Glutathione Reductase and a Mitochondrial Thioredoxin Play Overlapping Roles in Maintaining Iron-Sulfur Enzymes in Fission Yeast

Abstract

In the fission yeast Schizosaccharomyces pombe, the pgr1 ؉ gene encoding glutathione (GSH) reductase (GR) is essentially required for cell survival. Depletion of GR caused proliferation arrest at the G 1 phase of the cell cycle under aerobic conditions. Multicopy suppressors that restore growth were screened, and one effective suppressor was found to be the trx2 ؉ gene, encoding a mitochondrial thioredoxin. This suggests that GR is critically required for some mitochondrial function(s). We found that GR resides in both cytosolic and organellar fractions of the cell. Depletion of GR lowered the respiration rate and the activity of oxidation-labile Fe-S enzymes such as mitochondrial aconitase and cytosolic sulfite reductase. Trx2 did not reverse the high ratio of oxidized glutathione to GSH or the low respiration rate observed in GR-depleted cells. However, it brought the activity of oxidation-labile Fe-S enzymes to a normal level, suggesting that the maintenance of Fe-S enzymes is a critical factor in the survival of S. pombe. The activity of succinate dehydrogenase, an oxidationinsensitive Fe-S enzyme, however, was not affected by GR depletion, suggesting that GR is not required for the biogenesis of the Fe-S cluster. The total iron content was greatly increased by GR depletion and was brought to a nearly normal level by Trx2. These results indicate that the essentiality of GR in the aerobic growth of S. pombe is derived from its role in maintaining oxidation-labile Fe-S enzymes and iron homeostasis.

The Glutathione System of Aspergillus nidulans Involves a Fungus-specific Glutathione S-Transferase

“…These results indicated that the first methionine and Met 86 are responsible for GlrA localization in the mitochondria and cytosol, respectively, and that the 85 amino acid residues before Met 86 target GlrA into the mitochondria. Both cytosolic and mitochondrial localizations of A. nidulans GR are similar to the GR of S. cerevisiae and of S. pombe, which produce both mitochondrial and cytosolic GR from two translation initiation sites (10,36). However, the activity of the mitochondrial GR differed in that A. nidulans produced considerably less of it than S. cerevisiae, which produces ϳ35% more activity mg protein Ϫ1 in the mitochondria than in the cytosol (36).…”

“…We showed that exogenous oxidants induced glrA expression and inhibited cell growth and conidial germination more effectively in the DGR1 than in the wild type strain, indicating that GR (or GSH) functions as an antioxidant in A. nidulans as it does in yeasts and other organisms (7)(8)(9)(10). Menadione was the most powerful inducer of glrA expression among the tested oxidants.…”

“…The triple mutant (trx1, trx2, glr1) is also non-viable, indicating overlapping functions of thioredoxins and glutaredoxins for reducing protein disulfides (3). This seems to be true for S. pombe, in which multicopies of the thioredoxin gene (trx2 ϩ ) suppress the growth deficiency of GR mutants (10). Taken together, the difference in the species requirement for GR is presumed to be caused by the extent of activity of compensation mechanisms such as the thioredoxin system.…”

“…3B). This is likely due to the inactivation of Fe-S proteins in the mitochondrial respiratory chain as O 2 Ϫ inactivates Fe-S proteins by oxidizing their Fe-S cluster (10,14). The low respiration activity could explain the slow growth due to GlrA depletion (Fig.…”

“…2E). This indicated that GSH elimination induces de novo GSH synthesis in DGR1 (see below) as in yeasts (7,8,10). The WT strain contained 1.6 Ϯ 0.1 nmol mg cell Ϫ1 of reduced GSH which occupied most (94%) of the intracellular GSH/ GSSG pool (Fig.…”