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.     

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.     

Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu(2+)-deficient or an active Cu,Zn-superoxide dismutase

Yeast overexpressing SOD1, the gene for Cu,Zn-superoxide dismutase (Cu,Zn-Sod), was used to determine how Sod1p overexpression influences the chronological lifespan [the survival of non-dividing stationary (G0) phase cells over time], the replicative lifespan (the number of buds produced by actively dividing yeast cells) and stress resistance. Increasing the level of active Cu,Zn-Sod in yeast was found to require either growth in the presence of high copper, or the simultaneous overexpression of both SOD1 and CCS1 (the latter being the gene that encodes the chaperone dedicated to Cu(2+)-loading of Sod1p in vivo). Dual SOD1 + CCS1 overexpression elevated the levels of Cu,Zn-Sod activity six- to eight-fold in vegetative cultures. It also increased the optimized survival of stationary cells up to two-fold, showing this chronological lifespan is ultimately limited by oxidative stress. In contrast, several detrimental effects resulted when the SOD1 gene was overexpressed in the absence of either high copper or a simultaneous overexpression of CCS1. Both the chronological and the replicative lifespans were shortened; the cells displayed an abnormally high level of endogenous oxidative stress, resulting in a high rate of spontaneous mutation. Such harmful effects were all reversed through the overexpression of CCS1. It is apparent therefore that they relate to the incomplete Cu(2+)-loading of the overexpressed Sod1p, most probably accumulation of a Cu(2+)-deficient Sod1p to appreciable levels in vivo. The same events may generate the detrimental effects that are frequently, though not universally, observed when Cu,Zn-Sod overexpression is attempted in metazoans.     

Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Delta cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.     

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.     

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.     

Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance

Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real-time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro . ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro . The reduced viability of sod5 Δ/Δ and sod4 Δ/Δ sod5 Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox ^{−/ −} mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans , and perhaps many other microbial pathogens, can evade host immune surveillance in vivo .     

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.     

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.     

Phenotypic heterogeneity can enhance rare-cell survival in 'stress-sensitive' yeast populations

Individual cells within isogenic microbial cultures exhibit phenotypic heterogeneity, an issue that is attracting intense interest. Heterogeneity could confer benefits, in generating variant subpopulations that may be better equipped to persist during perturbation. We tested this hypothesis by comparing the survival of wild-type Saccharomyces cerevisiae with that of mutants which are considered stress-sensitive but which, we demonstrate, also have increased heterogeneity. The mutants (e.g. vma3, ctr1, sod1) exhibited the anticipated sensitivities to intermediate doses of nickel, copper, alkaline pH, menadione or paraquat. However, enhanced heterogeneity meant that the resistances of individual mutant cells spanned a broad range, and at high stress occasional-cell survival in most of these populations overtook that of the wild type. Green fluorescent protein (GFP) reporter studies showed that this heterogeneity-dependent advantage was not related to perturbation of buffered gene expression. Deletion strain screens combined with other approaches revealed that vacuolar alkalinization resulting from loss of Vma-dependent vacuolar H(+)-ATPase activity was not the cause of vma mutants' net stress sensitivities. An alternative Vma-dependent resistance mechanism was found to suppress an influence of variable vacuolar pH on the metal resistances of individual wild-type cells. In addition to revealing new mechanisms of heterogeneity generation, the results demonstrate experimentally a benefit under adverse conditions that arises specifically from heterogeneity, and in populations conventionally considered to be disadvantaged.     

Function of oxygen resistance proteins in the anaerobic,sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.     

Copper-sensitive mutant of Arabidopsis thaliana.

A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.     

Protection against oxidation during dehydration of yeast

Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined. Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration. In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod. To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2',7'-dichlorofluorescein, was used. Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration. Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation.     

Loss of SOD1 and LYS7 sensitizes Saccharomyces cerevisiae to hydroxyurea and DNA damage agents and downregulates MEC1 pathway effectors

Aerobic metabolism produces reactive oxygen species, including superoxide anions, which cause DNA damage unless removed by scavengers such as superoxide dismutases. We show that loss of the Cu,Zn-dependent superoxide dismutase, SOD1, or its copper chaperone, LYS7, confers oxygen-dependent sensitivity to replication arrest and DNA damage in Saccharomyces cerevisiae. We also find that sod1Delta strains, and to a lesser extent lys7Delta strains, when arrested with hydroxyurea (HU) show reduced induction of the MEC1 pathway effector Rnr3p and of Hug1p. The HU sensitivity of sod1Delta and lys7Delta strains is suppressed by overexpression of TKL1, a transketolase that generates NADPH, which balances redox in the cell and is required for ribonucleotide reductase activity. Our results suggest that the MEC1 pathway in sod1Delta mutant strains is sensitive to the altered cellular redox state due to increased superoxide anions and establish a new relationship between SOD1, LYS7, and the MEC1-mediated checkpoint response to replication arrest and DNA damage in S. cerevisiae.     

The effect of superoxide dismutase deficiency on cadmium stress

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.     

Protective role of mitochondrial superoxide dismutase against high osmolarity,heat and metalloid stress in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Superoxide dismutases, both cytosolic Cu, Zn-SOD encoded by SOD1 and mitochondrial Mn-SOD encoded by SOD2, serve Saccharomyces cerevisiae cells for defense against the superoxide radical but the phenotypes of sod1A and sod2delta mutant strains are different. Compared with the parent strain and the sod1delta mutant, the sod2delta mutant shows a much more severe growth defect at elevated salt concentrations, which is partially rescued by 2 mmol/L glutathione. The growth of all three strains is reduced at 37 degrees C, the sod2delta showing the highest sensitivity, especially when cultured in air. Addition of 1 mmol/L glutathione to the medium restores aerobic growth of the sod1delta mutant but has only a minor effect on the growth of the sod2delta strain at 37 degrees C. The sod2delta strain is also sensitive to AsIIl and AsV and its sensitivity is much more pronounced under aerobic conditions. These results suggest that, unlike the Sodlp protein, whose major role is oxidative stress defense, Sod2p also plays a role in protecting S. cerevisiae cells against other stresses--high osmolarity, heat and metalloid stress.     

Mutations in Saccharomyces cerevisiae iron-sulfur cluster assembly genes and oxidative stress relevant to Cu,Zn superoxide dismutase

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.     

Functional expression and cellular localization of the Na+/H+ exchanger Sod2 of the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the Na+/H+ exchanger, Sod2, plays a major role in the removal of excess intracellular sodium, and its disruption results in a sodium-sensitive phenotype. We examined the subcellular distribution and dynamics of Sod2 expression in S. pombe using a sod2-GFP fusion protein under the control of an attenuated version of the inducible nmt promoter. Sod2 was localized throughout the plasma membrane, the nuclear envelope, and some internal membrane systems. In exponentially growing cells, in which sod2-GFP was expressed and then the promoter turned-off, previously synthesized sod2-GFP was stable for long periods and found localized to the plasma membrane in the medial regions of the cell. It was not present at the actively growing cell ends. This suggests that these regions of the cell contain old plasma membrane protein vs. newly synthesized plasma membrane without Sod2 at the growing ends. Sod2 localization was not affected by salt stress. The results suggest that Sod2 is both a plasma membrane protein and is present in intracellular membranes. It is likely tethered within discrete regions of the plasma membrane and is not free to diffuse throughout the bilayer.