Cloning of Arabidopsis thaliana Glutathione Synthetase (GSH2) by Functional Complementation of a Yeast Gsh2 Mutant

Abstract

Glutathione (L-y-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Ambidopsis thaliunu was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Succharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced M,: 53 783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharonzyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The M , is similar to those of soluble animal enzymes, suggesting that the Amhidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectibility in mutants to about 50% of the wild-type levels in transformed cells.Keywords: glutathione synthetase; expression cloning; Arabidopsis thaliana; evolutionary relationships; cadmium.Glutathione (L-y-glutamyl-L-cysteinylglycine, GSH) is an ubiquitous tripeptide (Rennenberg, 1982;Fahey and Sundquist, 1991: Penninckx andElskens, 1993) that plays an important role in the protection of plants against various types of stress caused by reactive oxygen species and gazeous pollutants (Alscher, 19891, low temperature (De Kok and Oosterhuis, 1983) and heavy metals (Grill et al., 1985). GSH is also involved in the detoxification of xenobiotics through formation of conjugates via the action of' various GSH S-transferases (Timmerman, 1989). For some safeners, compounds which increase the selectivity of some herbicides, it was reported that the protection of the crop plant is partially due to a stimulation of GSH accumulation (Farago et al., 1994), followed by increased conjugation and sequestration into the vacuole (Gaillard et al., 1994). Furthermore, GSH, which is chemically stable, represents the most abundant form of reduced sulphur within the cell and, in plants, it appears to be involved in the control of uptake and xylem loading of sulfate (Herschbach and Rennenberg, 1994 Ahhreviations. GSH, glutathione; GSH-I, y-glutamylcysteine synthetase; GSHI, y-glutamylcysteine synthetase gene; GSH-11, glutathione synthetase; GSH2, glutathione synthetase gene.Enzymes. Glutathione synthetase, y-L-glutamyl-L-cysteine: glycine ligase (ADP forming) (EC 6.3.2.3); pglutamylcysteine synthetase (EC 6.3.2.2).Note. The nucleotide sequence mentioned here has been submitted to the EMBL sequence data bank and is available under accession number XX3411.GSH is syn...

Glutathione Participates in the Regulation of Mitophagy in Yeast

“…In the present report, we observed that manipulating the intracellular glutathione content modulated mitophagy: (i) nitrogen starvation was associated to an early decrease of GSH content; (ii) the addition of NAC maintained (and actually increased) the intracellular level of GSH and prevented mitophagy; (iii) the depletion of GSH by ethacrynic acid (48) reversed the protective effect of NAC on mitophagy; (iv) GSH2 inactivation (49,50) increased the rate of mitophagy; and (v) the addition of a cell-permeable form of glutathione had a similar effect as NAC.…”

“…Because mitophagy could be detected only in cells grown on a non-fermentable carbon source, the gsh1 mutant could not be used and we limited the study to the gsh2 mutant. The inactivation of the GSH2 gene led to the absence of detectable glutathione (47,48) and the accumulation of ␥-glutamylcysteine (␥-Glu-Cys) (49). The ⌬gsh2 strain displayed a significantly higher rate of nitrogen starvation-induced vacuolar delivery of mtGFP than the wild type (Fig.…”

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

“…The final step in GSH synthesis is the ligation of glycine with L-y-Glu-Cys catalyzed by GSH synthetase (Gsh2). Genes encoding Gsh2 have been identified in several bacterial and eukaryotic species (Gushima et al, 1984;Mutoh et al, 1991;Peters et al, 1992;Habenicht et al, 1993;Rawlins et al, 1995;Ullmann et al, 1996). However, prior to this study the Saccharomyces cerevisiae gene encoding Gsh2 had not been identified.…”

“…The yeast sequence contains the highly conserved glycine-rich domains (Figure 1) that have been suggested to play an essential role in the catalytic activity of GSH synthetases (Ullmann et al, 1996). However, the cysteine residue (Cys-302 in the Arabidopsis sequence), which is conserved in all other eukaryotic enzymes and may play a role in substrate binding (Oppenheimer et al, 1979;Ullmann et al, 1996), is absent from the yeast sequence. Similar to the other eukaryotic Gsh2 enzymes, hydropathy analysis of the yeast Gsh2 indicated a pattern typical of a soluble protein.…”

“…The yeast protein shares 44% similarity and 18% identity with the bacterial enzyme but this does not include the proposed ATP or y-glutamylcysteine binding sites (Yamaguchi et al, 1993;Fan et al, 1995;Hara et al, 1995). The yeast sequence contains the highly conserved glycine-rich domains (Figure 1) that have been suggested to play an essential role in the catalytic activity of GSH synthetases (Ullmann et al, 1996). However, the cysteine residue (Cys-302 in the Arabidopsis sequence), which is conserved in all other eukaryotic enzymes and may play a role in substrate binding (Oppenheimer et al, 1979;Ullmann et al, 1996), is absent from the yeast sequence.…”