Cell surface expression of the prion protein in yeast does not alter copper utilization phenotypes

Prion diseases are fatal neurodegenerative disorders that result from conversion of a normal, cell surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is thought to be infectious. Although a great deal is known about the role of PrP(Sc) in the disease process, the physiological function of PrP(C) has remained enigmatic. In this report, we have used the yeast Saccharomyces cerevisiae to test one hypothesized function of PrP(C), as a receptor for the uptake or efflux of copper ions. We first modified the PrP signal peptide by replacing its hydrophobic core with the signal sequence from the yeast protein dipeptidyl aminopeptidase B, so that the resulting protein was targeted cotranslationally to the secretory pathway when synthesized in yeast. PrP molecules with the modified signal peptide were efficiently glycosylated, glycolipid-anchored, and localized to the plasma membrane. We then tested whether PrP expression altered the growth deficiency phenotypes of yeast strains harboring deletions in genes that encode key components of copper utilization pathways, including transporters, chaperones, pumps, reductases, and cuproenzymes. We found that PrP did not rescue any of these mutant phenotypes, arguing against a direct role for the protein in copper utilization. Our results provide further clarification of the physiological function of PrP(C), and lay the groundwork for using PrP-expressing yeast to study other aspects of prion biology.     

Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Amino acid facilitates absorption of copper in the Caco-2 cell culture model

Aim

Copper deficiency could cause fatal hematological and neurological disorders or other diseases. Amino acids are involved in the absorption of copper ions. The purpose of this study is to evaluate the absorption of copper in amino acid complex forms and determine its mechanism in the Caco-2 cell culture model.

Main methods

The human colonic adenocarcinoma cell line Caco-2 culture model was used to determine the permeability of copper ions in inorganic form (CuSO₄) and the amino acid complex forms. Lysine and methionine, as well as carboplatin were used to determine the possible involvement of amino acid transporters or copper transporter 1 (CTR1).

Key findings

The results showed that all of the amino acid complex forms facilitated copper absorption. The apparent permeabilities of copper ions in these complex forms were at least 7.6 fold higher than those in the CuSO₄ form. The permeability rank order of copper in various amino acid complex forms was Cu-glutamate < Cu-lysine = Cu-aspartic acid = Cu methionine < Cu-arginine < Cu-(lysine/glutamate). Mechanistic studies revealed that the enhanced absorption of copper in copper amino acid complexes could be the result of enhanced uptake (as in Cu-methionine complex) or enhanced basolateral efflux (as in Cu-lysine complex). Copper transporter 1 (or CTR1) inhibitor carboplatin did not affect the absorption of copper in Cu-methionine complex, suggesting that the dominant pathway for copper amino acid complexes is not CTR1.

Significance

Enhanced absorption of copper ions in amino acid complex appears to be mediated by amino acid transporters.     

Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein.

The oxidative modification of low-density lipoprotein (LDL) is suggested to play an important role in the pathogenesis of atherosclerosis. The present study examined the role of the formation of LDL-copper (Cu) complex in the peroxidation of LDL. The amount of copper bound to LDL increased during incubation performed with increasing concentrations of CuSO4. More than 80% of the copper bound to the LDL particle was observed in the protein phase of LDL, suggesting that most of the copper ions formed complexes with the ligand-binding sites of apoprotein. The addition of histidine (1 mM), known to form a high affinity complex with copper, and EDTA (1 mM), a metal chelator, during the incubation of LDL with CuSO4 prevented the formation of both thiobarbituric acid-reactive substances (TBARS) and LDL-Cu complexes. EDTA inhibited the copper-catalyzed ascorbate oxidation whereas histidine had no effect, suggesting that the copper within the complex with histidine is available to catalyze the reaction, in contrast to EDTA. These observations indicate that the preventive effect of histidine on the copper-catalyzed peroxidation of LDL is not simply mediated by chelating free copper ions in aqueous phase. Evidence that copper bound to LDL particle still has a redox potential was provided by the observed increase in TBARS content during incubation of LDL-Cu complexes in the absence of free copper ions. The addition of either histidine or EDTA to LDL-Cu complexes inhibited the formation of TBARS by removing copper ions from the LDL forming the corresponding complexes. However, there still remained small amounts of copper in the LDL particles following the treatment of LDL-Cu complexes with histidine or EDTA. The copper ions remaining in the LDL particle lacked the ability to catalyze LDL peroxidation, suggesting that there may be two types of copper binding sites in LDL: tight-binding sites, from which the copper ions are not removed by chelation, and weak-binding sites, from which copper ions are easily removed by chelators. The formation of TBARS in the LDL preparation during incubation with CuSO4 was comparable to the incubation with FeSO4. In contrast, the formation of TBARS in the LDL-lipid micelles by CuSO4 was nearly eliminated even in the presence of ascorbate to promote metal-catalyzed lipid peroxidation, although a marked increase in TBARS content was observed in the LDL-lipid micelles with FeSO4, and with FeCl3 in the presence of ascorbate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure–function correlations in tyrosinases

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure–function correlations in tyrosinases along with comparison to other type-3 copper proteins.     

Menkes copper-translocating P-type ATPase (ATP7A): biochemical and cell biology properties,and role in Menkes disease

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.     

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

The stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-β(1-40) peptide (Aβ1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pH-dependent metal binding to Aβ1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Aβ1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of β-amyloid peptide. Some complexes of Aβ1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Aβ oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Aβ molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Aβ peptide to bind certain heavy metal ions. As a consequence, copper–silver interaction with the binding process to Aβ1-40 became highly complex. A competition between silver and nickel ions for Aβ1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion–peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion–Aβ complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Prion,amyloid beta-derived Cu(II) ions,or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate

Copper are generally bound to proteins, e.g. the prion and the amyloid beta proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid beta peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.     

Monocarboxylate Transporters are not Responsible for Cr3+ Transport from Endosomes

Cr(3+), similar to Fe(3+), is transported into cells primarily via endocytosis as the metal-transferrin complex. As Cr(3+) ions are not readily reduced under biological conditions, the ion cannot be transported from endosomes by the same mechanism as iron that utilized divalent metal ion transporters. Cr(3+) has been hypothesized to potentially be transported as small ligand complexes with a free carboxylate functionality by monocarboxylate transporters (MCT), in a similar fashion to that proposed for Al(3+). Consequently, mouse C2C12 muscle cells were utilized to determine if Cr(3+) is potentially transported by MCT by examining the effects of MCT inhibitors on Cr and Fe transport and subcellular distribution when the metals are added as their transferrin complexes. The results suggest that Cr is not primarily transported by MCT from the endosomes to the cytosol, and that another mechanism for this transport needs to be identified.     

Distorted copper homeostasis with decreased sensitivity to cisplatin upon chaperone <Emphasis Type="Italic">Atox1</Emphasis> deletion in <Emphasis Type="Italic">Drosophila</Emphasis>

Copper is an integral part of a number of proteins and thus an essential trace metal. However, free copper ions can be highly toxic and every organism has to carefully control its bioavailability. Eukaryotes contain three copper chaperones; Atx1p/Atox1 which delivers copper to ATP7 transporters located in the trans-Golgi network, Cox17 which provides copper to the mitochondrial cytochrome c oxidase, and CCS which is a copper chaperone for superoxide dismutase 1. Here we describe the knockout phenotype of the Drosophila homolog of mammalian Atox1 (ATX1 in yeast). Atox1−/− flies develop normally, though at reduced numbers, and the eclosing flies are fertile. However, the mutants are unable to develop on low-copper food. Furthermore, the intestinal copper importer Ctr1B, which is regulated by copper demand, fails to be induced upon copper starvation in Atox1−/− larvae. At the same time, intestinal metallothionein is upregulated. This phenotype, which resembles the one of the ATP7 mutant, is best explained by intestinal copper accumulation, combined with insufficient delivery to the rest of the body. In addition, compared to controls, Drosophila Atox1 mutants are relatively insensitive to the anticancer drug cisplatin, a compound which is also imported via Ctr1 copper transporters and was recently found to bind mammalian Atox1.     

Transporters of ligands for essential metal ions in plants

Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.     

Brain copper content and cuproenzyme activity do not vary with prion protein expression level

Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.     

The structural basis of secondary active transport mechanisms

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

The family of SMF metal ion transporters in yeast cells

Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis. The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles. In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes. Each null mutation exhibited sensitivity to metal ion chelators at different concentrations. The combination of null mutants DeltaSMF1 + DeltaSMF2 and the triple null mutant Delta3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively. Addition of 5 microm copper or 25 microm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent. The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein. Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Delta3SMF. Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes. The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3 and FET4 were inactivated. Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium. We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells.     

Functional differentiation in the mammalian metallothionein gene family: metal binding features of mouse MT4 and comparison with its paralog MT1

This paper reports on the characterization of the metal binding abilities of mammalian MT4 and their comparison with those of the well known MT1. Heterologous Escherichia coli expression in cultures supplemented with zinc, cadmium, or copper was achieved for MT4 and for its separate alphaMT4 and betaMT4 domains as well as for MT1 and its alphaMT1 domain in cadmium-enriched medium. The in vivo conformed metal complexes and the in vitro substituted zinc/cadmium and zinc/copper MT4 aggregates were characterized. Biosynthesis of MT4 and betaMT4 in Cd(II)-supplemented medium revealed that these peptides failed to form the same homometallic species as MT1, thus appearing less effective for cadmium coordination. Conversely, the entire MT4 and both of its domains showed better Cu(I) binding properties than MT1, affording Cu(10)-MT4, Cu(5)-alphaMT4 and Cu(7)-betaMT4, stoichiometries that make the domain dependence toward Cu(I) clear. Overall results allow consideration of MT4 as a novel copper-thionein, made up of two copper-thionein domains, the first of this class reported in mammals, and by extension in vertebrates. Furthermore, the in silico protein sequence analyses corroborated the copper-thionein nature of the MT4 peptides. As a consequence, there is the suggestion of a possible physiological role played by MT4 related with copper requirements in epithelial differentiating tissues, where MT4 is expressed.     

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.     

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe³⁺ ions.Erythrocytes were exposed to various concentrations of CuSO₄, Fe(NO₃)₃, and K₃Fe(CN)₆ for up to 5 h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe²⁺ to Cu²⁺. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO₃)₃. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO₄ increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 μM. This effect was decreased by potassium ferricyanide and Fe(NO₃)₃ only after 1 and 3 h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 μM CuSO₄ as well as 20 μM Fe(NO₃)₃ and 10 mM K₃Fe(CN)₆ induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K₃Fe(CN)₆. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO₃)₃.     

Quaternary structure and functional unit of energy coupling factor (ECF)-type transporters

ATP-binding cassette (ABC) transporters mediate transport of diverse substrates across membranes. We have determined the quaternary structure and functional unit of the recently discovered ECF-type (energy coupling factor) of ABC transporters, which is widespread among prokaryotes. ECF transporters are protein complexes consisting of a conserved energizing module (two peripheral ATPases and the integral membrane protein EcfT) and a non-conserved integral membrane protein responsible for substrate specificity (S-component). S-components for different substrates are often unrelated in amino acid sequence but may associate with the same energizing module. Here, the energizing module from Lactococcus lactis was shown to form stable complexes with each of the eight predicted S-components found in the organism. The quaternary structures of three of these complexes were determined by light scattering. EcfT, the two ATPases (EcfA and EcfA'), and the S-components were found to be present in a 1:1:1:1 ratio. The complexes were reconstituted in proteoliposomes and shown to mediate ATP-dependent transport. ECF-type transporters are the smallest known ABC transporters.