Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.     

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.     

Functional analysis of the Escherichia coli zinc transporter ZitB

The membrane transporter ZitB responsible for Zn(II) efflux in Escherichia coli was studied by site-directed mutagenesis to elucidate the function of individual amino acid residues. Substitutions of several charged or polar residues, H53, H159, D163 and D186, located in predicted transmembrane domains resulted in loss of ZitB function. In contrast, neither the amino-terminal nor the carboxy-terminal regions, both histidine-rich, were required for function.     

Probing metal ion substrate-binding to the E. coli ZitB exporter in native membranes by solid state NMR

Metal ion homeostasis is important for healthy cell function and is regulated by metal ion transporters and chaperones. To explore metal ion binding to membrane transport proteins we have used cadmium-113 as a solid state NMR probe of the Escherichia coli zinc exporter ZitB present in native membrane preparations. Competition experiments with other metal ions indicated that nickel and copper are also able to bind to this protein. Metal ion uptake studies were also performed using ZitB-reconstituted into proteoliposomes for a well established fluorescence assay. The results of both the solid state NMR and the uptake studies demonstrate that ZitB is potentially capable of transporting not only zinc but also cadmium, nickel and copper. The solid state NMR approach therefore offers great potential for defining the substrate spectrum of metal ion transporter proteins in their native membrane environments. Further, it should be useful for functional dissection of transporter mechanisms by facilitating the identification of functional residues by mutational studies.     

ZupT Is a Zn(II) Uptake System in Escherichia coli

Escherichia coli zupT (ygiE), encoding a ZIP family member, mediated zinc uptake. Growth of cells disrupted in both zupT and the znuABC operon was inhibited by EDTA at a much lower concentration than a single mutant or the wild type. Cells expressing ZupT from a plasmid exhibited increased uptake of (65)Zn(2+).     

Zinc uptake and incorporation into proteins in T4D bacteriophage-infected Escherichia coli.

The metabolism of Zn2+ in Escherichia coli infected with T4D bacteriophage and various T4D mutants has been examined. E. coli B infected with T4D, and all T4D mutants except T4D 12-, took up zinc ions at a rate identical to that of uninfected cells. E. coli B infected with T4D 12- had a markedly decreased rate of zinc uptake. The incorporation of zinc into proteins of infected cells has also been studied. T4D phage infection was found to shut off the synthesis of all bacterial host zinc metalloproteins while allowing the formation of viral-induced zinc proteins. The amount of zinc incorporated into viral proteins was affected by the absence of various T4D gene products. Cells infected with T4D 12-, and to a much less extent those infected with T4D 29-, incorporated the least amount of zinc into proteins, while cells infected with T4D 11- and T4D 51- incorporated increased amounts of zinc into the zinc metalloproteins. In cells infected with T4D 11- and 51- most of the zinc protein was found to be the product of gene 12. The marked effect of infection of E. coli with T4D 12- on both zinc uptake and zinc incorporation into protein supports the conclusion that T4D gene 12 protein is a zinc metalloprotein. Additionally, these observations have indicated that this metalloprotein interacts with host cell membrane.     

Heterologous production and functional and thermodynamic characterization of cation diffusion facilitator (CDF) transporters of mesophilic and hyperthermophilic origin

Abstract The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 μm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+ transport across membranes.     

The effect of cellular age on zinc levels in untreated and zinc-treated human diploid fibroblasts

Cellular aging is accompanied by increased cellular permeability to zinc(II). The intrinsic zinc content of human diploid fibroblast cells increases with cell age, so that it quadruples from early to late passage, on a Zn(II) per cell or per cell volume basis, but it remains constant on a Zn(II) per protein basis. When the cells are challenged with toxic concentrations (0.2 mM) of Zn(II), both the rate of zinc incorporation into the cells and the amount of zinc incorporated at equilibrium increases considerably with age (unless measured as zinc per protein). In terms of growth inhibition, Zn(II) is more toxic to the cell than Cu(II), Mn(II), or Mg(II).     

Expression of ZntA, a zinc-transporting P1-type ATPase, is specifically regulated by zinc and cadmium

All cells have developed various mechanisms to regulate precisely the availability of important micronutrients such as zinc and copper; in many cells, this regulation is mediated by P1-type ATPases. Most of the P1-ATPases have been described very recently, and little is known about their molecular mechanism and regulation. Here, we demonstrate that the expression of ZntA, a Zn,Cd-transporting P-type ATPase of Escherichia coli, is specifically regulated by the transported cations, cadmium and zinc. Nickel, cobalt, and copper did not induce the expression of ZntA, even when present at concentrations as high as 0.6-1 mM. The effect of zinc and cadmium on the ZntA expression is concentration dependent, the apparent Km for Cd (19 microM) being markedly lower than that for Zn (100 microM). This metal selectivity is opposite to the known metal selectivity of transport by ZntA. Thus, we speculate that, to maintain zinc concentrations in the cell in the presence of cadmium, ZntA probably interacts with other proteins that modulate the ZntA selectivity towards transported cations.     

Metallothionein can function as a chaperone for zinc uptake transport into prostate and liver mitochondria

In mammalian cells the cytosolic concentration of free Zn(2+) ions is extremely low (nM-fM range) and unlikely to provide an adequate pool for the uptake and accumulation of zinc in mitochondria. We previously identified a mitochondrial uptake transport process that effectively transports zinc directly from low molecular weight zinc ligands independent of and in the absence of available free Zn(2+) ions. Since metallothionein (MT) is an important ligand form of cellular zinc, we determined if Zn(7)-MT was an effective chaperone and donor for delivery and uptake of zinc by prostate and liver mitochondria. The results reveal that both intact mitochondria and mitoplasts effectively accumulated zinc from Zn(7)-MT. The study confirms and extends our previous report that the putative zinc transporter is associated with the inner mitochondrial membrane and involves a direct exchange of zinc from the ligand to the transporter. The ventral prostate cells contain no detectable MT; so that ligands (such as citrate, aspartate) other than MT are zinc donors for mitochondrial zinc accumulation. However, in liver and perhaps other cells, Zn(7)-MT is probably important in the cytosolic trafficking of zinc to the mitochondria for the uptake of zinc into the mitochondrial matrix by the putative zinc uptake transporter.     

Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions

We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E. coli cells using a 4-step 16-fold purification procedure, a yield of 60%. We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity. The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo. Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used. The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate). The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.     

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC)

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.     

Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles. Each amino acid was altered in both E. coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined. Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions. H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+). The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC. The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion. Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.     

A Zn(II)-translocating P-type ATPase from Proteus mirabilis.

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.     

The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli.

The function of the CzcABC protein complex, which mediates resistance to Co2+, Zn2+, and Cd2+ in Alcaligenes eutrophus by cation efflux, was investigated by using everted membrane vesicles of Escherichia coli and an acridine orange fluorescence quenching assay. Since metal cation uptake could not be measured with inside-out membrane vesicles prepared from A. eutrophus and since available E. coli strains did not express the Czc-mediated resistance to cobalt, zinc, and cadmium salts, mutants of E. coli which exhibited a Czc-dependent increase in heavy metal resistance were isolated. E. coli mutant strain EC351 constitutively accumulated Co2+, Zn2+, and Cd2+. In the presence of Czc, net uptake of these heavy metal cations was reduced to the wild-type level. Inside-out vesicles prepared from E. coli EC351 cells displayed a Czc-dependent uptake of Co2+, Zn2+, and Cd2+ and a cation-triggered acridine orange fluorescence increase. The czc-encoded protein complex CzcABC was shown to be a zinc-proton antiporter.     

The effects of transformation and ZnT-1 silencing on zinc homeostasis in cultured cells

We have previously demonstrated that reducing the availability of zinc with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) promotes efflux of (65)Zn from rat primary hepatocytes and pituitary cells, but increases retention of label in rat hepatoma (H4IIE) and anterior pituitary tumor (GH3) cell lines. To further understand this differential response between primary cells and the corresponding cancer cell lines, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Rat primary hepatocytes were electroporated with the SV40 large T-antigen-coding plasmid pSV3-neo and selected for neomycin resistance. This resulted in cell division of the normally quiescent hepatocytes. When these cells were prelabeled with (65)Zn, DTPA decreased efflux of (65)Zn, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of dividing cells and be mediated by alterations in the activity of zinc transporter ZnT-1, which is responsible for zinc efflux. To further understand the mechanism of DTPA-induced zinc retention, we down-regulated the expression of ZnT-1 in rat hepatoma cells using vector-based short hairpin RNA interference. Expression of ZnT-1 protein was reduced to approximately 50%. Down-regulation of ZnT-1 resulted in greater retention of (65)Zn in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT-1 is required for the decreased efflux in response to DTPA. We conclude that ZnT-1 expression is crucial for maintaining zinc homeostasis, in particular, for the enhanced retention of zinc in transformed cells when subjected to zinc deprivation.     

Exploring the microbial metalloproteome using MIRAGE

The microbial metalloproteome has been largely unexplored. Using the metalloproteomics approach MIRAGE (Metal Isotope native RadioAutography in Gel Electrophoresis) we have been able to explore the soluble Fe and Zn metalloproteome of Escherichia coli. The protein identification by MS/MS typically resulted in several overlapping proteins for each metal containing spot. Using the E. coli genome annotation the proteins relevant to the iron and zinc proteome were selected. Superoxide dismutase (SodB) was found to be the major iron protein after cultivation with a normal iron concentration of 6 μM. Upon an elevated iron concentration of 40 μM, ferritin (FtnA) became dominant. Under both conditions 90% of the iron was associated with just three different proteins: superoxide dismutase (SodB), ferritin (FtnA) and bacterioferritin (Bfr). The uncharacterized proteins YgfK and XdhD were found to be significant iron containing proteins under elevated iron conditions. The zinc proteome of E. coli experiencing zinc stress was dominated by ZraP, a putative zinc storage protein.     

Kinetic identification of a mitochondrial zinc uptake transport process in prostate cells

Prostate cells accumulate high cellular and mitochondrial concentrations of zinc, generally 3-10-fold higher than other mammalian cells. However, the mechanism of mitochondrial import and accumulation of zinc from cytosolic sources of zinc has not been established for these cells or for any mammalian cells. Since the cytosolic concentration of free Zn(2+) ions is negligible (estimates vary from 10(-9) to 10(-15) M), we postulated that loosely bound zinc-ligand complexes (Zn-Ligands) serve as zinc donor sources for mitochondrial import. Zinc chelated with citrate (Zn-Cit) is a major form of zinc in prostate and represents an important potential cytosolic source of transportable zinc into mitochondria. The mitochondrial uptake transport of zinc was studied with isolated mitochondrial preparations obtained from rat ventral prostate. The uptake rates of zinc from Zn-Ligands (citrate, aspartate, histidine, cysteine) and from ZnCl(2) (free Zn(2+)) were essentially the same. No zinc uptake occurred from either Zn-EDTA, or Zn-EGTA. Zinc uptake exhibited Michaelis-Menten kinetics and characteristics of a functional energy-independent facilitative transporter associated with the mitochondrial inner membrane. The uptake and accumulation of zinc from various Zn-Ligand preparations with logK(f) (formation constant) values less than 11 was the same as for ZnCl(2;) and was dependent upon the total zinc concentration independent of the free Zn(2+) ion concentration. Zn-Ligands with logK(f) values greater than 11 were not zinc donors. Therefore the putative zinc transporter exhibits an effective logK(f) of approximately 11 and involves a direct exchange of zinc from Zn-Ligand to transporter. The uptake of zinc by liver mitochondria exhibited transport kinetics similar to prostate mitochondria. The results demonstrate the existence of a mitochondrial zinc uptake transporter that exists for the import of zinc from cytosolic Zn-Ligands. This provides the mechanism for mitochondrial zinc accumulation from the cytosol which contains a negligible concentration of free Zn(2+). The uniquely high accumulation of mitochondrial zinc in prostate cells appears to be due to their high cytosolic level of zinc-transportable ligands, particularly Zn-Cit.