Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase

Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1 Delta yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1 Delta mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPR-detectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.     

Evidence for a novel role of copper-zinc superoxide dismutase in zinc metabolism

The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SOD1 gene). In yeast lacking Lys7 (lys7Delta), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Delta) and lys7Delta yeast show very similar phenotypes, namely poor growth in air and aerobic auxotrophies for lysine and methionine. Here, we demonstrate certain phenotypic differences between these strains: 1) lys7Delta cells are slightly less sensitive to paraquat than sod1Delta cells, 2) EPR-detectable or "free" iron is dramatically elevated in sod1Delta mutants but not in lys7Delta yeast, and 3) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SOD1 polypeptide, we overexpressed Mn-SOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.     

An Intracellular Iron Chelator Pleiotropically Suppresses Enzymatic and Growth Defects of Superoxide Dismutase-Deficient Escherichia coli

Mutants of Escherichia coli that lack cytoplasmic superoxide dismutase (SOD) exhibit auxotrophies for sulfur-containing, branched-chain, and aromatic amino acids and cannot catabolize nonfermentable carbon sources. A secondary-site mutation substantially relieved all of these growth defects. The requirement for fermentable carbon and the branched-chain auxotrophy occur because superoxide (O2-) leaches iron from the [4Fe-4S] clusters of a family of dehydratases, thereby inactivating them; the suppression of these phenotypes was mediated by the restoration of activity to these dehydratases, evidently without changing the intracellular concentration of O2-. Cloning, complementation, and sequence analysis identified the suppressor mutation to be in dapD, which encodes tetrahydrodipicolinate succinylase, an enzyme involved in diaminopimelate and lysine biosynthesis. A block in dapB, which encodes dihydrodipicolinate reductase in the same pathway, conferred similar protection. Genetic analysis indicated that the protection stems from the intracellular accumulation of tetrahydro- or dihydrodipicolinate. Heterologous expression in the SOD mutants of the dipicolinate synthase of Bacillus subtilis generated dipicolinate and similarly protected them. Dipicolinates are excellent iron chelators, and their accumulation in the cell triggered derepression of the Fur regulon and a large increase in the intracellular pool of free iron, presumably as a dipicolinate chelate. A fur mutation only partially relieved the auxotrophies, indicating that Fur derepression assists but is not sufficient for suppression. It seems plausible that the abundant internal iron permits efficient reactivation of superoxide-damaged iron-sulfur clusters. This result provides circumstantial evidence that the sulfur and aromatic auxotrophies of SOD mutants are also directly or indirectly linked to iron metabolism.     

Metabolic alterations in yeast lacking copper-zinc superoxide dismutase

Yeast lacking copper-zinc superoxide dismutase (sod1?) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression in which mitochondrial respiration is deemphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is reemphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1? cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1? cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1-lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress.     

Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.     

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.     

Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase.

The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae. Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it. Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise. Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage. Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage.     

The overlapping roles of manganese and Cu/Zn SOD in oxidative stress protection

In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.     

Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles.

Oxygen toxicity in Saccharomyces cerevisiae lacking the copper/zinc superoxide dismutase (SOD1) can be suppressed by overexpression of the S. cerevisiae ATX2 gene. Multiple copies of ATX2 were found to reverse the aerobic auxotrophies of sod1(delta) mutants for lysine and methionine and also to enhance the resistance of these yeast strains to paraquat and atmospheric levels of oxygen. ATX2 encodes a novel 34.4-kDa polypeptide with a number of potential membrane-spanning domains. Our studies indicate that Atx2p localizes to the membrane of a vesicular compartment in yeast cells reminiscent of the Golgi apparatus. With indirect immunofluorescence microscopy, Atx2p exhibited a punctate pattern of staining typical of the Golgi apparatus, and upon subcellular fractionation, Atx2p colocalized with a biochemical marker for the yeast Golgi apparatus. We demonstrate here that this vesicle protein normally functions in the homeostasis of manganese ions and that this role in metal metabolism is necessary for the ATX1 suppression of SOD1 deficiency. First, overexpression of ATX2 caused cells to accumulate increased levels of manganese. Second, a deletion in ATX2 caused a decrease in the apparent available level of intracellular manganese and caused sod1(delta) mutants to become dependent upon exogenous manganese for aerobic growth. Third, ATX2 was incapable of suppressing oxidative damage in cells depleted of manganese ions or lacking the plasma membrane transporter for manganese. The effect of ATX2 overexpression on manganese accumulation and oxygen resistance is similar to what we have previously reported for mutations in PMR1, which encodes a manganese-trafficking protein that also resides in a vesicular compartment. Our studies are consistent with a model in which Atx2p and Pmr1p work in opposite directions to control manganese homeostasis.     

Mitochondrial protein oxidation in yeast mutants lacking manganese-(MnSOD) or copper- and zinc-containing superoxide dismutase (CuZnSOD): evidence that MnSOD and CuZnSOD have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage

Saccharomyces cerevisiae expresses two forms of superoxide dismutase (SOD): MnSOD, encoded by SOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded by SOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type or sod mutant strains. Moreover, mitochondrial protein carbonylation levels in sod1, sod2, and sod1sod2 mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither MnSOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly in sod1 and sod1sod2 mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated in sod1 and sod2 mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in a sod1 mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in a sod2 mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that MnSOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.     

Isolation and characterization of Arabidopsis thaliana ISU1 gene

We describe the isolation of a cDNA encoding Arabidopsis thaliana ISU1 (AtISU1), which regulates iron homeostasis in the mitochondria. The AtISU1 gene contained an open reading frame that encoded 167 amino acid residues. Northern blot analysis demonstrated that AtISU1 gene was ubiquitously expressed in plant tissues examined. The yeast seo5-1, which harbors a single base-pair deletion in ScISU1, is a suppressor of oxidative damage in sod1-deficient mutant. Based on comparative expression analyses using yeast ISU1 gene (ScISU1) in seo5-1 mutant, we found that AtISU1 acts as a counterpart of ScISU1.     

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression of SSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.     

Protective role of superoxide dismutases against ionizing radiation in yeast

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.     

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.     

Yeast lacking superoxide dismutase. Isolation of genetic suppressors.

Null mutants of superoxide dismutase (SOD) in Saccharomyces cerevisiae are associated with a number of biochemical defects. In addition to being hypersensitive to oxygen toxicity, strains containing deletions in both the SOD1 (encoding Cu/Zn-SOD) and SOD2 (encoding Mn-SOD) genes are defective in sporulation, are associated with a high mutation rate, and are unable to biosynthesize lysine and methionine. The sod-linked defect in lysine metabolism was explored in detail and was found to occur at an early step in lysine biosynthesis, evidently at the level of the alpha-amino adipate transaminase. To better understand the role of SOD in cell metabolism, our laboratory has isolated yeast suppressors that have bypassed the SOD defect ("bsd" strains), that is, S. cerevisiae cells lacking SOD, yet resistant to oxygen toxicity. Two nuclear bsd complementation groups have been identified, and both suppress a variety of biological defects associated with sod1 and sod2 null mutants. These results demonstrate that a single gene mutation can alleviate the requirement for SOD in cell growth. Both bsd complementation groups are unable to utilize many non-fermentable carbon sources, suggesting a possible suppressor-linked defect in electron transport.     

Mitochondrial superoxide decreases yeast survival in stationary phase

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.     

SOD1 mutations cause hypersensitivity to high-pressure-induced oxidative stress in Saccharomyces cerevisiae

Living organisms are subject to various mechanical stressors, such as high hydrostatic pressure. Empirical evidence shows that under high pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. Here, we demonstrate that superoxide dismutase 1 (Sod1) plays a role in resisting high pressure for cell growth. Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. When these mutant cells were cultured under 25 MPa, their intracellular O₂^?– levels increased while sod1? mutant genome stability was unaffected. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O₂^?– generator paraquat. The sod1? mutant is known to exhibit methionine and lysine auxotrophy. However, excess methionine addition or overexpression of the lysine permease gene LYP1 did not counteract high-pressure sensitivity in the sod1 mutants, suggesting that their amino acid availability might be intact under 25 MPa. Interestingly, an exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria appeared to partially restore the high-pressure growth ability in the sod1 mutants. Taken these results together, we suggest that high pressure enhances O₂^?– production and Sod1 within the IMS plays a role in scavenging O₂^?– allowing the cells to grow under high pressure.BackgroundEmpirical evidence shows that under high hydrostatic pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. In the current study, we aimed to explore the role of superoxide dismutase 1 (Sod1) in yeast able to grow under high pressure.MethodsWild type and sod1 mutant cells were cultured in high-pressure chambers under 25 MPa (~250 kg/cm²). The SOD activity in whole cell extracts and 6His-tagged Sod1 recombinant proteins was analyzed using an SOD assay kit. The O₂^?– generation in cells was estimated by fluorescence staining.ResultsMutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O₂^?– generator paraquat. Exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria partially restored the high-pressure growth ability in the sod1 mutants.ConclusionsHigh pressure enhances O₂^?– production and Sod1 within the IMS plays a role in scavenging O₂^?– allowing the cells to grow under high pressure.General significanceUnlike external free radical-generating compounds, high-pressure treatment appeared to increase endogenous O₂^?– levels in yeast cells. Our experimental system offers a unique approach to investigating the physiological responses to mechanical and oxidative stresses in human body.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.