Zinc Transporter Proteins

Zinc, which is involved in the structure of all enzyme classes, is a micro nutrient element and necessary for growth and development. The ability of zinc to function without causing toxic effects is depends on the protection of its homeostasis. Zinc transporter proteins are responsible for keeping zinc at certain concentrations. Based on their predicted membrane topology, Zn transporters are divided into two major families, SLC39s/ZIPs and SLC30s/ZnTs, which transport Zn in opposite directions through cellular and intracellular membranes. ZIPs increases the zinc concentration in the cytosol. For this, the ZIPs carries the zinc from extracellular and intracellular compartments to the cytosol. ZnTs, reduces the concentration of zinc in the cytosol. For this, ZnTs carries the zinc from the cytosol to extracellular and intracellular compartments. After being transported to the cell, 50% of the zinc is found in the cytoplasm, 30–40% in the nucleus, and 10% in the plasma and organelle membranes. The expression of many zinc transporter proteins in the cell is depending on the concentration of zinc and the physiological problems. The aim of this study is to give information about association of zinc transporter proteins with physiological events and health problems.     

Zinc signalling and subcellular distribution: emerging targets in type 2 diabetes

A finely tuned subcellular distribution of zinc (Zn), through the coordinated action of Zn transporters (ZnTs) and metallothioneins (MTs), is crucial for optimal cell function. Dysfunctions of these proteins might act as key causative or promoting factors in several chronic pathologies. Evidence of their involvement in the pathogenesis of type 2 diabetes (DM2) is emerging. The association of single nucleotide polymorphisms in genes encoding ZnT-8 and MT with DM2 has drawn attention to the relevance of Zn homeostasis for insulin secretory capacity and responsiveness. Here, we propose that potential mechanisms leading to altered subcellular Zn distribution rather than deficiency might be important in DM2. Increasing knowledge of the mechanisms of Zn homeostasis and signalling should promote the development of targeted interventions with the potential to reduce the burden of disease.     

Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T(3) and T(4) levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn(++) in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthyroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell-specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis

Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals.     

A novel member of a zinc transporter family is defective in acrodermatitis enteropathica

The rare inherited condition acrodermatitis enteropathica (AE) results from a defect in the absorption of dietary zinc. Recently, we used homozygosity mapping in consanguineous Middle Eastern kindreds to localize the AE gene to an approximately 3.5-cM region on 8q24. In this article, we identify a gene, SLC39A4, located in the candidate region and, in patients with AE, document mutations that likely lead to the disease. The gene encodes a histidine-rich protein, which we refer to as "hZIP4," which is a member of a large family of transmembrane proteins, some of which are known to serve as zinc-uptake proteins. We show that Slc39A4 is abundantly expressed in mouse enterocytes and that the protein resides in the apical membrane of these cells. These findings suggest that the hZIP4 transporter is responsible for intestinal absorption of zinc.     

Zinc transporter Slc30a1 regulates melanocyte development by interacting with mt2 in zebrafish

The essential trace element zinc is involved in multiple biological processes including development and metabolism, while its role in melanocyte formation is still unclear. Slc30a1a and Slc30a1b are zinc exporters in zebrafish. Here, we found that melanocytes were increased in slc30a1a and slc30a1b double mutant zebrafish. SMART-seq data revealed that genes involved in the melanoma pathway and the gene mt2, which encodes zinc-binding protein, were significantly upregulated in the mutants. In addition, the expression of mt2 was specifically increased in mutant melanocytes, as detected by in situ hybridization, suggesting an essential role of this gene in the tissue. Mechanistically, we demonstrated that elevated zinc levels resulting from Slc30a1 deficiency promoted melanocyte proliferation and that mt2 played a protective role in the process of Slc30a1/zinc-mediated melanocyte hyperplasia. This study uncovered the critical function of Slc30a1-mediated zinc homeostasis in melanocyte development and suggests that accumulated zinc in melanocytes would be a risk for inducing melanoma and that mt2 is a potential target for controlling diseases related to abnormal melanocyte development.     

Identification of autophagy genes participating in zinc-induced necrotic cell death in Saccharomyces cerevisiae

Eukaryotes use a common set of genes to perform two mechanistically similar autophagic processes. Bulk autophagy harvests proteins nonselectively and reuses their constitutents when nutrients are scarce. In contrast, different forms of selective autophagy target protein aggregates or damaged organelles that threaten to interfere with growth. Yeast uses one form of selective autophagy, called cytoplasm-to-vacuole targeting (Cvt), to engulf two vacuolar enzymes in Cvt vesicles ("CVT-somes") within which they are transported to vacuoles for maturation. While both are dispensable normally, bulk and selective autophagy help sustain life under stressful conditions. Consistent with this view, knocking out several genes participating in Cvt and specialized autophagic pathways heightened the sensitivity of Saccharomyces cerevisiae to inhibitory levels of Zn(2+). The loss of other autophagic genes, and genes responsible for apoptotic cell death, had no such effect. Unexpectedly, the loss of members of a third set of autophagy genes heightened cellular resistance to zinc as if they encoded proteins that actively contributed to zinc-induced cell death. Further studies showed that both sensitive and resistant strains accumulated similar amounts of H2O2 during zinc treatments, but that more sensitive strains showed signs of necrosis sooner. Although zinc lethality depended on autophagic proteins, studies with several reporter genes failed to reveal increased autophagic activity. In fact, microscopy analysis indicated that Zn(2+) partially inhibited fusion of Cvt vesicles with vacuoles. Further studies into how the loss of autophagic processes suppressed necrosis in yeast might reveal whether a similar process could occur in plants and animals.     

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn²⁺ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn²⁺ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp⁵⁹⁹ and His⁴⁵¹, with alanine was sufficient to block Zn²⁺ transport. These findings indicate, for the first time, that Zn²⁺ transport mediated by a mammalian ZnT is catalyzed by H⁺/Zn²⁺ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.     

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.     

A potential role for zinc alterations in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests that the etiology of AD may involve disruptions of zinc (Zn) homeostasis. This review discusses current evidence supporting a potential role of Zn and zinc transporters (ZnTs) in processing of the amyloid beta protein precursor (APP) and amyloid beta (Aβ) peptide generation and aggregation.     

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.     

The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.     

Slc39a1 to 3 (subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency

Subfamily II of the solute carrier (Slc)39a family contains three highly conserved members (ZIPs 1-3) that share a 12-amino acid signature sequence present in the putative fourth transmembrane domain and function as zinc transporters in transfected cells. The physiological significance of this genetic redundancy is unknown. Here we report that the complete elimination of all three of these Zip genes, by targeted mutagenesis and crossbreeding mice, causes no overt phenotypic effect. When mice were fed a zinc-adequate diet, several indicators of zinc status were indistinguishable between wild-type and triple-knockout mice, including embryonic morphogenesis and growth, alkaline phosphatase activity in the embryo, ZIP4 protein in the visceral yolk sac, and initial rates (30 min) of accumulation/retention of (67)Zn in liver and pancreas. When mice were fed a zinc-deficient diet, embryonic membrane-bound alkaline phosphatase activity was reduced to a much greater extent, and 80% of the embryos of the triple-knockout mice developed abnormally compared with 12% of the embryos of wild-type mice. During zinc deficiency, the accumulation/retention (3 h) of (67)Zn in the liver and pancreas of weanlings was significantly impaired in the triple-knockout mice compared with wild-type mice. Thus none of these three mammalian Zip genes apparently plays a critical role in zinc homeostasis when zinc is replete, but they play important, noncompensatory roles when this metal is deficient.     

Serum protein and zinc levels in patients with thoracic empyema

The element Zn is the metal component or activator of many important enzymes. The tissue concentrations and activities of Zn metalloenzymes direct the rate of protein and nucleic acid syntheses, thereby influencing tissue growth and reperative processes. Most of the serum Zn is normally bound to circulating proteins. Low serum Zn concentrations might result from depletion of Zn-binding proteins. Serum protein and Zn concentrations have been reported to be depressed in patients with acute and chronic diseases. We compare the serum protein and Zn values of patients with thoracic empyema (n=20) with those of a control group (n=20). The values obtained in the empyema group were significantly lower than those in the control group before the study. Test group administered 220 mg zinc sulfate (ZnSO₄. 7H₂O) over 20 d and there was a significant increase in the values for serum protein and Zn after the oral administration of the zinc sulfate.     

Effects of dietary zinc on gene expression of antioxidant enzymes and heat shock proteins in hepatopancreas of abalone Haliotis discus hannai

The expression patterns of different genes encoding antioxidant enzymes and heat shock proteins were investigated, in present study, by real-time quantitative PCR in the hepatopancreas of abalone Haliotis discus hannai fed with different levels of dietary zinc (6.69, 33.8, 710.6 and 3462.5 mg/kg) for 20 weeks. The antioxidant enzymes include Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), mu-glutathione-s-transferase (mu-GST) and thioredoxin peroxidase (TPx). The results showed that the mRNA expression of these antioxidant enzymes increased and reached the maximum at the dietary zinc level of 33.8 mg/kg, and then dropped progressively. Expression levels of the heat shock proteins (HSP26, HSP70 and HSP90) firstly increased at 33.8 mg/kg dietary Zn level, and reached to the maximum at 710.6 mg/kg, then dropped at 3462.5 mg/kg (p<0.05). Excessive dietary Zn (710.6 and 3462.5 mg/kg) significantly increases the Zn content and significantly decreases the total antioxidant capacity (T-AOC) in hepatopancreas (p<0.05). These findings showed that dietary Zn (33.8 mg/kg) could highly trigger the expression levels of antioxidant enzymes and heat shock proteins, but excessive dietary Zn (710.6 and 3462.5 mg/kg) induces a high oxidative stress in abalone.     

The oxidative stress of zinc deficiency

Zinc is an essential catalytic and structural cofactor for many enzymes and other proteins. While Zn2+ is not redox active under physiological conditions, it has been known for many years that zinc deficiency causes increased oxidative stress and, consequently, increased oxidative damage to DNA, proteins, and lipids. These results have indicated that zinc plays an indirect antioxidant role and that dietary inadequacy may contribute to human diseases such as cancer. Recent studies are helping to identify the primary sources of oxidative stress in low zinc. In addition, through studies of the model eukaryotic cell, Saccharomyces cerevisiae, we are now beginning to understand the strategies cells use to limit this stress and reduce its damage.     

Heterodimerization,Altered Subcellular Localization,and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.