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• W2171471851 abstract "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 sod1Δ yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Δ yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependentsod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Δ 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 sod1Δ 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 sod1Δ 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 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 sod1Δ yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Δ yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependentsod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Δ 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 sod1Δ 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 sod1Δ 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. superoxide dismutase Aerobic organisms are chronically exposed to potentially harmful reactive oxygen species generated as by-products of cellular metabolism. One antioxidant enzyme Cu,Zn-superoxide dismutase 1 (Sod1p) plays an important role in detoxifying superoxide radicals (O⨪2). Superoxide radicals can oxidize iron-sulfur cluster proteins liberating iron (1Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 2Flint D. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, 3Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar). Thus, Sod1p-deficient yeast (sod1Δ yeast) may have increased pools of reactive iron (5Liochev S.I. Fridovich I. Free Rad. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (361) Google Scholar) as has been shown for SOD1-deficientEscherichia coli (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar). “Free” iron can react with hydrogen peroxide (H2O2), and possibly other reactive oxygen species, to generate toxic hydroxyl radicals (OH−) by Fenton chemistry (6Fenton H.J.H. J. Chem. Soc. 1894; 65: 899-903Crossref Google Scholar, 7Haber F. Weiss J. Proc. R. Soc. London. 1934; 147: 332-351Crossref Google Scholar). Hydroxyl radicals have the potential to damage proteins, nucleic acids, and membranes (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar, 8Bostek C. J. Am. Assoc. Nur. Anes. 1989; 57: 231-237Google Scholar, 9Halliwell B. Gutteridge J.M.C. Lancet. 1984; 1: 1396-1398Abstract PubMed Scopus (675) Google Scholar, 10Halliwell B. Gutteridge J.M. Hum. Toxicol. 1988; 7: 7-13Crossref PubMed Scopus (151) Google Scholar, 11Davies K.J. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 12Imlay J. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1646) Google Scholar).In recent years, insight into oxidative defense, and SOD1 in particular, has come from studies using the unicellular eukaryote,Saccharomyces cerevisiae. Yeast lacking Sod1p grow slowly in air (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar), are sensitive to superoxide generating agents (e.g. paraquat) (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar, 14Gralla E. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar), and exhibit as yet poorly understood metabolic and biosynthetic defects (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar, 14Gralla E. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar, 15Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (137) Google Scholar, 16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). To further our understanding of oxidative damage to the cell, we examined the effects of superoxide dismutase deficiency on overall cell structure and organelle morphology and asked whether any such abnormalities may be tied into iron-mediated toxicity. We report here that oxidative stress to sod1Δ yeast results in fragmentation of the vacuole, an organelle analogous to the lysosome and thought to play a role in iron homeostasis (Refs. 18Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar and 19Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar). Furthermore, we find lowering iron availability significantly reduces damage to the vacuole, consistent with disruption of iron handling as part of the pathway through which Sod1p deficiency causes vacuole fragmentation.DISCUSSIONBecause mitochondria are the primary intracellular sources of superoxide radicals, we anticipated mitochondrial damage insod1Δ yeast. However, the most obvious sod1Δdefects were not in the mitochondria, but rather in the vacuole. Oxidative stress to Sod1p-deficient yeast leads to vacuolar fragmentation through an iron-dependent process. Evidence that the vacuole fragmentation is oxygen (presumably superoxide radical) mediated includes the findings that only aerobically grownsod1Δ yeast exhibit aberrant vacuoles and genetic supressors (such as pmr1Δ) capable of rescuing all other known aerobic sod1Δ deficits can also rescue this fragmentation phenotype. Iron deprivation (as in fet3Δ sod1Δ strains) also alleviates the fragmentation without providing resistance to O⨪2 generating reagents or ameliorating other oxygen-dependent phenotypes of sod1Δyeast. Thus, reactive oxygen species arising in the absence of Sod1p, in combination with iron, provoke damage to vacuoles.The notion of iron exacerbating damage under conditions of oxidative stress has been raised previously in other contexts. Fe2+can react with physiological levels of H2O2(and possibly other reactive oxygen species) to produce toxic hydroxyl radicals (reviewed in Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In vivo, iron is not found typically in a free state but is sequestered as iron complexes or is bound protectively to enzymes, proteins, or iron carriers (for review, see Refs. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar and 52Kaplan J. O'Halloran T.V. Science. 1996; 271: 1510-1512Crossref PubMed Scopus (124) Google Scholar). However, superoxide can alter this cellular iron homeostasis. Several groups have found in vivo evidence that superoxide radicals oxidize labile iron-sulfur clusters to liberate iron (1Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 2Flint D. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, 3Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 46Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 47Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 1478-1483Abstract Full Text PDF PubMed Google Scholar) although the fate and redox state of the “liberated” iron is not known. Additionally, genetic screens have found that mutations in iron-sulfur cluster assembly proteins lessen oxidative damage in sod1Δ yeast, suggesting that reducing the number of iron-sulfur targets for oxygen radical damage lessens damage in these oxidatively stressed cells (53Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Similarly, superoxide radicals have been shown to oxidize iron storage components to liberate iron which is released in a form capable of catalyzing hydroxyl radical production (54Biemond P. Eijk H.G.v. Swaak A.J. Koster J.F. J. Clin. Invest. 1984; 73: 1576-1579Crossref PubMed Scopus (327) Google Scholar). More generally, Wisnicka et al. (40Wisnicka R. Krzepilko A. Wawryn J. Krawiec Z. Bilinski T. Biochem. Mol. Biol. Int. 1998; 44: 635-641PubMed Google Scholar) have suggested that superoxide radicals (in sod1Δ yeast) may influence the redox status of cellular iron pools by reducing Fe3+ to the more reactive Fe2+ state. Although total iron is not elevated in sod1Δyeast, 2V. C. Culotta, unpublished results. the cellular distribution of iron, the ratio of ferrous to ferric iron, and the level of free iron may be altered. In any case, much evidence does suggest that oxygen radicals liberate iron which in turn may exacerbate oxidative damage.In yeast, the vacuole plays a role in iron homeostasis. Subcellular fractionation of iron-loaded cells suggests that the vacuole is the major site of iron sequestration (18Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar) and yeast lacking a recognizable vacuole structure are unable to accumulate high levels of iron (19Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar). Thus, an iron-enriched yeast vacuole may be particularly susceptible to iron-mediated damage in superoxide-rich sod1Δ yeast. One possibility is that the vacuole damage may result from a disruption of the iron storage state in the vacuole. Under the influence of increased superoxide levels (which perhaps may be found throughout the Sod1p-deficient cell), vacuolar iron pools may be converted to a more accessible and active Fe2+ state that can catalyze hydroxyl radical production and damage macromolecules in the immediate vicinity of the vacuole. A second, not mutually exclusive, possibility is that alterations in iron handling and homeostasis within the cell may result in an increased trafficking of free iron to the vacuole. Increased vacuolar iron may result in increased iron-mediated oxidative reactions within the vacuole or alternatively, the increased iron may simply bind nonproductively to various sites in the vacuole (i.e.sulfur, nitrogen, or oxygen containing ligands). We cannot rule out the possibility that the primary damage actually occurs to cytosolic components that then secondarily afflict vacuolar structure and function. Indeed, we should stress, however, that there is not a global defect in trafficking to the vacuole or vacuole assembly as judged by normal carboxypeptidase Y trafficking results. In any case, both reactive oxygen species and iron contribute to vacuole changes.Iron-mediated damage occurs not just in the yeast vacuole but also in the mammalian analogue, the lysosome. Similar to the yeast vacuole, the lysosome plays a role in intracellular iron storage, homeostasis, and detoxification (55Radisky D.C. Kaplan J. Biochem. J. 1998; 336: 201-205Crossref PubMed Scopus (121) Google Scholar, 56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Iron overload in the rat liver has been shown to increase lysosomal fragility (56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Although lysosomal fragility has been partially attributed to lipid peroxidation, we should stress that similar lipid membrane damage is not the cause of sod1Δvacuole abnormalities because S. cerevisiae do not synthesize the polyunsaturated fatty acids that are susceptible to lipid peroxidation (57Bilinski T. Litwinska J. Blaszczynski M. Bajus A. Biochim. Biophys. Acta. 1989; 1001: 102-106Crossref PubMed Scopus (38) Google Scholar). Damage to vacuolar membrane proteins (as well as lysosomal membrane proteins) is a more likely possibility.By examining structural changes in cellular architecture caused by Sod1p deficiency, we have uncovered a plausible explanation for various puzzling defects in the sod1Δ yeast: some of the pleiotropic sod1Δ phenotypes may arise as secondary consequences of a compromised vacuole. For example, sod1Δyeast have a sporulation defect (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar) and an increased death rate upon entering stationary phase (17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Previously, this sensitivity to adverse conditions was attributed to mitochondrial insufficiency (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). However, although sod1Δ yeast do not grow as robustly on non-fermentable carbon sources as wild-type yeast, they nevertheless are respiration proficient (e.g. Ref. 15Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (137) Google Scholar). Instead, we suggest these sod1Δ abnormalities may be, in part, vacuole related. When yeast are exposed to such adverse conditions, they normally up-regulate vacuolar hydrolases to turnover intravacuolar reserves and cytosolic macromolecules in order to recycle necessary nutrients (36Achstetter T. Wolf D.H. Yeast. 1985; 1: 139-157Crossref PubMed Scopus (92) Google Scholar, 37Jones E.W. Annu. Rev. Genet. 1984; 18: 233-270Crossref PubMed Scopus (113) Google Scholar, 38Teichert U. Mechler B. Muller H. Wolf D.H. J. Biol. Chem. 1989; 264: 16037-16045Abstract Full Text PDF PubMed Google Scholar). The fact that the sod1Δ yeast cannot survive through such starvation conditions may be an indication that either vacuolar nutrient storage or the vacuolar-dependent macromolecular turnover processes are compromised. Indeed, many known vacuolar mutants also cannot survive adverse conditions (39Preston R.A. Manolson M.F. Becherer K. Weidenhammer E. Kirkpatrick D. Wright R. Jones E.W. Mol. Cell. Biol. 1991; 11: 5801-5812Crossref PubMed Scopus (51) Google Scholar). Furthermore, we demonstrate that sod1Δ yeast have a sensitivity to pH which cannot easily be explained without invoking the role of the vacuole in pH homeostasis. Likewise, an increased sensitivity to transition metals may be directly or indirectly related to vacuole abnormalities. Thus, some of the curious metabolic and biochemical abnormalities in sod1Δ yeast may in fact be attributable to vacuolar aberrations.In summary, we demonstrate that oxidative stress to sod1Δyeast results in vacuolar fragmentation and either removal of oxygen or iron can ameliorate the damage. This may have medical relevance in that oxidative damage and alterations in iron homeostasis have been implicated in a number of disease states including atherosclerosis, neurodegeneration, arthritis, and aging (for review, see Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In some cases oxidative insult is thought to be the primary cause of damage. For example, brain and spinal cord deterioration after ischemic or traumatic injury often appears excessive for the level of trauma (58Halliwell B. Gutteridge J.M.C. Trends Neurosci. 1985; 8: 22Abstract Full Text PDF Scopus (787) Google Scholar). One explanation put forth is that iron-binding capacity in the central nervous system and in the cerebral spinal fluid bathing the central nervous system is particularly low. Thus, release of iron by oxidatively or mechanically damaged cells, organelles, and proteins may catalyze toxic oxidative side reactions causing further cell injury. Consistent with this concept, preliminary trials of treatment with iron-chelating agents have had success in diminishing post-traumatic degeneration of brain and spinal cord (59Hall E.D. J. Neurosurg. 1988; 68: 462-465Crossref PubMed Scopus (139) Google Scholar, 60Hall E.D. Yonkers P.A. McCall J.M. Braughler J.M. J. Neurosurg. 1988; 68: 456-461Crossref PubMed Scopus (252) Google Scholar). Thus, iron-mediated oxidative damage is likely a common event in aerobic organisms, and dissection and understanding of the process (and ways of prevention) in yeast may give insight into human disease. Aerobic organisms are chronically exposed to potentially harmful reactive oxygen species generated as by-products of cellular metabolism. One antioxidant enzyme Cu,Zn-superoxide dismutase 1 (Sod1p) plays an important role in detoxifying superoxide radicals (O⨪2). Superoxide radicals can oxidize iron-sulfur cluster proteins liberating iron (1Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 2Flint D. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, 3Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar). Thus, Sod1p-deficient yeast (sod1Δ yeast) may have increased pools of reactive iron (5Liochev S.I. Fridovich I. Free Rad. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (361) Google Scholar) as has been shown for SOD1-deficientEscherichia coli (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar). “Free” iron can react with hydrogen peroxide (H2O2), and possibly other reactive oxygen species, to generate toxic hydroxyl radicals (OH−) by Fenton chemistry (6Fenton H.J.H. J. Chem. Soc. 1894; 65: 899-903Crossref Google Scholar, 7Haber F. Weiss J. Proc. R. Soc. London. 1934; 147: 332-351Crossref Google Scholar). Hydroxyl radicals have the potential to damage proteins, nucleic acids, and membranes (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (678) Google Scholar, 8Bostek C. J. Am. Assoc. Nur. Anes. 1989; 57: 231-237Google Scholar, 9Halliwell B. Gutteridge J.M.C. Lancet. 1984; 1: 1396-1398Abstract PubMed Scopus (675) Google Scholar, 10Halliwell B. Gutteridge J.M. Hum. Toxicol. 1988; 7: 7-13Crossref PubMed Scopus (151) Google Scholar, 11Davies K.J. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 12Imlay J. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1646) Google Scholar). In recent years, insight into oxidative defense, and SOD1 in particular, has come from studies using the unicellular eukaryote,Saccharomyces cerevisiae. Yeast lacking Sod1p grow slowly in air (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar), are sensitive to superoxide generating agents (e.g. paraquat) (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar, 14Gralla E. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar), and exhibit as yet poorly understood metabolic and biosynthetic defects (13Bilinski T. Krawiec Z. Liczmanski L. Litwinska J. Biochem. Biophys. Res. Commun. 1985; 130: 533-539Crossref PubMed Scopus (132) Google Scholar, 14Gralla E. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar, 15Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (137) Google Scholar, 16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). To further our understanding of oxidative damage to the cell, we examined the effects of superoxide dismutase deficiency on overall cell structure and organelle morphology and asked whether any such abnormalities may be tied into iron-mediated toxicity. We report here that oxidative stress to sod1Δ yeast results in fragmentation of the vacuole, an organelle analogous to the lysosome and thought to play a role in iron homeostasis (Refs. 18Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar and 19Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar). Furthermore, we find lowering iron availability significantly reduces damage to the vacuole, consistent with disruption of iron handling as part of the pathway through which Sod1p deficiency causes vacuole fragmentation. DISCUSSIONBecause mitochondria are the primary intracellular sources of superoxide radicals, we anticipated mitochondrial damage insod1Δ yeast. However, the most obvious sod1Δdefects were not in the mitochondria, but rather in the vacuole. Oxidative stress to Sod1p-deficient yeast leads to vacuolar fragmentation through an iron-dependent process. Evidence that the vacuole fragmentation is oxygen (presumably superoxide radical) mediated includes the findings that only aerobically grownsod1Δ yeast exhibit aberrant vacuoles and genetic supressors (such as pmr1Δ) capable of rescuing all other known aerobic sod1Δ deficits can also rescue this fragmentation phenotype. Iron deprivation (as in fet3Δ sod1Δ strains) also alleviates the fragmentation without providing resistance to O⨪2 generating reagents or ameliorating other oxygen-dependent phenotypes of sod1Δyeast. Thus, reactive oxygen species arising in the absence of Sod1p, in combination with iron, provoke damage to vacuoles.The notion of iron exacerbating damage under conditions of oxidative stress has been raised previously in other contexts. Fe2+can react with physiological levels of H2O2(and possibly other reactive oxygen species) to produce toxic hydroxyl radicals (reviewed in Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In vivo, iron is not found typically in a free state but is sequestered as iron complexes or is bound protectively to enzymes, proteins, or iron carriers (for review, see Refs. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar and 52Kaplan J. O'Halloran T.V. Science. 1996; 271: 1510-1512Crossref PubMed Scopus (124) Google Scholar). However, superoxide can alter this cellular iron homeostasis. Several groups have found in vivo evidence that superoxide radicals oxidize labile iron-sulfur clusters to liberate iron (1Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 2Flint D. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, 3Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 46Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 47Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 1478-1483Abstract Full Text PDF PubMed Google Scholar) although the fate and redox state of the “liberated” iron is not known. Additionally, genetic screens have found that mutations in iron-sulfur cluster assembly proteins lessen oxidative damage in sod1Δ yeast, suggesting that reducing the number of iron-sulfur targets for oxygen radical damage lessens damage in these oxidatively stressed cells (53Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Similarly, superoxide radicals have been shown to oxidize iron storage components to liberate iron which is released in a form capable of catalyzing hydroxyl radical production (54Biemond P. Eijk H.G.v. Swaak A.J. Koster J.F. J. Clin. Invest. 1984; 73: 1576-1579Crossref PubMed Scopus (327) Google Scholar). More generally, Wisnicka et al. (40Wisnicka R. Krzepilko A. Wawryn J. Krawiec Z. Bilinski T. Biochem. Mol. Biol. Int. 1998; 44: 635-641PubMed Google Scholar) have suggested that superoxide radicals (in sod1Δ yeast) may influence the redox status of cellular iron pools by reducing Fe3+ to the more reactive Fe2+ state. Although total iron is not elevated in sod1Δyeast, 2V. C. Culotta, unpublished results. the cellular distribution of iron, the ratio of ferrous to ferric iron, and the level of free iron may be altered. In any case, much evidence does suggest that oxygen radicals liberate iron which in turn may exacerbate oxidative damage.In yeast, the vacuole plays a role in iron homeostasis. Subcellular fractionation of iron-loaded cells suggests that the vacuole is the major site of iron sequestration (18Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar) and yeast lacking a recognizable vacuole structure are unable to accumulate high levels of iron (19Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar). Thus, an iron-enriched yeast vacuole may be particularly susceptible to iron-mediated damage in superoxide-rich sod1Δ yeast. One possibility is that the vacuole damage may result from a disruption of the iron storage state in the vacuole. Under the influence of increased superoxide levels (which perhaps may be found throughout the Sod1p-deficient cell), vacuolar iron pools may be converted to a more accessible and active Fe2+ state that can catalyze hydroxyl radical production and damage macromolecules in the immediate vicinity of the vacuole. A second, not mutually exclusive, possibility is that alterations in iron handling and homeostasis within the cell may result in an increased trafficking of free iron to the vacuole. Increased vacuolar iron may result in increased iron-mediated oxidative reactions within the vacuole or alternatively, the increased iron may simply bind nonproductively to various sites in the vacuole (i.e.sulfur, nitrogen, or oxygen containing ligands). We cannot rule out the possibility that the primary damage actually occurs to cytosolic components that then secondarily afflict vacuolar structure and function. Indeed, we should stress, however, that there is not a global defect in trafficking to the vacuole or vacuole assembly as judged by normal carboxypeptidase Y trafficking results. In any case, both reactive oxygen species and iron contribute to vacuole changes.Iron-mediated damage occurs not just in the yeast vacuole but also in the mammalian analogue, the lysosome. Similar to the yeast vacuole, the lysosome plays a role in intracellular iron storage, homeostasis, and detoxification (55Radisky D.C. Kaplan J. Biochem. J. 1998; 336: 201-205Crossref PubMed Scopus (121) Google Scholar, 56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Iron overload in the rat liver has been shown to increase lysosomal fragility (56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Although lysosomal fragility has been partially attributed to lipid peroxidation, we should stress that similar lipid membrane damage is not the cause of sod1Δvacuole abnormalities because S. cerevisiae do not synthesize the polyunsaturated fatty acids that are susceptible to lipid peroxidation (57Bilinski T. Litwinska J. Blaszczynski M. Bajus A. Biochim. Biophys. Acta. 1989; 1001: 102-106Crossref PubMed Scopus (38) Google Scholar). Damage to vacuolar membrane proteins (as well as lysosomal membrane proteins) is a more likely possibility.By examining structural changes in cellular architecture caused by Sod1p deficiency, we have uncovered a plausible explanation for various puzzling defects in the sod1Δ yeast: some of the pleiotropic sod1Δ phenotypes may arise as secondary consequences of a compromised vacuole. For example, sod1Δyeast have a sporulation defect (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar) and an increased death rate upon entering stationary phase (17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Previously, this sensitivity to adverse conditions was attributed to mitochondrial insufficiency (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). However, although sod1Δ yeast do not grow as robustly on non-fermentable carbon sources as wild-type yeast, they nevertheless are respiration proficient (e.g. Ref. 15Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (137) Google Scholar). Instead, we suggest these sod1Δ abnormalities may be, in part, vacuole related. When yeast are exposed to such adverse conditions, they normally up-regulate vacuolar hydrolases to turnover intravacuolar reserves and cytosolic macromolecules in order to recycle necessary nutrients (36Achstetter T. Wolf D.H. Yeast. 1985; 1: 139-157Crossref PubMed Scopus (92) Google Scholar, 37Jones E.W. Annu. Rev. Genet. 1984; 18: 233-270Crossref PubMed Scopus (113) Google Scholar, 38Teichert U. Mechler B. Muller H. Wolf D.H. J. Biol. Chem. 1989; 264: 16037-16045Abstract Full Text PDF PubMed Google Scholar). The fact that the sod1Δ yeast cannot survive through such starvation conditions may be an indication that either vacuolar nutrient storage or the vacuolar-dependent macromolecular turnover processes are compromised. Indeed, many known vacuolar mutants also cannot survive adverse conditions (39Preston R.A. Manolson M.F. Becherer K. Weidenhammer E. Kirkpatrick D. Wright R. Jones E.W. Mol. Cell. Biol. 1991; 11: 5801-5812Crossref PubMed Scopus (51) Google Scholar). Furthermore, we demonstrate that sod1Δ yeast have a sensitivity to pH which cannot easily be explained without invoking the role of the vacuole in pH homeostasis. Likewise, an increased sensitivity to transition metals may be directly or indirectly related to vacuole abnormalities. Thus, some of the curious metabolic and biochemical abnormalities in sod1Δ yeast may in fact be attributable to vacuolar aberrations.In summary, we demonstrate that oxidative stress to sod1Δyeast results in vacuolar fragmentation and either removal of oxygen or iron can ameliorate the damage. This may have medical relevance in that oxidative damage and alterations in iron homeostasis have been implicated in a number of disease states including atherosclerosis, neurodegeneration, arthritis, and aging (for review, see Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In some cases oxidative insult is thought to be the primary cause of damage. For example, brain and spinal cord deterioration after ischemic or traumatic injury often appears excessive for the level of trauma (58Halliwell B. Gutteridge J.M.C. Trends Neurosci. 1985; 8: 22Abstract Full Text PDF Scopus (787) Google Scholar). One explanation put forth is that iron-binding capacity in the central nervous system and in the cerebral spinal fluid bathing the central nervous system is particularly low. Thus, release of iron by oxidatively or mechanically damaged cells, organelles, and proteins may catalyze toxic oxidative side reactions causing further cell injury. Consistent with this concept, preliminary trials of treatment with iron-chelating agents have had success in diminishing post-traumatic degeneration of brain and spinal cord (59Hall E.D. J. Neurosurg. 1988; 68: 462-465Crossref PubMed Scopus (139) Google Scholar, 60Hall E.D. Yonkers P.A. McCall J.M. Braughler J.M. J. Neurosurg. 1988; 68: 456-461Crossref PubMed Scopus (252) Google Scholar). Thus, iron-mediated oxidative damage is likely a common event in aerobic organisms, and dissection and understanding of the process (and ways of prevention) in yeast may give insight into human disease. Because mitochondria are the primary intracellular sources of superoxide radicals, we anticipated mitochondrial damage insod1Δ yeast. However, the most obvious sod1Δdefects were not in the mitochondria, but rather in the vacuole. Oxidative stress to Sod1p-deficient yeast leads to vacuolar fragmentation through an iron-dependent process. Evidence that the vacuole fragmentation is oxygen (presumably superoxide radical) mediated includes the findings that only aerobically grownsod1Δ yeast exhibit aberrant vacuoles and genetic supressors (such as pmr1Δ) capable of rescuing all other known aerobic sod1Δ deficits can also rescue this fragmentation phenotype. Iron deprivation (as in fet3Δ sod1Δ strains) also alleviates the fragmentation without providing resistance to O⨪2 generating reagents or ameliorating other oxygen-dependent phenotypes of sod1Δyeast. Thus, reactive oxygen species arising in the absence of Sod1p, in combination with iron, provoke damage to vacuoles. The notion of iron exacerbating damage under conditions of oxidative stress has been raised previously in other contexts. Fe2+can react with physiological levels of H2O2(and possibly other reactive oxygen species) to produce toxic hydroxyl radicals (reviewed in Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In vivo, iron is not found typically in a free state but is sequestered as iron complexes or is bound protectively to enzymes, proteins, or iron carriers (for review, see Refs. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar and 52Kaplan J. O'Halloran T.V. Science. 1996; 271: 1510-1512Crossref PubMed Scopus (124) Google Scholar). However, superoxide can alter this cellular iron homeostasis. Several groups have found in vivo evidence that superoxide radicals oxidize labile iron-sulfur clusters to liberate iron (1Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 2Flint D. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, 3Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 46Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 47Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 1478-1483Abstract Full Text PDF PubMed Google Scholar) although the fate and redox state of the “liberated” iron is not known. Additionally, genetic screens have found that mutations in iron-sulfur cluster assembly proteins lessen oxidative damage in sod1Δ yeast, suggesting that reducing the number of iron-sulfur targets for oxygen radical damage lessens damage in these oxidatively stressed cells (53Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Similarly, superoxide radicals have been shown to oxidize iron storage components to liberate iron which is released in a form capable of catalyzing hydroxyl radical production (54Biemond P. Eijk H.G.v. Swaak A.J. Koster J.F. J. Clin. Invest. 1984; 73: 1576-1579Crossref PubMed Scopus (327) Google Scholar). More generally, Wisnicka et al. (40Wisnicka R. Krzepilko A. Wawryn J. Krawiec Z. Bilinski T. Biochem. Mol. Biol. Int. 1998; 44: 635-641PubMed Google Scholar) have suggested that superoxide radicals (in sod1Δ yeast) may influence the redox status of cellular iron pools by reducing Fe3+ to the more reactive Fe2+ state. Although total iron is not elevated in sod1Δyeast, 2V. C. Culotta, unpublished results. the cellular distribution of iron, the ratio of ferrous to ferric iron, and the level of free iron may be altered. In any case, much evidence does suggest that oxygen radicals liberate iron which in turn may exacerbate oxidative damage. In yeast, the vacuole plays a role in iron homeostasis. Subcellular fractionation of iron-loaded cells suggests that the vacuole is the major site of iron sequestration (18Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar) and yeast lacking a recognizable vacuole structure are unable to accumulate high levels of iron (19Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar). Thus, an iron-enriched yeast vacuole may be particularly susceptible to iron-mediated damage in superoxide-rich sod1Δ yeast. One possibility is that the vacuole damage may result from a disruption of the iron storage state in the vacuole. Under the influence of increased superoxide levels (which perhaps may be found throughout the Sod1p-deficient cell), vacuolar iron pools may be converted to a more accessible and active Fe2+ state that can catalyze hydroxyl radical production and damage macromolecules in the immediate vicinity of the vacuole. A second, not mutually exclusive, possibility is that alterations in iron handling and homeostasis within the cell may result in an increased trafficking of free iron to the vacuole. Increased vacuolar iron may result in increased iron-mediated oxidative reactions within the vacuole or alternatively, the increased iron may simply bind nonproductively to various sites in the vacuole (i.e.sulfur, nitrogen, or oxygen containing ligands). We cannot rule out the possibility that the primary damage actually occurs to cytosolic components that then secondarily afflict vacuolar structure and function. Indeed, we should stress, however, that there is not a global defect in trafficking to the vacuole or vacuole assembly as judged by normal carboxypeptidase Y trafficking results. In any case, both reactive oxygen species and iron contribute to vacuole changes. Iron-mediated damage occurs not just in the yeast vacuole but also in the mammalian analogue, the lysosome. Similar to the yeast vacuole, the lysosome plays a role in intracellular iron storage, homeostasis, and detoxification (55Radisky D.C. Kaplan J. Biochem. J. 1998; 336: 201-205Crossref PubMed Scopus (121) Google Scholar, 56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Iron overload in the rat liver has been shown to increase lysosomal fragility (56LeSage G.D. Kost L.J. Barham S.S. LaRusso N.F. J. Clin. Invest. 1986; 77: 90-97Crossref PubMed Scopus (79) Google Scholar). Although lysosomal fragility has been partially attributed to lipid peroxidation, we should stress that similar lipid membrane damage is not the cause of sod1Δvacuole abnormalities because S. cerevisiae do not synthesize the polyunsaturated fatty acids that are susceptible to lipid peroxidation (57Bilinski T. Litwinska J. Blaszczynski M. Bajus A. Biochim. Biophys. Acta. 1989; 1001: 102-106Crossref PubMed Scopus (38) Google Scholar). Damage to vacuolar membrane proteins (as well as lysosomal membrane proteins) is a more likely possibility. By examining structural changes in cellular architecture caused by Sod1p deficiency, we have uncovered a plausible explanation for various puzzling defects in the sod1Δ yeast: some of the pleiotropic sod1Δ phenotypes may arise as secondary consequences of a compromised vacuole. For example, sod1Δyeast have a sporulation defect (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar) and an increased death rate upon entering stationary phase (17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Previously, this sensitivity to adverse conditions was attributed to mitochondrial insufficiency (16Liu X.F. Elashvili I. Gralla E.B. Valentine J.S. Lapinskas P. Culotta V.C. J. Biol. Chem. 1992; 267: 18298-18302Abstract Full Text PDF PubMed Google Scholar, 17Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). However, although sod1Δ yeast do not grow as robustly on non-fermentable carbon sources as wild-type yeast, they nevertheless are respiration proficient (e.g. Ref. 15Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (137) Google Scholar). Instead, we suggest these sod1Δ abnormalities may be, in part, vacuole related. When yeast are exposed to such adverse conditions, they normally up-regulate vacuolar hydrolases to turnover intravacuolar reserves and cytosolic macromolecules in order to recycle necessary nutrients (36Achstetter T. Wolf D.H. Yeast. 1985; 1: 139-157Crossref PubMed Scopus (92) Google Scholar, 37Jones E.W. Annu. Rev. Genet. 1984; 18: 233-270Crossref PubMed Scopus (113) Google Scholar, 38Teichert U. Mechler B. Muller H. Wolf D.H. J. Biol. Chem. 1989; 264: 16037-16045Abstract Full Text PDF PubMed Google Scholar). The fact that the sod1Δ yeast cannot survive through such starvation conditions may be an indication that either vacuolar nutrient storage or the vacuolar-dependent macromolecular turnover processes are compromised. Indeed, many known vacuolar mutants also cannot survive adverse conditions (39Preston R.A. Manolson M.F. Becherer K. Weidenhammer E. Kirkpatrick D. Wright R. Jones E.W. Mol. Cell. Biol. 1991; 11: 5801-5812Crossref PubMed Scopus (51) Google Scholar). Furthermore, we demonstrate that sod1Δ yeast have a sensitivity to pH which cannot easily be explained without invoking the role of the vacuole in pH homeostasis. Likewise, an increased sensitivity to transition metals may be directly or indirectly related to vacuole abnormalities. Thus, some of the curious metabolic and biochemical abnormalities in sod1Δ yeast may in fact be attributable to vacuolar aberrations. In summary, we demonstrate that oxidative stress to sod1Δyeast results in vacuolar fragmentation and either removal of oxygen or iron can ameliorate the damage. This may have medical relevance in that oxidative damage and alterations in iron homeostasis have been implicated in a number of disease states including atherosclerosis, neurodegeneration, arthritis, and aging (for review, see Ref. 48Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar). In some cases oxidative insult is thought to be the primary cause of damage. For example, brain and spinal cord deterioration after ischemic or traumatic injury often appears excessive for the level of trauma (58Halliwell B. Gutteridge J.M.C. Trends Neurosci. 1985; 8: 22Abstract Full Text PDF Scopus (787) Google Scholar). One explanation put forth is that iron-binding capacity in the central nervous system and in the cerebral spinal fluid bathing the central nervous system is particularly low. Thus, release of iron by oxidatively or mechanically damaged cells, organelles, and proteins may catalyze toxic oxidative side reactions causing further cell injury. Consistent with this concept, preliminary trials of treatment with iron-chelating agents have had success in diminishing post-traumatic degeneration of brain and spinal cord (59Hall E.D. J. Neurosurg. 1988; 68: 462-465Crossref PubMed Scopus (139) Google Scholar, 60Hall E.D. Yonkers P.A. McCall J.M. Braughler J.M. J. Neurosurg. 1988; 68: 456-461Crossref PubMed Scopus (252) Google Scholar). Thus, iron-mediated oxidative damage is likely a common event in aerobic organisms, and dissection and understanding of the process (and ways of prevention) in yeast may give insight into human disease. We thank Steve Gould for advice on EM analysis of yeast and Scott Emr for advice and reagents to analyze vacuoles. We also thank Dan Kosman (SUNY, Buffalo) for providing yeast strain YPH250-fet3Δ." @default.
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• W2171471851 title "Oxidative Stress and Iron Are Implicated in Fragmenting Vacuoles of Saccharomyces cerevisiae Lacking Cu,Zn-Superoxide Dismutase" @default.
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