Exogenous Copper Research Articles

Cu(ii) ion coordination to SPARC: a model study on short peptide fragments

SPARC (secreted protein, acidic and rich in cysteine) is a glycoprotein of the extracellular matrix that mediates the cell-matrix interactions. It plays also a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. The human SPARC consists of three distinct modules. Module II is follistatin-like and its hydrolysis gives rise to a number of oligopeptides that can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. In order to completely understand the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the three SPARC122–126, SPARC121–126 and SPARC120–126 fragments, protected at both their amino and carboxylic ends, is reported. These peptides (Ac-HKLHL-NH2, Ac-GHKLHL-NH2 and Ac-KGHKLHL-NH2, respectively) constitute good models for the strong copper-binding site of the protein. The behaviour of the three ligands is very similar: complex formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles promotes amide ionization in the physiological pH range and this can explain SPARC binding to the Cu(II) ion.

Ctr6, a Vacuolar Membrane Copper Transporter inSchizosaccharomyces pombe

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6(+) gene. The transcription of ctr6(+) is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6Delta allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6+ gene is overexpressed from the thiamine-inducible nmt1(+) promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4(+) gene expression. By using a functional ctr6(+) epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase.

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.

Cu(ii) ion coordination to the pentadecapeptide model of the SPARC copper-binding site

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein with many biological functions: it mediates the interactions between cells and the extracellular matrix, playing a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. Proteolysis of SPARC gives rise to a number of oligopeptides which can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. Human SPARC consists of three distinct modules. Module II is follistatin-like and contains two copper binding sites, the strongest of which—the cationic region 2 (amino acids 114–130)—contains the sequence Gly–His–Lys. In order to shed more light on the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the SPARC114–128 fragment, protected at both its amino and carboxylic ends, is reported. This peptide (Ac–TLEGTKKGHKLHLDY–NH2) constitutes a good model to the strong copper-binding site of the protein. The whole experimental data suggest that complex-formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles is able to promote amide ionisation in the physiological pH range and this could be the key to the SPARC affinity for Cu(II) ion.

Copper-associated liver disease in North Ronaldsay sheep: a possible animal model for non-Wilsonian hepatic copper toxicosis of infancy and childhood.

Indian childhood cirrhosis (ICC), endemic Tyrolean infantile cirrhosis (ETIC) and idiopathic copper toxicosis (ICT), are clinically and pathologically indistinguishable liver disorders of infants and young children linked with exogenous copper and with increasing evidence for a genetic predisposition. North Ronaldsay sheep are a primitive breed which have adapted to a copper impoverished environment (<5 ppm) and display an abnormal sensitivity to copper poisoning when transferred to a copper replete (11 ppm) habitat. The aetiological parallels prompted a study of copper-associated liver disease in North Ronaldsay sheep (RCT) to see if the pathology could contribute to the understanding of the childhood disorder. A retrospective study was performed in which the livers of 22 mainland-bred North Ronaldsay sheep were compared with three island-bred sheep and categorized for liver copper content and pathomorphology. It was found that all the mainland sheep had accumulated liver copper (>300 microg/g), in contrast to the island sheep, although 10 sheep with increased liver copper (mean 600 SD 270 microg/g) showed no evidence of liver damage. A further 10 sheep with liver copper (mean 1276 SD 508 microg/g) exhibited periportal to panlobular histochemical copper retention, a periportal and/or panlobular pericellular fibrosis, a mixed inflammatory infiltrate and cholangioplasia. Steatosis was absent and regeneration was in abeyance. Finally, two sheep (liver copper >2000 microg/g) had a more active hepatitis with a florid pericellular, panlobular fibrosis and cirrhosis. Electron microscopy identified large numbers of collagen-producing hepatic stellate (Ito) cells in periportal regions. The pathological findings were sufficiently reminiscent of ICC, ETIC and ICT to warrant further exploration of RCT as a putative animal model. The North Ronaldsay sheep liver may be a useful tool for the investigation of copper-induced fibrogenesis.

Effects of KE-758; an active metabolite of the new anti-rheumatic drug KE-298, d-penicillamine, bucillamine and auranofin on the proliferation of murine lymphocytes, and the production of nitric oxide by murine macrophages

2-Mercaptomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-758), which is the active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), is a novel sulphydryl anti-rheumatic drug. In this study we analyzed the effect of KE-758 on the proliferation of murine lymphocytes, and on the production of nitric oxide (NO) by RAW264.7 murine macrophage cells. We compared its effect with other sulphydryl drugs such as d-penicillamine, bucillamine and auranofin. The proliferation of lymphocytes was measured by 3H-thymidine incorporation assay. Nitrite was measured using Griess Reagent. In the absence of copper ions, KE-758, d-penicillamine and bucillamine rarely affected the proliferation of concanavarin A (ConA) activated murine splenocytes. However, in the presence of copper, pharmacological concentrations of KE-758 but not d-penicillamine and bucillamine suppressed the proliferation of murine splenocytes through a hydrogen peroxide-dependent mechanism. Auranofin markedly suppressed the proliferation regardless of the presence of copper ions by reducing the cellular viability. Furthermore, only KE-758 markedly suppressed the proliferation of phorbol myristate acetate (PMA) plus ionomycin activated murine whole blood lymphocytes (WBL) even in the absence of exogenous copper ions by a hydrogen peroxide-independent mechanism. Meanwhile, lipopolysaccharide (LPS) or LPS plus interferon-gamma (IFN-γ) induced NO production by RAW264.7 cells were suppressed by KE-758 and auranofin but not by d-penicillamine and bucillamine. In conclusion, KE-758 is a novel immunosuppressive drug, which inhibits both lymphocyte and macrophage functions and its unique anti-rheumatic profile is distinct from that of d-penicillamine, bucillamine and auranofin.

Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

Role of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity.

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4). These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level.

The antioxidant and metal-chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serum-depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o-phenanthroline, bathophenanthroline disulfonic acid, and N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC-induced endothelial cell death was greater than that of copper. Zn-EDTA did not block PDTC-induced cell death, whereas Ca-EDTA and Cu-EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn-EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC-induced cerebral endothelial cell death.

Effects of Long-term Sublethal Exposure to Copper on Subsequent Uptake and Distribution of Metal in the Shore Crab Carcinus Maenas

As in many aquatic organisms, the gill epithelium is the main site of the uptake of copper and toxicity in the shore crab. Long term pre-exposure to sublethal doses of water-borne copper has been shown to lead after some weeks to progressive recovery from the initial toxic effects. We report experimental results that shed light on some of the mechanisms underlying these acclimatory effects. The metal adaptation seems related to an important reduction of copper uptake from the ambient medium and to a better efficiency of metal transfer from hemolymph to the tissues. As a consequence, steady-state hemolymph levels of exogenous copper are strongly decreased during a further lethal exposure in pre-acclimated crabs compared to controls. A 17-day sublethal pre-acclimation period appears, however, to be too short to improve the resistance of the animals to a further challenge at usually lethal doses of copper.

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription.

The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly for acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution of redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Accumulation of metallothionein transcripts in response to iron, copper and zinc: Metallothionein and metal-chelate reductase

It has been proposed that plant metallothionein (MT) sequesters excess copper, and possibly zinc, thereby preventing adverse metal-protein interactions. These metals can accumulate either gratuitously in response to other nutritional deficiencies or in plants grown in either copper- or zinc-enriched medium. Data are presented which confirm that in pea roots grown in low available iron there is increased (i) copper accumulation, (ii) MT transcript abundance, (iii) ferric-chelate reductase activity and (iv) cupric-chelate reductase activity. It is also shown that in roots grown in iron supplemented medium MT transcripts accumulate in response to elevated exogenous zinc. However, contrary to expectations, depletion of exogenous copper below normal micronutrient levels also confers an increase in the abundance of MT transcripts.

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron‐responsive gene transcription

The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions. © 1997 John Wiley & Sons, Ltd.

Effect of Metal on 2,4,5-Trihydroxyphenylalanine (Topa) Quinone Biogenesis in the Hansenula polymorpha Copper Amine Oxidase

Previous studies of wild-type and mutant forms of a recombinant copper amine oxidase from Hansenula polymorpha, expressed in Saccharomyces cerevisiae, have indicated a self-processing mechanism for 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis involving the active site copper (Cai, D., and Klinman, J. P. (1994) J. Biol. Chem. 269, 32039-32042). In contrast to prokaryotic copper amine oxidases, however, it has not been possible to initiate topa quinone formation by the addition of exogenous copper to precursor H. polymorpha amine oxidase lacking copper. Metal analysis of copper-depleted wild-type enzyme reveals 0.2-0.3 mol copper, together with 0.6 mol zinc. Despite changes in the zinc and copper levels in growth media, the level of zinc in purified enzyme remains fairly constant. Further, we have been unable to displace protein-bound zinc by exogenously added copper. The H. polymorpha amine oxidase gene was subsequently expressed in Escherichia coli and found to be almost completely free of copper and zinc. In vitro reconstitution of this apoprotein confirms that zinc binds to H. polymorpha amine oxidase and prevents reconstitution with copper. By contrast, addition of copper first to apoprotein leads to formation of topa quinone and stable activity in the presence of added zinc. These findings indicate efficient binding of either zinc or copper to a site that undergoes little or no exchange. The data confirm that topa quinone biogenesis in the H. polymorpha system is catalyzed by copper and occurs in the absence of added factors. We conclude that the mechanisms of cofactor biogenesis in pro- and eukaryotic systems are likely to be similar or identical. The results described herein imply different pathways for the in vivo assembly of heterologously expressed amine oxidases in S. cerevisiae and E. coli.

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver.

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identified in the liver of rats made copper-deficient by dietary restriction. In homogenates of such livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoretic profile with respect to the native enzyme. When rat liver extracts were treated with exogenous copper an electrophoretic pattern resembling the native one was observed. Enzyme purified by chromatography on DE-52 resin shows two major components, one corresponding to genuine, native enzyme and another one, eluting at higher ionic strength. The latter protein (Fraction II) consists of several isoforms which show the same characteristics of the native superoxide dismutase as far as immunoreactivity and molecular weight are concerned, but with decreased contents of copper and zinc. Its catalytic constant, referring to copper content, was 15 times lower than that obtained for the native enzyme. Moreover, the catalytic power of purified Fraction II was not regained upon incubation with copper. The occurrence of a superoxide dismutase void of metals confirms the hypothesis that this protein plays a dual physiological role: in metal metabolism and in superoxide anion dismutation.

Detoxification of exogenous copper by binding to hemolymph proteins in the shore crab, Carcinus maenas

Copper of exogenous origin is present in crab hemolymph mainly in a protein-bound form. Various approaches were used to assess the toxicity of this protein-bound exogenous metal. Mortality due to copper directly injected into the hemolymph space was apparent at circulating concentrations of non-hemocyanic copper much higher than that (about 150 μM) attained during exposure to a lethal level of waterborne copper (31.5 μM in water). No cytological damage to the gill epithelium was apparent in crabs injected with the metal to attain a hemolymph exogenous copper level of 150 μM, in contrast to those observed at similar hemolymph concentrations during waterborne metal exposure. The cytotoxicity of copper added to an in vitro hepatopancreatic cell preparation was greatly diminished in the presence of hemolymph proteins, probably in relation to a reduced copper uptake by these cells. Thus, binding of copper to hemolymph proteins and particularly hemocyanin plays a significant role, not only for transport and distribution, but also for detoxification of the metal within the organism.

Haemolymph transport and tissue accumulation of exogenous copper in the shore crab, Carcinus maenas

Exogenous (i.e. non-haemocyanic) copper was quantified in the haemolymph of shore crabs Carcinus maenas and the fate of the metal was studied in animals contaminated either by exposure to waterborne copper or by injection into the haemolymph space. Direct contamination from the ambient water resulted in a relatively steady level of haemolymph exogenous copper by 2–3 days. Following a single injection, exogenous copper disappeared rapidly from haemolymph, following an exponential time course (half time 22.4 h). Insignificant amounts of the metal were excreted to the ambient water but dose-dependent tissue uptake could be demonstrated in vivo and by using an in vitro hepatopancreatic cell preparation. Almost all haemolymph exogenous copper was protein-bound and non-filterable. By ultracentrifugation and gel filtration experiments, the only protein binding exogenous copper was shown to be the respiratory pigment haemocyanin. These results emphasize the role of haemolymph and haemocyanin as an efficient copper transport system in the crustaceans.

Detergent Solubilization of Membrane-Bound Methane Monooxygenase Requires Plastoquinol Analogs as Electron Donors

Quinols can provide reducing equivalents for the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membranes. Furthermore, quinols are effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl analog of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) provide the greatest methane monooxygenase activity in whole cells and membrane suspensions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytically active methane monooxygenase. Optimal pMMO activity in the detergent-solubilized fraction is obtained with a ratio of ∼1.7 mg of detergent per milligram of membrane protein, independent of protein concentration. The detergent-solubilized pMMO retains its sensitivity to inhibition by cyanide, acetylene, and EDTA. It is also stimulated by exogenous copper, as in isolated membrane fractions. Reaction of the detergent-solubilized enzyme with [ 14C]acetylene results in labeling of a 26-kDa peptide, analogous to the behavior observed for isolated membrane suspensions. The selectivity of pMMO for duroquinol and decyl-plastoquinol, relative to other structurally similar quinols, suggests that the enzyme obtains reducing equivalents directly from a quinol (probably plastoquinol) in vivo.

An Electron Paramagnetic Resonance Method for Quantification of Copper Isotopes in Proteins: Application to a Mixed-Isotope Derivative of Laccase

We have developed a method of denaturing a copper-containing protein and sequestering the copper in the form of the bis(acetylacetonato)copper(II) complex. Because of the excellent resolution of the hyperfine structure in the resulting EPR spectrum, the method is useful for determining the 65Cu/ 63Cu isotope ratio in proteins such as tree laccase. Here we analyzed a mixed isotope sample prepared by reconstituting 63Cu-enriched type 2-depleted laccase with 65Cu(I) and found that it contained only 25% 65Cu. This proves that reconstitution of the type 2 copper is possible under conditions that permit little or no exchange between resident copper and the external pool. However, exchange does occur under reducing conditions when we expose the holoprotein to a large excess of exogenous copper.

The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa polypeptide exhibiting significant similarity and identity to various bacterial metal transporters. Potential ATX1 homologues were also identified in multicellular eukaryotes, including the plants Arabidopsis thaliana and Oryza sativa and the nematode Caenorhabditis elegans. In yeast cells, ATX1 evidently acts in the transport and/or partitioning of copper, and this role in copper homeostasis appears to be directly relevant to the ATX1 suppression of oxygen toxicity: ATX1 was incapable of compensating for SOD when cells were depleted of exogenous copper. Strains containing a deletion in the chromosomal ATX1 locus were generated. Loss of ATX1 function rendered both mutant and wild-type SOD strains hypersensitive toward paraquat (a generator of superoxide anion) and was also associated with an increased sensitivity toward hydrogen peroxide. Hence, ATX1 protects cells against the toxicity of both superoxide anion and hydrogen peroxide.