Abstract

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.

Highlights

  • Glutaredoxins (Grx)1 are small (ϳ10 kDa) thiol disulfide oxidoreductases that catalyze glutathione (GSH)-disulfide redox reactions [1, 2]

  • Grx5 is located at the mitochondrial matrix and participates in the biogenesis of iron/sulfur (Fe/S) clusters [29, 30]

  • We have shown that when either Grx3 or Grx4 are targeted to the mitochondria they can substitute for Grx5 function, unlike dithiol glutaredoxins

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Summary

Introduction

Glutaredoxins (Grx) are small (ϳ10 kDa) thiol disulfide oxidoreductases that catalyze glutathione (GSH)-disulfide redox reactions [1, 2] They reduce protein disulfides through a dithiol mechanism that involves two cysteine residues in the active site of the glutaredoxin molecule, or they deglutathionylate GSH-protein mixed disulfides through a monothiol mechanism of action involving a single cysteine at the active site [2]. The thioredoxin and glutaredoxin systems are present in yeasts (18 –20) Saccharomyces cerevisiae contains both a cytosolic and a mitochondrial thioredoxin system [21, 22] whose functions may overlap with those of the glutaredoxin system [23]. S. cerevisiae contains two glutaredoxins (Grx and Grx2) with conserved CPYC motifs in their active sites [24] Both are cytosolic, though a minor fraction of Grx molecules is located at the mitochondria [25]. Wild type Wild type Ref. 29 Ref. 29 As MML100, but derived from W301–1B Integration of linear pMM227 in W303–1A Integration of linear pMM229 in W303–1A Integration of linear pMM225 in W303–1B Spore from a cross MML241 ϫ MML443 Spore from a cross MML241 ϫ MML446 Spore from a cross MML100 ϫ MML458 Integration of linear pMM338 in W303–1B Integration of linear pMM340 in W303–1B Integration of linear pMM342 in W303–1B Spore from a cross MML100 ϫ MML488 Spore from a cross MML100 ϫ MML490 Spore from a cross MML100 ϫ MML492 Integration of linear pMM344 in W303–1B Integration of linear pMM349 in W303–1B Spore from a cross MML100 ϫ MML509 Spore from a cross MML100 ϫ MML507 Integration of linear pMM387 in W303–1B Spore from a cross MML100 ϫ MML563 Integration of linear pMM221 in W303–1A Spore from a cross MML241 ϫ MML607

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