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.

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/ sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys 60 and Gly 61 are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly 115 and Gly 116 could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys 117 . Substitution of Phe 50 alters the ␤-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.Glutaredoxins are thiol oxidireductases that catalyze redox reactions involving reduced glutathione as a hydrogen donor for the reduction of protein disulfides (dithiol mechanism of action) or glutathione-protein-mixed disulfides (monothiol mechanism of action) (see Refs. 1 and 2 for review). Previously described glutaredoxins are small proteins (about 10 kDa) with a conserved active site that includes two cysteine residues (Cys-Pro-Tyr-Cys). Site-directed mutagenesis (3-5) has demonstrated that both cysteine residues in the active site are required for the dithiol reaction. In contrast, the amino-terminal cysteine is sufficient to catalyze the deglutathionylation of the reduced glutathione-mixed disulfides that are formed under oxidative stress conditions (5).Three-dimensional structures of oxidized and reduced forms of viral, bacterial, and mammalian glutaredoxins and also of reduced glutathione-glutaredoxin complexes have been identified using x-ray crystallography (6, 7) or nuclear magnetic resonance spectroscopy (8 -14). These studies have revealed which residues, apart from those at the active site, are important for stable interactions between glutathione and the glutaredoxin molecule (10,13,14). Dithiol glutaredoxins are members of the thioredoxin superfamily (15, 16) along with at least five other classes of proteins that interact with cysteine-containing substrates (thioredoxins, DbsA, protein disulfide isomerases, glutathione S-transferases, and glutathione peroxidases). This superfamily shares a structural motif (called the thioredoxin fold or ␣␤␣ fold) formed by a four or fivestranded ␤-sheet (with parallel and antiparallel strands) surrounded by three or more ␣-helices distributed on ei...

In order to characterize new yeast genes regulating cell proliferation, a number of overexpression-sensitive clones have been isolated from a Saccharomyces cerevisiae cDNA library in a multicopy vector under the control of the GAL1 promoter, on the basis of growth arrest phenotype under galactose-induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis-related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression-sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression of MCM1 (whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose-induced overexpression of MCM1 leads to rapid growth arrest at the G1 or S cell cycle stages, with many morphologically-abnormal cells. Several of the other clones also exhibit a G1 arrest terminal phenotype when overexpressed.

Alcohol dehydrogenase 1 (Adh1)p catalyses the conversion of acetaldehyde to ethanol, regenerating NAD + . In Saccharomyces cerevisiae, Adh1p is oxidatively modified during ageing and, consequently, its activity becomes reduced. To analyse whether maintaining this activity is advantageous for the cell, a yeast strain with an extra copy of the ADH1 gene (2¾ADH1) was constructed, and the effects on chronological and replicative ageing were analysed. The strain showed increased survival in stationary phase (chronological ageing) due to induction of antioxidant enzymes such as catalase and superoxide dismutases. In addition, 2¾ADH1 cells displayed an increased activity of silent information regulator 2 (Sir2)p, an NAD + -dependent histone deacetylase, due to a higher NAD + /NADH ratio. As a consequence, a 30 % extension in replicative life span was observed. Taken together, these results suggest that the maintenance of enzymes that participate in NAD + /NADH balancing is important to chronological and replicative life-span parameters. INTRODUCTIONOne of the features of ageing in Saccharomyces cerevisiae, as well as in other organisms, is the oxidative modification of specific protein targets, whose functions are impaired by such modification. A mild oxidation contributes to marking the protein for degradation (Stadtman & Oliver, 1991), while strong oxidation can promote protein aggregation, making proteins unavailable for proteasome degradation (Grune et al., 2004). The accumulation of these modified proteins in a cell contributes to the ageing phenotype (Harman, 1981;Stadtman, 1992;Stadtman & Levine, 2000;Levine, 2002; Nyström, 2005). In yeasts, replicative ageing refers to the number of cell divisions occurring in a mother yeast cell . Chronological ageing refers to the ability of cells to maintain viability in stationary phase (Herman, 2002).During the last decade investigations of ageing have uncovered physiological and molecular mechanisms involved in this process, as well as providing some clues towards understanding life-span lengthening (Bordone & Guarente, 2005). Calorie restriction is one of the mechanisms that has been consistently proven to extend life span in organisms ranging from yeasts to mammals (Sohal & Weindruch, 1996). In yeast, the replicative life span can be increased by shifting the cells from 2 to 0.5 % glucose (calorie restriction) (Lin et al., , 2004. One of the key molecules is the silent information regulator 2 (Sir2)p, a class III NAD + -dependent histone deacetylase, which is involved in several physiologically important functions such as silencing telomeres and rDNA, maintaining genome integrity, and ageing (Bitterman et al., 2003). Strains that have an extra copy of the SIR2 gene have a life span extended by 40 %, while deletion of SIR2 shortens life span by 50 % (Kaeberlein et al., 1999). Under calorie restriction, the oxygen consumption increases, thus raising the NAD + /NADH ratio by lowering the concentration of NADH. Since NADH has been described as a Sir2p inhibitor, calorie res...