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 (lys7Δ), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Δ) andlys7Δ 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)lys7Δ cells are slightly less sensitive to paraquat thansod1Δ cells, 2) EPR-detectable or “free” iron is dramatically elevated in sod1Δ mutants but not inlys7Δ yeast, and 3) although sod1Δ mutants show increased sensitivity to extracellular zinc, thelys7Δ 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 sod1Δ 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. 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 (lys7Δ), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Δ) andlys7Δ 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)lys7Δ cells are slightly less sensitive to paraquat thansod1Δ cells, 2) EPR-detectable or “free” iron is dramatically elevated in sod1Δ mutants but not inlys7Δ yeast, and 3) although sod1Δ mutants show increased sensitivity to extracellular zinc, thelys7Δ 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 sod1Δ 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 dismutase wild type. With the appearance of molecular oxygen (O2) in the earth's atmosphere, all aerobic organisms evolved methods to utilize O2 for energy production. However, reactive byproducts of O2 metabolism (also known as reactive oxygen species) can be deleterious, and aerobic life forms have developed several systems to combat metabolic and environmental sources of oxygen toxicity. One major constituent in protecting cellular components against reactive oxygen species is superoxide dismutase (SOD).1 As a part of the primary line of defense against reactive oxygen species, SODs use metal ions to catalyze the disproportionation of superoxide (O⨪2) to hydrogen peroxide (H2O2) and O2 (1Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2731) Google Scholar). Eukaryotes, including Saccharomyces cerevisiae, contain two distinct SOD enzymes. Mn-SOD, encoded by the SOD2 gene, resides in the mitochondrial matrix; CuZn-SOD, encoded by the SOD1 gene, is localized mainly in the cytoplasm and nucleus as well as in the mitochondrial intermembrane space. Mutant derivatives of S. cerevisiae that lack either of the SODs exhibit phenotypic deficiencies which are particularly extreme in the sod1Δ mutants. Even though they are viable, sod1Δ strains grow poorly in air, are extremely sensitive to redox-cycling drugs, die quickly in the stationary phase, and display aerobic lysine and methionine auxotrophies. It is generally believed that the loss of superoxide-scavenging activity, which is due to the absence ofSOD1 gene product, leads to a serious burden of oxidative stress and causes havoc in cellular metabolism and growth processes (2Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (138) Google Scholar,3Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar). Employing in vivo whole-cell Fe(III) EPR methodology, Keyer and Imlay (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (693) Google Scholar) observed an increase in loosely bound or “free” iron 2It should be noted that there is no such chemical entity as free iron in an intracellular environment; it would most certainly exist only in complexes with other as yet undefined cellular components. We use “free” iron in quotes to indicate this fact. in sodmutants of E. coli. This iron is present in the Fe(II) state, as evidenced by the fact that it is only detectable by EPR at g = 4.3 after treatment of the cells with the iron chelator desferrioxamine, which converts EPR-silent Fe(II) to EPR-detectable Fe(III). A potential source of this increased pool of “free” iron includes a class of enzymes containing exposed [4Fe-4S] clusters, which are superoxide-sensitive (5Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (364) Google Scholar). The classic example of this type of enzyme is aconitase (6Flint D.H. Allen R.M. Chem. Rev. 1996; 96: 2315-2334Crossref PubMed Scopus (225) Google Scholar). Attack by O⨪2 oxidizes the [4Fe-4S] cluster, resulting in the release of at least one iron ion and inactivation of the enzyme. In E. coli, this process was proposed to be the source of the “free” iron that built up in mutants lacking SOD (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (693) Google Scholar). Recently, adapting the same EPR method, we demonstrated that yeast sod1Δ mutants also have greatly elevated levels of “free” or “EPR-detectable” iron (iron detectable at g = 4.3 by EPR). In yeast, unlike in E. coli, the iron is present in the Fe(III) state (7Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Studies of yeast mutants lacking the SOD1 gene (sod1Δ) have revealed intriguing links connecting CuZn-SOD with transition metal metabolism. Addition of manganese or copper salts to the medium in modest amounts can improve the growth of these strains (reviewed in Ref. 8Gralla E.B. Scandalios J. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1997: 495-525Google Scholar). More recently, we have shown thatsod1Δ yeast have an increased requirement for iron in aerobic growth, and under certain growth conditions, they display a moderate increase in iron uptake and accumulation (9De Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Moreover, a variety of second site genetic suppressors of the sod1Δ phenotype have been isolated (10Liu 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, 11Liu X.F. Culotta V.C. Mol. Cell. Biol. 1994; 14: 7037-7045Crossref PubMed Scopus (60) Google Scholar, 12Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar, 13Strain 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 (169) Google Scholar), and although the gene products involved are located in different cellular compartments and organelles, they all participate in the homeostasis of transition metal ions: copper, manganese, or iron. Out of this scientific endeavor has also come the discovery of the copper chaperone for SOD1 (CCS). This protein is responsible for the delivery of copper to CuZn-SOD and is encoded by the LYS7gene in S. cerevisiae (14Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Crossref PubMed Scopus (683) Google Scholar, 15Gamonet F. Lauquin G.J. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar). Because the SOD1 polypeptide in lys7Δ mutants lacks copper in its active site, it is incompetent to catalyze the removal of O⨪2. lys7Δ and sod1Δ strains exhibit very similar phenotypic defects (including poor growth in air and aerobic auxotrophies for methionine and lysine), but they are not identical, as we report here. Certain differences exist between these two mutant derivatives, most notably the following two characteristics: 1) sod1Δ mutants display a 5-fold increase in the levels of EPR-detectable iron relative to lys7Δ and wild-type yeast and 2) sod1Δ mutants are sensitive to zinc while lys7Δ yeast are not. These dissimilarities strongly suggest an in vivo role forSOD1 in metal metabolism, and we have provided further evidence that the SOD1 polypeptide may play a role in cellular resistance to elevated levels of zinc ions. High purity sulfate salts of zinc, copper, and nickel were obtained from Sigma. Methyl viologen, commonly known as paraquat, and the antibiotic G418 were also purchased from Sigma. The yeast strains used in this study are described in Table I. Strains BY4741, JW202, and JW203 were purchased from Research Genetics (Huntsville, AL). The lys7Δ strain JW101 was obtained by deleting the chromosomal LYS7 locus of EG103 using thelys7::LEU2 construct pSL2527 as described previously (16Horecka J. Kinsey P.T. Sprague Jr., G.F. Gene (Amst.). 1995; 162: 87-92Crossref PubMed Scopus (34) Google Scholar). Using oursod1Δ::URA3 construct, JW102 (lys7Δsod1Δ) was prepared by sequential gene deletion starting with the lys7Δ strain. Southern blot analysis was performed to verify the correct deletion of theLYS7 and SOD1 genes (data not shown). The plasmids pEMBLyex4 and pSALSOD1 (referred to herein as pMn-SOD and carrying the Bacillus stearothermophilus gene for Mn-SOD) were kindly provided by Dr. C. Bowler (17Bowler C. Van Kaer L. Van Camp W. Van Montagu M. Inze D. Dhaese P. J. Bacteriol. 1990; 172: 1539-1546Crossref PubMed Google Scholar) and introduced into JW201 (the sod1 null yeast in the BY4741 background) by lithium acetate transformation (18Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar) to create strains JW203 and JW204. EG118 (sod1Δ) yeast cells were transformed with YEp351, YEp600, or YEp351-yH46C to yield strains HH101, HH102, or HH103, respectively. YEp351 is a yeast multicopy shuttle vector with a LEU2 selectable marker (19Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1082) Google Scholar). YEp600 contains a 1.1-kilobase genomic fragment containing the wild-type SOD1 gene with its own promoter and carrying an engineered NcoI site at the translation start site (20Nishida C.R. Gralla E.B. Valentine J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9906-9910Crossref PubMed Scopus (84) Google Scholar). YEp351-yH46C was generated by inserting the yH46C fragment (NcoI-SmaI) from pGEM-3Zf(+)-yH46C (21Lu Y. Roe J.A. Bender C.J. Peisach J. Banci L. Bertini I. Gralla E.B. Valentine J.S. Inorganic Chem. 1996; 35: 1692-1770Crossref Scopus (53) Google Scholar) into YEp600 digested withNcoI-PstI/blunt and introduced into the specified yeast strain by lithium acetate transformation (18Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Expression was verified by Western blot analysis.Table IYeast strains used in this studyStrainRelevant genotypeComplete genotypeSourceEG103Wild typeMATα leu2 his3 trp1 ura3Ref. 3Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google ScholarEG118sod1ΔMATα leu2 his3 trp1 ura3 sod1Δ∷URA3Ref. 3Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google ScholarJW101lys7ΔMATα leu2 his3 trp1 ura3 lys7Δ∷LEU2This workJW102lys7Δsod1ΔMATα leu2 his3 trp1 ura3 lys7Δ∷LEU2 sod1Δ∷URA3This workBY4741Wild typeMATa his3 leu2 met15 ura3PurchasedJW201sod1ΔMATa his3 leu2 met15 ura3 sod1Δ∷KANRPurchasedJW202lys7ΔMATa his3 leu2 met15 ura3 lys7Δ∷KANRPurchasedJW203sod1Δ/pEMBLJW201 with pEMBLyex4-URA3This workJW204sod1Δ/pMnSODJW201 with pSALSOD-URA3This workHH101sod1Δ/YEp351EG118 with YEp351This workHH102sod1Δ/ySOD1EG118 with YEp600This workHH103sod1Δ/yH46CEG118 with YEp351-yH46C SODThis work Open table in a new tab For experiments, yeast without plasmids were cultured in synthetic complete medium with 2% glucose (SDC) or galactose (SGalC) as mentioned. Yeast with plasmids were cultured in the appropriate dropout medium (SD-Leu for HH101, HH102, and HH103 and SD-Ura or SGal-Ura for JW203 and JW204). SDC medium was composed as described (18Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar) except that the supplements Leu, His, Trp, Met, Ura, and Ade were increased 4-fold. Overnight startup cultures were grown from a single colony in the same type of medium that was used for further growth except when the medium contained galactose. For growth in galactose-containing medium, the startup culture in glucose was washed twice with sterile water prior to inoculation in galactose-containing medium. For EPR studies, EG103, EG118, JW101, and JW102 were seeded at an optical density at 600 nm (A600) of 0.1 in 50 ml of SDC medium and grown for 72 h at 30 °C. After growth, cells were centrifuged and then briefly treated with desferrioxamine, which binds to labile iron and converts it from Fe(II) (EPR-silent) to Fe(III) prior to washing with 20 mm Tris-Cl, pH 7.4. After washing, the pellet was resuspended in 200 μl of 20 mm Tris-Cl, pH 7.4, containing 20% glycerol, and 200 μl of the resuspended cells was packed in EPR tubes. The tubes were stored at −70 °C until EPR measurements were performed. EPR spectra were recorded using a Bruker X-band spectrometer using a finger Dewar filled with liquid nitrogen attached to the cavity of the instrument to maintain low temperature. Parameters used for low temperature Fe(III) EPR were as follows: center field, 1500 G; sweep width, 500 G; microwave power, 20 milliwatts; attenuation, 10 dB; modulation amplitude, 20.2 G; modulation frequency, 100 kHz. Further details regarding sample preparation, instrument parameters, and data analysis used for the whole-cell low temperature Fe(III) EPR measurements are delineated in our previous study (7Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Paraquat, zinc, copper, or nickel was added to the indicated medium at the indicated concentration. Experimental cultures in dextrose-containing medium were inoculated at an A600 of 0.03 to 0.05. Experimental cultures in medium containing galactose were seeded at an A600 of 0.05 to 0.07. To evaluate the effect of excess zinc ion, the starting pH of the growth medium was at 4 instead of 6 to prevent the precipitation of zinc as zinc hydroxide at pH 6. (This pH change had no obvious effect on growth. Data not shown.) Cultures were grown at 30 °C with shaking at 200 rpm for a maximum of 24 h. Total cell growth was determined by measuring turbidity at A600. AnA600 of 1 indicates a culture density of 1 × 107 cells/ml. Cells were grown as described above. Typically, duplicate samples containing 15 to 20 × 107 cells from 25-ml stationary phase cultures were harvested by centrifugation. The cell pellets were washed once in 5 ml of 10 mm EDTA, pH 8, followed by three washes in 5 ml of metal-free water. The final cell pellets were resuspended in 1 ml of 10% ultrapure nitric acid, and digestion was allowed to take place at 98 °C for 18 h. After complete digestion, 950 μl was diluted to 7 ml in metal-free water, and these samples were analyzed using a Thermo-Jarrel Ash Iris 1000 inductively coupled plasma-atomic emission spectrometer. lys7Δ yeast have normal SOD1 expression, but the SOD1 polypeptide lacks enzymatic activity because the copper is not incorporated into the active site (14Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Crossref PubMed Scopus (683) Google Scholar, 15Gamonet F. Lauquin G.J. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar). Therefore, one would expectlys7Δ and sod1Δ strains to possess comparable antioxidant capacity and thus to share similar phenotypes. With thelys7Δ and sod1Δ strains constructed from our laboratory wild-type yeast (EG103 background), we have observed numerous similarities between these two strains, such as slow growth, aerobic auxotrophies for lysine and methionine, poor growth in the presence of non-fermentable carbon source, and temperature sensitivity at 37 °C. However, further characterization of the lys7null strain led to the discovery of certain differences between these two yeast mutants. First, we examined the effect of paraquat, a known generator of intracellular superoxide, on wild-type,lys7Δ, sod1Δ, andlys7Δsod1Δ yeast by culturing cells in the presence of varying concentrations of paraquat for 24 h. As shown in Fig. 1 A, at 10 and 25 μm of paraquat, concentrations that are toxic for bothsod1Δ and lys7Δsod1Δ mutant strains, lys7Δ cells still grew as well as wild-type cells, but 100 μm paraquat was toxic. In contrast, wild-type EG103 can survive in 1 to 10 mm paraquat. Identical experiments were conducted in another genetic background, BY4741, with similar results (Fig. 1 B). In the BY4741 background, lys7Δ cells are resistant to the toxic effects of paraquat at concentrations as high as 100 μm(83.2 ± 2.9% control growth), whereas sod1Δ yeast exhibit significant levels of growth retardation at 25 μm(8.0 ± 4.8% control growth). Because it is postulated that an elevated superoxide level is connected to an increase in cellular “free” iron (4Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (693) Google Scholar, 9De Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Corson L.B. Folmer J. Strain J.J. Culotta V.C. Cleveland D.W. J. Biol. Chem. 1999; 274: 27590-27596Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we wonder whether the decreased sensitivity to paraquat for lys7Δ yeast correlated with a diminished level of “free” iron as measured by whole-cell Fe(III) EPR spectroscopy. Wild-type, lys7Δ, sod1Δ, andlys7Δsod1Δ cells were grown to stationary phase (72 h) in SDC, and EPR samples were prepared and evaluated as described under “Experimental Procedures.” As shown in Fig.2, lys7Δ cells had a basal level of EPR-detectable iron of 10.5 ± 3.2 μm, similar to that observed for wild-type yeast. However, the isogenicsod1Δ and lys7Δsod1Δ yeast had levels that were ∼5-fold higher (56.8 ± 14.3 μmand 47.9 ± 6.0 μm, respectively). We also prepared EPR samples of the aforementioned strains without desferrioxamine treatment and observed similar results (data not shown), suggesting that the “free” iron present in all strains was in the ferric state and that the intracellular redox status was similar. Interestingly, in these experiments, elevation of the “free” iron insod1Δ mutants was not accompanied by an increase in the total iron content (see below). One of the more puzzling aspects of the phenotypes of sod1Δ mutants has been their heightened sensitivity to extracellular zinc. We discovered this effect several years ago, and since then, we have sought for an explanation. Wild-type cells tolerate 5 or even 10 mm zinc in the medium (data not shown). On the other hand, cells lacking CuZn-SOD exhibit growth retardation at zinc levels as low as 1 mm. Thus, we decided to assess the ability of excess zinc ions to inhibit growth inlys7Δ yeast as well. In the experiment shown in Fig.3 A, lys7Δ,sod1Δ, lys7Δsod1Δ, and the parental wild-type strain (EG103) were cultured in the presence of increasing concentrations of ZnSO4. Unlike thesod1Δ mutant, which displayed marked growth retardation at 0.5 mm ZnSO4 (∼73% control growth),lys7Δ cells behaved like wild type at all concentrations of ZnSO4 tested (up to 10 mm, Fig. 3 and data not shown). To ascertain that this phenotypic difference betweenlys7Δ and sod1Δ null strains is not a strain-dependent phenomenon, we tested the effect of excess zinc ions on these two mutants made in the BY4741 background. As shown in Fig. 3 B, the resistance of lys7Δ to elevated levels of ZnSO4 mirrored that observed for the EG103 background. In two genetic suppressors of sod1Δ mutants,pmr1 and bsd2, increased sensitivity toward manganese and copper ions was found to correlate with the accumulation of intracellular manganese and copper, respectively (11Liu X.F. Culotta V.C. Mol. Cell. Biol. 1994; 14: 7037-7045Crossref PubMed Scopus (60) Google Scholar, 12Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). Thus, we proceeded to explore whether the sensitivity to excess zinc in thesod1Δ cells was associated with elevated levels of total cellular zinc. The total cellular level of zinc in thesod1Δ mutant was compared with those in isogeniclys7Δ and wild-type strains. In the experiment shown in Fig. 4, zinc levels were measured in cells after 72 h of growth in SDC medium. The sod1Δ, as well as the lys7Δsod1Δ, mutants accumulate ∼50% higher levels of zinc than do the wild-type andlys7Δ strains, further implicating SOD1 in the regulation of heavy metal metabolism. To evaluate the effects of other transition metal ions on wild-type,lys7Δ, and sod1Δ yeast, the growth of these strains was tested in the presence of CuSO4 and NiSO4. As shown in Fig.5 A, the extent of growth inhibition is comparable for all three strains. As has been observed previously, low concentrations of CuSO4 (0.5 mm) enhanced the growth of sod1Δ andlys7Δ mutants, but higher concentrations were equally toxic to mutant and wild-type cells. In medium supplemented with [Ni2+] ranging from 0 to 1.0 mm,lys7Δ and sod1Δ demonstrate similar growth potential (Fig. 5 B). Interestingly, low medium [Ni2+] seemed to have a more pronounced effect on the growth of wild-type (EG103) yeast. It is possible that the weakened antioxidant capacity of lys7 and sod1 null mutants have caused a constitutive up-regulation of various cellular stress genes, thus rendering these cells more resistant to other types of environmental stresses. Overall, the experimental evidence depicted in Fig. 5 suggests that the ability of both lys7Δ andsod1Δ cells to sequester other transition metal ions probably remains intact on a gross level and that the sensitivity ofsod1Δ mutants to excess metal ions is specific to zinc. To assess the relative contributions of the enzymatic superoxide dismutase activity and the zinc binding capacity of CuZn-SOD, we restored the superoxide resistance of sod1Δ yeast by overexpressing a bacterial Mn-SOD in their cytoplasm. Because Mn-SOD is expected to restore SOD activity but not zinc binding capacity in vivo, we were interested to see whether its expression would have any effect on the zinc sensitivity of the sod1Δ mutant. The sod1 null mutant in the BY4741 background was transformed with either pSALSOD (pMn-SOD), a yeast expression vector containing the B. stearothermophilus Mn-SOD gene under the control of the GAL-CYC1 promoter, or pEMBLyex4 (pEMBL), the corresponding vector control (17Bowler C. Van Kaer L. Van Camp W. Van Montagu M. Inze D. Dhaese P. J. Bacteriol. 1990; 172: 1539-1546Crossref PubMed Google Scholar). Growth of thesod1Δ/pMn-SOD strain in galactose resulted in the appearance of Mn-SOD activity and loss of the aerobic lysine auxotrophy (data not shown and Ref. 17Bowler C. Van Kaer L. Van Camp W. Van Montagu M. Inze D. Dhaese P. J. Bacteriol. 1990; 172: 1539-1546Crossref PubMed Google Scholar). We tested the paraquat sensitivity of the plasmid-containing strains and found thatsod1Δ/pMn-SOD cells behave very much like wild-type yeast, resisting paraquat concentrations as high as 1.0 mm(41.1 ± 11.4% control growth forsod1Δ/pMn-SOD compared with 52.8 ± 2.2% for wild type (WT)), whereas the sod1Δ/pEMBL strain was much more sensitive (Fig. 6 A). Possibly because of the galactose in the medium, somewhat higher levels (0.2 and 0.5 mm) of paraquat are required to cause toxicity in the sod1Δ/pEMBL as compared with the non-plasmid-containing sod1Δ strain. Nevertheless, we can conclude that the prokaryotic Mn-SOD efficiently strengthened the antioxidant capacity of the sod1Δ yeast as evidenced by greatly increased resistance to paraquat, a superoxide-generating chemical. We then tested whether the transformed strains were resistant to zinc (Fig. 6 B). For medium [Zn2+] below 2.0 mm, all four strains behaved similarly (data not shown). Thus, higher concentrations of ZnSO4 than were previously used were required to evaluate the ability of these transformants to resist zinc toxicity, again possibly because of the use of galactose-based medium. As can be seen in Fig. 6 B, expression of cytoplasmic Mn-SOD does not restore zinc resistance to wild-type levels, supporting a role for CuZn-SOD in zinc homeostasis. In a converse experiment, to provide further mechanistic insight into the zinc sensitivity observed in sod1Δ cells, we overexpressed yH46C (a mutant SOD1 protein lacking catalytic activity but capable of binding metal ions) in sod1Δyeast. Growth of the sod1Δ/yH46C strain was tested in medium supplemented with ZnSO4. As shown in Fig.7, sod1Δ/yH46C grew as well as sod1Δ/ySOD1 in the presence of 1.0 and 2.0 mm ZnSO4, but sod1Δ/YEp351 (vector control) exhibited significant growth inhibition at those concentrations. Expression of this mutant SOD had no effect on the resistance to paraquat, confirming that it is catalytically inactive. (At a concentration of 25 μm paraquat, growth ofsod1Δ/yH46C was just 2.1 ± 0.5% control growth.) In this study, we demonstrate that certain differences exist between lys7Δ and sod1Δ yeast.lys7Δ yeast are not as sensitive to the redox-cycling drug paraquat as the sod1Δ mutant, although they are still much less resistant than the isogenic wild-type cells. In addition,lys7Δ cells exhibit wild-type levels of “free” iron as measured by Fe(III) EPR, whereas sod1Δ mutants display a 5-fold increase (Fig. 2). Moreover, the lys7Δ strain grows as well as wild type in elevated zinc, while the sod1Δ strain fails to thrive (Fig. 3). The increased zinc sensitivity of thesod1Δ strain may be due to the fact that it accumulates excess zinc relative to the wild-type and lys7Δ strains (Fig. 4). To explain the phenotypic differences betweensod1Δ and lys7Δ yeast, two different (albeit not mutually exclusive) hypotheses have been considered. The first hypothesis is that the lys7Δ strain retains an extremely small amount of SOD activity, below the detection limit of currently available assays. The second is that the copper-free SOD1 protein, which is present in lys7Δ but not in thesod1Δ strain, plays a role in zinc metabolism either by acting as a depot for zinc ions or through some other mechanism. To distinguish among these possibilities, we performed experiments designed to separate SOD enzymatic activity from the zinc binding activity. We found that cytoplasmic expression of a bacterial Mn-SOD fully restored the paraquat resistance of sod1Δ strains but did not fully restore the zinc sensitivity (Fig. 6). Conversely, expression of H46C, a mutant CuZn-SOD with no enzymatic activity, fully restored zinc resistance (Fig. 7) but had no effect on paraquat resistance. The first hypothesis (that there may be a residual amount of SOD activity in lys7Δ yeast) is supported by the facts that the lys7Δ strains appear slightly more resistant to oxidative stress (e.g. paraquat resistance is a little higher in the lys7Δ mutants than in the sod1Δstrains) and that the zinc sensitivity of sod1Δ strains is only observed in aerobic growth. However, another possible explanation for these phenotypic differences is that paraquat resistance is conferred by superoxide dismutase activity plus some other antioxidant function of the SOD1 polypeptide. For example, inactive apoSOD1 polypeptide might be able to physically protect some vulnerable site(s). (If this is the case, the SOD activity is clearly the more important function because expression of active SOD in the cytoplasm increases paraquat resistance 100-fold or more, while expression of SOD1 made inactive by the absence of Lys7 only increases paraquat resistance a few fold.) The main evidence against the residual activity hypothesis is the fact that SOD activity is undetectable inlys7Δ strains by any assay we have tried. The second hypothesis, which we favor, is that the SOD1 protein plays a role in zinc metabolism or homeostasis. CuZn-SOD is an abundant enzyme, and thus it has the capacity to bind substantial amounts of copper and zinc. CuZn-SOD can help protect against copper toxicity in yeast lacking Cup1 (copper metallothionein) (23Culotta V.C. Joh H.D. Lin S.J. Slekar K.H. Strain J. J. Biol. Chem. 1995; 270: 29991-29997Crossref PubMed Scopus (140) Google Scholar). That it participates in protection against zinc toxicity is supported by several pieces of evidence: 1) zinc resistance was lost only when the SOD1 polypeptide was missing (as in the sod1Δ mutant, whether or not it was expressing cytoplasmic Mn-SOD); 2) paraquat resistance was lost only when SOD activity was missing (as in the lys7Δ mutant and the sod1Δ mutant, whether or not they were expressing the inactive H46C mutant SOD; and 3) a null mutation in the yeastSOD2 gene, which encodes the mitochondrial manganese SOD, is not associated with an increased sensitivity toward zinc (data not shown). Therefore, the presence of SOD1 polypeptide, either inactive or enzymatically active, is required to confer zinc tolerance. The simplest explanation is that SOD1 acts as a “zinc sink,” and its absence causes the cell interior to be exposed to levels of zinc in excess of what can be readily tolerated. However, the data showing elevated zinc levels in the sod1Δ strain are puzzling in this regard. It may be that the lack of SOD1 somehow interferes with zinc sensing by Zap1 or with zinc transport or storage, leading to a buildup or mislocalization of zinc stores. We are currently exploring these possibilities experimentally. Another level of complexity is that zinc resistance of thesod1Δ strain was somewhat improved by cytoplasmic expression of the heterologous Mn-SOD, although it was not fully restored. This may indicate that zinc toxicity has an oxidative stress component that is exacerbated when SOD activity is missing. It is possible that this is related to the differences in “free” iron shown in Fig. 2. Alternatively, it may simply be that a SOD-competent cell is better able to defend itself against other stresses or that cellular antioxidant defenses assist in managing disturbances in metal homeostasis. Numerous studies have implicated the vacuole in metal ion storage and homeostasis (24MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (306) Google Scholar, 25Nishimura K. Igarashi K. Kakinuma Y. J. Bacteriol. 1998; 180: 1962-1964Crossref PubMed Google Scholar, 26Pearce D.A. Sherman F. J. Bacteriol. 1999; 181: 4774-4779Crossref PubMed Google Scholar, 27Radisky D.C. Snyder W.B. Emr S.D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5662-5666Crossref PubMed Scopus (78) Google Scholar), and mutants defective in vacuole function or acidification are sensitive to various metal ions, including copper, nickel, and zinc (28Ramsay L.M. Gadd G.M. FEMS Microbiol. Lett. 1997; 152: 293-298Crossref PubMed Google Scholar, 29Szczypka M.S. Zhu Z. Silar P. Thiele D.J. Yeast. 1997; 13: 1423-1435Crossref PubMed Scopus (80) Google Scholar). Because sod1Δ mutants display aberrant vacuolar morphology (22Corson L.B. Folmer J. Strain J.J. Culotta V.C. Cleveland D.W. J. Biol. Chem. 1999; 274: 27590-27596Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), the possibility remained that the zinc sensitivity was caused by vacuole malfunction in thesod1Δ yeast. We reasoned that if the zinc sensitivity in our strain was due to defective vacuolar function, the ability of the cells to handle elevated levels of transition metal ions other than zinc would be affected as well. The experimental results depicted in Fig. 5 clearly demonstrate that sod1Δ cells are as resistant as wild-type cells to increased levels of Cu2+ or Ni2+. That is, their ability to detoxify metal ions other than zinc remains intact, and the defect responsible for the zinc sensitivity in sod1Δ strains is not one of general vacuolar function. Taken together, these data strongly suggest that the copper-free SOD present in lys7Δ cells as well as the normally metallated CuZn-SOD present in WT cells participate in zinc ion homeostasis. A relationship between zinc metabolism and iron metabolism is also possible. The zinc sensitivity of sod1Δ mutants may be an indirect consequence of their aberrant iron metabolism. Yeastsod1Δ mutants exhibit increased levels of “free” iron, which may also come from the destruction of vulnerable [Fe-S] clusters (7Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and its accumulation may be exacerbated by the increased cellular transport of iron under conditions of superoxide stress (9De Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Because iron(II) and Zn(II) are similar chemically with respect to their coordination chemistry, they are transported by some of the same cellular transporters and can be bound by some of the same cellular proteins. Increased iron uptake may result in increased zinc uptake as well (30Askwith C.C. de Silva D. Kaplan J. Mol. Microbiol. 1996; 20: 27-34Crossref PubMed Scopus (103) Google Scholar, 31Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (244) Google Scholar). Thus, attempts by the sod1Δ yeast to increase iron may backfire in two ways: increased formation of hydroxyl radical due to increased Fenton chemistry (32Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Third Ed. Oxford University Press, New York1999Google Scholar) and the accumulation of toxic intracellular levels of zinc. Here we report thatlys7Δ yeast, unlike sod1Δ yeast, do not exhibit altered iron metabolism as evidenced by their wild-type-like level of EPR-detectable “free” iron (Fig. 2), and they do not accumulate increased levels of zinc inside the cells. Further work will be required to determine whether increased levels of “free” iron and of zinc are a result of the same process or of different ones, but the relationship is intriguing. In summary, our work has revealed unexpected differences betweenlys7Δ and sod1Δ mutant yeast that imply connections between superoxide toxicity, zinc metabolism, and iron metabolism. Our results indicate that the SOD1 polypeptide participates somehow in zinc homeostasis and that superoxide stress clearly involves iron metabolism. Continued investigations will shed more light on these relationships. We thank Drs. Barney L. Bales and Miroslav Peric at California State University, Northridge for the use of an EPR instrument and many helpful interactions.
Spontaneous coronary artery dissection is a rare cause of acute myocardial infarction and sudden death. It typically, but not always, occurs in healthy postpartum women without traditional risk factors for atherosclerosis. Moreover, the site of dissection usually involves the proximal, major coronary arteries: left main coronary artery and/or the left anterior descending artery, and in men, more often the right coronary artery. We report a case of sudden death caused by dissection of the obtuse marginal branch of the left circumflex artery, in a 49-year-old man, a very rare site of fatal coronary dissection.
502 Background: The 70-gene MammaPrint (MP) assay predicts risk of distant recurrence (DR) in hormone-receptor positive early-stage breast cancer and classifies cancers as Low Risk or High Risk. NSABP B-42 evaluated ELT in patients (pts) who had completed 5 yrs of adjuvant endocrine therapy (tx). The primary objective was to determine the utility of MP to identify pts enrolled in NSABP B-42 who are likely to benefit from ELT. Methods: A total of 1,866 pts from B-42 had available MP results. Primary endpoint is DR. Secondary endpoints are disease-free survival (DFS) and breast cancer-free interval (BCFI). For the primary analysis, pts were classified as High Risk (MP-H) (MP score ≤0.000) or Low Risk (MP-L) (MP score > 0.000). Exploratory analyses were performed for MP-L subcategories: MP Ultralow Risk (MP-UL) (MP score > 0.355) and MP-L but not MP-UL (MP-LNUL) (MP score > 0.000, ≤0.355). Likelihood ratio test based on stratified Cox proportional hazards (PH) model was used for treatment by risk group interaction. Stratified log-rank test was used to compare treatment groups. Hazard ratios and 95% CI were computed based on the stratified Cox PH model. Results: Among 1,866 pts, 706 (38%) were MP-H and 1,160 (62%) were MP-L. Of the MP-L, 252 (22%) were MP-UL. There were no significant differences in the distribution of patient and tumor characteristics between the MP group and the rest of the B-42 cohort, except for HER2 status. ELT effect was more pronounced in the MP cohort than in the overall B-42 population. For DR, there was statistically significant ELT benefit in MP-L (HR = 0.43, 95% CI 0.25-0.74, p = 0.002), but not MP-H (HR = 0.65, 0.34-1.24, p = 0.19) (interaction p = 0.38). For DFS, there was statistically significant ELT benefit in MP-L, but not MP-H (interaction p = 0.015). Similar findings were observed for BCFI (interaction p = 0.006). Within subcategories of MP-L, there was statistically significant ELT benefit in MP-LNUL, but not in MP-UL for all three endpoints, however the power in MP-UL was limited due to low number of pts (Table). Clinical trial information: 00382070. Conclusions: Statistically significant ELT benefit was observed for MP-L, but not MP-H. The treatment by risk group interaction was not statistically significant for DR, but it was for DFS and BCFI. The benefit appears to be stronger in MP-LNUL than in MP-UL. NCT: 00382070. Support: U10CA180868, -180822, U24CA196067; Novartis; Agendia.[Table: see text]
