]Cys, using internal standards of GSH to correct for losses during derivatization (Cummins et al., 1997
All purification steps were carried out at 4°C. For in vitro thiolation studies, Arabidopsis cells (10 g) were homogenized in 0.1 M Tris-Cl, pH 7.5, containing 1 mM EDTA, 2 mM DTT, and, after centrifugation (13,000g, 20 min), protein precipitated by addition of (NH4)2SO4 to 80% saturation. Following recentrifugation, the protein pellet was desalted in 20 mM Tris-Cl, pH 7.5 (18 mL), using a HiTrap desalting column (Amersham Biosciences, Chalfont St. Giles, UK). GSSG-biotin (10 µM) was added to and the sample incubated for 10 min, prior to precipitation of the proteins with 80% (NH4)2SO4. The pellet was then washed with buffer A (20 mM Tris-Cl, 0.5 M NaCl, 1 mM EDTA, pH 6.8) containing 80% (NH4)2SO4, to remove unreacted GSSG-biotin, prior to desalting the protein in buffer A (12 mL). For in vivo thiolation, protein was extracted in 0.1 M Tris-Cl, pH 7.5, containing 1 mM EDTA and then treated as above, except that the initial desalting, GSSG-biotin treatment, and subsequent precipitation steps were omitted. To purify thiolated proteins, 750 µL of streptavidin-agarose resin (Amersham Biosciences), prewashed with buffer A, was added to the extract and mixed gently for 10 min. The matrix was pelleted by centrifugation (700g, 2 min) and washed four times with 40 mL buffer A. The matrix was then resuspended in buffer A (4 mL) containing 10 mM DTT for 15 min at 20°C to release proteins that had formed mixed disulfides with biotinylated GSH. The filtered protein extract was precipitated with acetone (16 mL) at –20°C for 16 h and the pellet washed with 80% acetone. As a control, the above was repeated using nonbiotinylated GSSG to detect proteins that bound nonspecifically to the streptavidin matrix. For in vitro thiolation studies, experiments were also performed with the inclusion of an extra wash procedure to remove proteins that were interacting with the thiolated proteins bound to the streptavidin rather than being disulfide bonded to the biotinylated GSH. After binding the thiolated protein mixture, the matrix was treated with 2.5 mL of buffer B (20 mM Tris-Cl, 6 M urea, pH 6.8). Following a 15-min incubation at 20°C, the displaced protein solution was separated from the matrix by filtration, with the streptavidin-agarose resin then washed two times with 25 mL buffer A and the disulfide-bound protein recovered with buffer A containing DTT.
Affinity-purified thiolated proteins were analyzed by 2D-PAGE. Acetone-precipitated proteins were redissolved in 340 µL isoelectric focusing (IEF) buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 40 mM DTT, 0.5% pH 3-10NL ampholytes, 0.002% [w/v] bromphenol blue) and subjected to IEF on 18-cm pH 3-10NL Immobiline DryStrips (Amersham Biosciences) using a Multiphor II flatbed electrophoresis system as recommended by the manufacturer. Following IEF, strips were washed first in second dimension buffer (50 mM Tris-Cl, pH 8.8, 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS, 0.002% [w/v] bromphenol blue) containing 1% (w/v) DTT (15 min, 20°C), then in second dimension buffer containing 2.5% (w/v) iodoacetamide (15 min, 20°C). Strips were run on ExcelGel 2D homogeneous 12.5% PAGE gels (Amersham Biosciences), then stained with SYPRO Ruby protein gel stain (Invitrogen, Paisley, UK) or with silver stain. Major spots were picked, trypsin digested, and analyzed on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Warrington, UK) as described (Chivasa et al., 2002
The coding sequences of thiolated proteins were PCR amplified from cDNA prepared from an Arabidopsis ethanol-treated root culture (Dixon et al., 2002
Direct modification of purified proteins by thiol reagents and subsequent electrospray-MS analysis was performed using a Micromass LCT mass spectrometer as described previously (Dixon et al., 2002 After incubating for 20 min at 4°C, Ni-NTA magnetic agarose beads (50 µL; Qiagen, Crawley, UK) were added to purify the His-tagged recombinant proteins, which were then analyzed by SDS-PAGE under nonreducing conditions (DTT omitted from loading buffer). Thiolation was detected by western blotting onto polyvinylidene difluoride membrane and probing for proteins containing biotinylated GSH using alkaline phosphatase-linked streptavidin (Sigma-Aldrich, St. Louis). Recombinant protein recovery was monitored by anti-His C-terminal antibody (Invitrogen) and horseradish-peroxidase-linked anti-mouse secondary antibody using ECL+ detection using a Typhoon 9400 imaging system (Amersham Biosciences). Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Received December 23, 2004; returned for revision March 23, 2005; accepted May 3, 2005.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. 12/P13738). 
2 These authors contributed equally to the paper.
3 Present address: Smith and Nephew Research Centre, York Science Park, Heslington, York YO1 5DF, UK. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058917. * Corresponding author; e-mail robert.edwards{at}durham.ac.uk; fax 0044–191–334–1201.
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ABSTRACT
RESULTS
-tubulin or Suc synthase. Since many of the polypeptides identified were smaller than expected, this suggested that the GSSG-biotin/BHP feeding study had elicited large-scale proteolysis. To extend these thiolation studies with GSSG-biotin using conditions that would not induce protein degradation, a more sensitive in vitro labeling system was developed. This had the additional benefit of increasing the likelihood of identifying a more comprehensive range of proteins undergoing thiolation since in the in vivo system it was likely that many proteins may already have been disulfide modified prior to treatment with GSSG-biotin. 
-subunit rings and two 
