The role of zinc transporters in cadmium and manganese transport in mammalian cells

To understand the mechanism of cadmium accumulation, it is important to know the precise mechanisms of transport systems for other metals. Recently, utilization of genomics and metallomics has clarified the involvement of specific metal transporter(s) in cadmium uptake. Studies with metallothionein (MT)-null cadmium-resistant cells have revealed the involvement of the manganese/zinc transport system in cadmium uptake. Genomic studies of strain differences in sensitivity to cadmium-induced testicular hemorrhage revealed that a zinc transporter, Zrt-, Irt-related protein (ZIP) 8 encoded by slc39a8, is responsible for the strain difference. Ectopic expression of ZIP8 in various cells enhanced the uptake of cadmium, manganese, and zinc. ZIP8-transgenic mice showed high expression of ZIP8 in the vasculature of testis and apical membrane of proximal tubules in kidney, and exhibited enhanced cadmium accumulation and toxicity when treated with cadmium. The expression of ZIP8 was found to be down-regulated in MT-null cadmium-resistant cells, in which the uptake rates of both cadmium and manganese were decreased. These data suggest that ZIP8 plays an important role in the uptake of both cadmium and manganese in mammalian cells. The role of ZIP14 in the uptake of cadmium and manganese is also discussed.     

The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific,zinc-regulated zinc transporter in mice

The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.     

Zinc deficiency-inducible OsZIP8 encodes a plasma membrane-localized zinc transporter in rice

Zinc is an essential micronutrient for several physiological and biochemical processes. To investigate its transport in rice, we characterized OsZIP8, a rice ZIP (Zrt, Irt-like Protein) gene that is strongly up-regulated in shoots and roots under Zn deficiency. OsZIP8 could complement the growth defect of Zn-uptake yeast mutant. The OsZIP8-GFP fusion proteins were localized to the plasma membrane, suggesting that OsZIP8 is a plasma membrane zinc transporter in rice. Activation and overexpression of this gene disturbed the zinc distribution in rice plants, resulting in lower levels in shoots and mature seeds, but an increase in the roots. Field-grown transgenic plants were shorter than the WT. Under treatment with excess zinc, transgenics contained less zinc in their shoots but accumulated more in the roots. Altogether, these results demonstrate that OsZIP8 is a zinc transporter that functions in Zn uptake and distribution. Furthermore, zinc homeostasis is important to the proper growth and development of rice.     

A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.     

锌转运蛋白基因研究进展

锌作为一种重要的微量元素参与了植物体内广泛的生理和生化过程,本文详细介绍了涉及Zn^2＋吸收转运的ZIP基因家族（ZRT／IRT相关蛋白）和CDF（Cation　diffusion　facilitator）家族。ZIP家族转运蛋白主要负责将Zn^2＋等二价阳离子跨膜转运进细胞内,以完成细胞内多种生理生化反应。CDF家族转运蛋白主要负责将过量Zn^2＋运出细胞,或者将细胞内过量Zn^2＋进行区室化隔离,降低Zn^2＋对细胞的危害作用。ZIP家族转运蛋白和CDF家族转运蛋白的相互协调使得Zn^2＋在细胞和有机体水平上维持着稳态,进而为细胞内各种生理生化反应的进行供一种保障机制。     

Extreme Population Differences in the Human Zinc Transporter ZIP4 (SLC39A4) Are Explained by Positive Selection in Sub-Saharan Africa

Extreme differences in allele frequency between West Africans and Eurasians were observed for a leucine-to-valine substitution (Leu372Val) in the human intestinal zinc uptake transporter, ZIP4, yet no further evidence was found for a selective sweep around the ZIP4 gene (SLC39A4). By interrogating allele frequencies in more than 100 diverse human populations and resequencing Neanderthal DNA, we confirmed the ancestral state of this locus and found a strong geographical gradient for the derived allele (Val372), with near fixation in West Africa. In extensive coalescent simulations, we show that the extreme differences in allele frequency, yet absence of a classical sweep signature, can be explained by the effect of a local recombination hotspot, together with directional selection favoring the Val372 allele in Sub-Saharan Africans. The possible functional effect of the Leu372Val substitution, together with two pathological mutations at the same codon (Leu372Pro and Leu372Arg) that cause acrodermatitis enteropathica (a disease phenotype characterized by extreme zinc deficiency), was investigated by transient overexpression of human ZIP4 protein in HeLa cells. Both acrodermatitis mutations cause absence of the ZIP4 transporter cell surface expression and nearly absent zinc uptake, while the Val372 variant displayed significantly reduced surface protein expression, reduced basal levels of intracellular zinc, and reduced zinc uptake in comparison with the Leu372 variant. We speculate that reduced zinc uptake by the ZIP4-derived Val372 isoform may act by starving certain pathogens of zinc, and hence may have been advantageous in Sub-Saharan Africa. Moreover, these functional results may indicate differences in zinc homeostasis among modern human populations with possible relevance for disease risk.     

In vivo zinc toxicity phenotypes provide a sensitized background that suggests zinc transport activities for most of the Drosophila Zip and ZnT genes

Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 Drosophila Zip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.     

Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the spondylocheiro dysplastic Ehlers-Danlos syndrome

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.     

The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.     

Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone.

The glandular epithelial cells of the human prostate gland have the unique capability and function of accumulating the highest zinc levels of any soft tissue in the body. Zinc accumulation in the prostate is regulated by prolactin and testosterone; however, little information is available concerning the mechanisms associated with zinc accumulation and its regulation in prostate epithelial cells. In the present studies the uptake and accumulation of zinc were determined in the human malignant prostate cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and PC-3 cells possess the unique capability of accumulating high levels of zinc. Zinc accumulation in both cell types is stimulated by physiological concentrations of prolactin and testosterone. The studies reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under physiological conditions that reflect the total and mobile zinc levels in circulation. Correspondingly, genetic studies demonstrate the expression of a ZIP family zinc uptake transporter in both LNCaP and PC-3 cells. The rapid zinc uptake transport process is stimulated by treatment of cells with physiological levels of prolactin and testosterone, which possibly is the result of the regulation of the ZIP-type zinc transporter gene. These zinc-accumulating characteristics are specific for prostate cells. The studies support the concept that these prostate cells express a unique hormone-responsive, plasma membrane-associated, rapid zinc uptake transporter gene associated with their unique ability to accumulate high zinc levels.     

Structure, function, and regulation of a subfamily of mouse zinc transporter genes

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258-22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474-33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.     

Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis

Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP), is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites

Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal‐ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn²⁺ was the preferred LiZIP3 substrate (K_0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell‐surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short‐lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis.     

Genome Wide Identification,Phylogeny and Expression of Zinc Transporter Genes in Common Carp

Background

Zinc is an essential trace element in organisms, which serves as a cofactor for hundreds of enzymes that are involved in many pivotal biological processes including growth, development, reproduction and immunity. Therefore, the homeostasis of zinc in the cell is fundamental. The zinc transporter gene family is a large gene family that encodes proteins which regulate the movement of zinc across cellular and intracellular membranes. However, studies on teleost zinc transporters are mainly limited to model species.

Methodology/Principal Findings

We identified a set of 37 zinc transporters in common carp genome, including 17 from SLC30 family (ZnT), and 20 from SLC39 family (ZIP). Phylogenetic and syntenic analysis revealed that most of the zinc transporters are highly conserved, though recent gene duplication and gene losses do exist. Through examining the copy number of zinc transporter genes across several vertebrate genomes, thirteen zinc transporters in common carp are found to have undergone the gene duplications, including SLC30A1, SLC30A2, SLC30A5, SLC30A7, SLC30A9, SLC30A10, SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7 and SLC39A9. The expression patterns of all zinc transporters were established in various tissues, including blood, brain, gill, heart, intestine, liver, muscle, skin, spleen and kidney, and showed that most of the zinc transporters were ubiquitously expressed, indicating the critical role of zinc transporters in common carp.

Conclusions

To some extent, examination of gene families with detailed phylogenetic or orthology analysis could verify the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. The gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp zinc transporters provides an important genomic resource for future biochemical, toxicological and physiological studies of zinc in teleost.     

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.