Stress-Induced Protein <em>S</em>-Glutathionylation in Arabidopsis<sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
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 [S]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; γ-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.
Notes
This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. 12/P13738).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058917.