ZIP8 Is an Iron and Zinc Transporter Whose Cell-surface Expression Is Up-regulated by Cellular Iron Loading

ZIP8 (SLC39A8) belongs to the ZIP family of metal-ion transporters. Among the ZIP proteins, ZIP8 is most closely related to ZIP14, which can transport iron, zinc, manganese, and cadmium. Here we investigated the iron transport ability of ZIP8, its subcellular localization, pH dependence, and regulation by iron. Transfection of HEK 293T cells with ZIP8 cDNA enhanced the uptake of ⁵⁹Fe and ⁶⁵Zn by 200 and 40%, respectively, compared with controls. Excess iron inhibited the uptake of zinc and vice versa. In RNA-injected Xenopus oocytes, ZIP8-mediated ⁵⁵Fe²⁺ transport was saturable (K_0.5 of ∼0.7 μm) and inhibited by zinc. ZIP8 also mediated the uptake of ¹⁰⁹Cd²⁺, ⁵⁷Co²⁺, ⁶⁵Zn²⁺ > ⁵⁴Mn²⁺, but not ⁶⁴Cu (I or II). By using immunofluorescence analysis, we found that ZIP8 expressed in HEK 293T cells localized to the plasma membrane and partially in early endosomes. Iron loading increased total and cell-surface levels of ZIP8 in H4IIE rat hepatoma cells. We also determined by using site-directed mutagenesis that asparagine residues 40, 88, and 96 of rat ZIP8 are glycosylated and that N-glycosylation is not required for iron or zinc transport. Analysis of 20 different human tissues revealed abundant ZIP8 expression in lung and placenta and showed that its expression profile differs markedly from ZIP14, suggesting nonredundant functions. Suppression of endogenous ZIP8 expression in BeWo cells, a placental cell line, reduced iron uptake by ∼40%, suggesting that ZIP8 participates in placental iron transport. Collectively, these data identify ZIP8 as an iron transport protein that may function in iron metabolism.     

Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells

Chronic exposure to cadmium causes preferential accumulation of cadmium in the kidney, leading to nephrotoxicity. In the process of renal cadmium accumulation, the cadmium bound to a low-molecular-weight metal-binding protein, metallothionein, has been considered to play an important role in reabsorption by epithelial cells of proximal tubules in the kidney. However, the role and mechanism of the transport of Cd(2+) ions in proximal tubule cells remain unclear. Zinc transporters such as Zrt, Irt-related protein 8 (ZIP8) and ZIP14, and divalent metal transporter 1 (DMT1) have been reported to have affinities for Cd(2+) and Mn(2+). To examine the roles of these metal transporters in the absorption of luminal Cd(2+) and Mn(2+) into proximal tubule cells, we utilized a cell culture system, in which apical and basolateral transport of metals can be separately examined. The uptake of Cd(2+) and Mn(2+) from the apical side of proximal tubule cells was inhibited by simultaneous addition of Mn(2+) and Cd(2+), respectively. The knockdown of ZIP8, ZIP14 or DMT1 by siRNA transfection significantly reduced the uptake of Cd(2+) and Mn(2+) from the apical membrane. The excretion of Cd(2+) and Mn(2+) was detected predominantly in the apical side of the proximal tubule cells. In situ hybridization of these transporters revealed that ZIP8 and ZIP14 are highly expressed in the proximal tubules of the outer stripe of the outer medulla. These results suggest that ZIP8 and ZIP14 expressed in the S3 segment of proximal tubules play significant roles in the absorption of Cd(2+) and Mn(2+) in the kidney.     

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.     

Effect of dietary iron deficiency and overload on the expression of ZIP metal-ion transporters in rat liver

The mammalian ZIP (Zrt-, Irt-like Protein) family of transmembrane transport proteins consists of 14 members that share considerable homology. ZIP proteins have been shown to mediate the cellular uptake of the essential trace elements zinc, iron, and manganese. The aim of the present study was to determine the effect of dietary iron deficiency and overload on the expression of all 14 ZIP transporters in the liver, the main site of iron storage. Weanling male rats (n = 6/group) were fed iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets in two independent feeding studies. In study 1, diets were based on the TestDiet 5755 formulation and contained iron at 9 ppm (FeD), 215 ppm (FeA), and 27,974 ppm (3% FeO). In study 2, diets were based on the AIN-93G formulation and contained iron at 9 ppm Fe (FeD), 50 ppm Fe (FeA), or 18916 ppm (2% FeO). After 3 weeks, the FeD diets depleted liver non-heme iron stores and induced anemia, whereas FeO diets resulted in hepatic iron overload. Quantitative RT-PCR revealed that ZIP5 mRNA levels were 3- and 8-fold higher in 2% FeO and 3% FeO livers, respectively, compared with FeA controls. In both studies, a consistent downregulation of ZIP6, ZIP7, and ZIP10 was also observed in FeO liver relative to FeA controls. Studies in H4IIE hepatoma cells further documented that iron loading affects the expression of these ZIP transporters. Overall, our data suggest that ZIP5, ZIP6, ZIP7, and ZIP10 are regulated by iron, indicating that they may play a role in hepatic iron/metal homeostasis during iron deficiency and overload.     

The ZIP family of metal transporters

Members of the ZIP gene family, a novel metal transporter family first identified in plants, are capable of transporting a variety of cations, including cadmium, iron, manganese and zinc. Information on where in the plant each of the ZIP transporters functions and how each is controlled in response to nutrient availability may allow the manipulation of plant mineral status with an eye to (1) creating food crops with enhanced mineral content, and (2) developing crops that bioaccumulate or exclude toxic metals.     

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism.     

The prion-ZIP connection: From cousins to partners in iron uptake

ABSTRACT

Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrP^C), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) isoform. Biochemical evidence indicates that PrP^C facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrP^C and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrP^C with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrP^C and ZIP14 suggest that PrP^C functions as a FR partner for certain members of this family. The connection between PrP^C and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrP^C in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains.     

High sensitivity of RBL-2H3 cells to cadmium and manganese: an implication of the role of ZIP8

Cellular incorporation of Cd involves multiple transport systems for other metals such as Fe, Zn, Mn, and Ca. Metal transporters including divalent metal transporter 1, Zrt/Irt-related protein (ZIP) 8, and ZIP14, and certain types of voltage-dependent Ca channels have been shown to be involved in cellular Cd uptake. However, tissue- or cell-specific roles of these metal transporters in the accumulation and toxicity of Cd remains unclear. In the present study, we compared the sensitivity to and accumulation of Cd, Mn, and Zn among four types of rat cell lines. Rat basophilic leukemia RBL-2H3 cells showed the highest sensitivity to Cd and Mn due to the highest accumulation of Cd and Mn among the four cell lines. The high accumulation of Cd and Mn was caused by high uptake rates of Cd and Mn. Since relatively high expression of ZIP8 and ZIP14 was found in RBL-2H3 cells, siRNAs of ZIP8 and ZIP14 were transfected into RBL-2H3 cells. The knockdown of ZIP8, but not of ZIP14, significantly reduced the uptake rates of Cd and Mn in RBL-2H3 cells, especially in the presence of bicarbonate. These results suggest that the high expression of ZIP8, which is known to have affinities for both Cd and Mn, resulted in high accumulation of Cd and Mn, leading to high sensitivity to these metals in RBL-2H3 cells. Thus, RBL-2H3 cells may serve as a good model for clarifying the mechanisms of Cd and Mn transport via ZIP8.     

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn²⁺, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca²⁺-uptake transporter thought to support Golgi uptake of Mn²⁺ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca²⁺ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca²⁺ levels. In turn, we found increased cytoplasmic Ca²⁺ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in ⁵⁴Mn²⁺ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca²⁺ and ⁵⁴Mn²⁺ accumulation. While addition of Ca²⁺ positively regulated ZIP-mediated ⁵⁴Mn²⁺ uptake, we show chelation of Ca²⁺ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn²⁺ and other divalent metal ions–mediated ZIP metal transporters.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

A distinctive sequence motif in the fourth transmembrane domain confers ZIP13 iron function in Drosophila melanogaster

The zinc/iron permease (ZIP/SLC39A) family plays an important role in metal ion transport and is essential for diverse physiological processes. Members of the ZIP family function primarily in the influx of transition metal ions zinc and iron, into cytoplasm from extracellular space or intracellular organelles. The molecular determinants defining metal ion selectivity among ZIP family members remain unclear. Specifically, we reported before that the Drosophila ZIP family member ZIP13 (dZIP13), functions as an iron exporter and was responsible for pumping iron into the secretory pathway. ZIP13 protein is unique in that it differs from the other LIV-1 subfamily members at transmembrane domain IV (TM4), wherein relative positions of the conserved H and D residues in the HNXXD sequence motif are switched, generating a DNXXH motif. In this study, we undertook an in vivo approach to explore the significance of this D/H exchange. Comparative functional analysis of mutants revealed that the relative positions of D and H are critical for the physiological roles of dZIP13 and its close homologue dZIP7. Swapping D/H position of this DNXXH sequence in dZIP13 resulted in loss of iron activity; normal dZIP13 could not complement dZIP7 loss, but swapping the two relative amino acid positions D and H in dZIP13 was sufficient to make it functionally analogous to its close homologue dZIP7. This work provides the first in vivo functional analysis of a structural motif required to differentiate different transporting functions of ZIPs.     

植物ZIP基因家族铁载体蛋白基因研究进展

主要概述了植物ZIP基因家族铁载体蛋白基因研究的最新进展。从结构和功能上介绍了铁载体蛋白基因IRT1、IRT2、LeIRT1、LeIRT2、P5RIT1和O5IRT1。应用Clustal X序列分析软件,对6个铁载体蛋白基因在蛋白质水平上比较后发现,与IRT1基因蛋白质序列有较高的同源性。植物ZIP基因家族铁载体蛋白基因主要受缺铁胁迫条件的诱导,在根部表达。表达的量与环境中的铁含量、时间、温度、光照等因素有关。铁载体蛋白基因在转录和转录后水平上被环境中的铁含量和植物体内的铁营养水平综合调控。转铁载体蛋白基因植物表现出较强的抗缺铁能力,预示其在农业生产上有广阔的应用前景。     

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.     

The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals

Zinc is the second most abundant transition metal in the body. Despite the fact that hundreds of biomolecules require zinc for proper function and/or structure, the mechanism of zinc transport into cells is not well-understood. The ZIP (Zrt- and Irt-like proteins; SLC39A) family of proteins acts to increase cytosolic concentrations of zinc. Mutations in one member of the ZIP family of proteins, the human ZIP4 (hZIP4; SLC39A4) protein, can result in the disease acrodermatitis enteropathica (AE). AE is characterized by growth retardation and diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, and residues involved in cation translocation are unresolved questions. Therefore, we have established a high signal-to-noise zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that zinc, copper(II), and nickel can be transported by hZIP4 when the cation concentration is in the micromolar range. We have also identified a nanomolar binding affinity where copper(II) and zinc can be transported. In contrast, under these conditions, nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or diethylpyrocarbonate indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results of our experiments identify at least two coordination sites for divalent cations and provide a new framework for investigating the ZIP family of proteins.     

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.     

Zinc transport mediated by barley ZIP proteins are induced by low pH

It is estimated that nearly 50% of the world''s population is at risk of zinc (Zn) deficiency. The challenge is therefore to increase the Zn content in edible plant parts in order to improve the nutritional value of staple foods. We recently reported the identification and characterization of three barley genes encoding zinc transport proteins belonging to the ZIP protein family. These proteins are believed to be involved in cellular uptake of Zn²⁺. In this addendum, the Zn²⁺ transport capacity of ZIP proteins isolated from barley roots was investigated in response to various pH levels. We show that a lowering of pH induces a better growth at low Zn²⁺ concentrations of yeast cells expressing ZIP proteins. However, no significant change in transport capacity (V_max) could be observed for HvIRT1, whereas lowering of pH from 5.5 to 4.2 increased the V_max value with 64% for HvZIP5. These results indicate that proton activity has an important role in regulating the Zn²⁺ transport capacity of Zn²⁺ specific ZIP transport proteins. This information will increase the understanding of ZIP proteins and facilitate engineering of genotypes able to grow efficiently on marginal soils.Key words: ZIP proteins, barley, zinc transport, pH     

Heterogeneous Immunolocalisation of Zinc Transporters ZIP6, ZIP10 and ZIP14 in Human Normo- and Asthenozoospermic Spermatozoa

Zinc (in the form of Zn²⁺) is necessary for male fertility. Both Zn²⁺ quantity and its localisation have been detected in seminal plasma and ejaculated spermatozoa, suggesting its active uptake via zinc import transporters (ZIPs). Immunofluorescence was used to characterise the expression and localisation of three distinct types of ZIP transporters in ejaculated spermatozoa of normo- and asthenozoospermic sperm samples. ZIP6, ZIP10 and ZIP14 showed heterogeneous sperm cell expression and different compartmental distribution. In both types of sperm samples, ZIP6 and ZIP14 were predominantly localised in the sperm head, while ZIP10 was found along the sperm tail. Compartmental localisation of ZIPs in asthenozoospermia was not changed. However, regarding sub-compartmental localisation in sperm head regions, for ZIP6 asthenozoospermia only decreased its acorn/crescent-like pattern. In contrast, ZIP14 immunostaining was altered in favour of crescent-like, as opposed to acorn-like and acorn/crescent-like patterns. The specific ZIPs localisation may reflect their different roles in sperm cell integrity and motility and may change over time. This is the first report of their specific compartmental and sub-compartmental localisation in ejaculated human sperm cells. Further research will lead to a greater understanding of the roles of ZIPs in sperm cell biology, which could positively influence procedures for human infertility therapy.     

Drosophila fear of intimacy encodes a Zrt/IRT-like protein (ZIP) family zinc transporter functionally related to mammalian ZIP proteins

Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm; however, little is known about their in vivo roles. Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila. Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI. Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes.     

Sequence Analyses and Phylogenetic Characterization of the ZIP Family of Metal Ion Transport Proteins

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, α-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins. Received: 31 March 1997/Revised: 14 May 1998     

Functional characterization of the twin ZIP/SLC39 metal transporters, NpunF3111 and NpunF2202 in Nostoc punctiforme

The ZIP family of metal transporters is involved in the transport of Zn²⁺ and other metal cations from the extracellular environment and/or organelles into the cytoplasm of prokaryotes, eukaryotes and archaeotes. In the present study, we identified twin ZIP transporters, Zip11 (Npun_F3111) and Zip63 (Npun_F2202) encoded within the genome of the filamentous cyanobacterium, Nostoc punctiforme PCC73120. Sequence-based analyses and structural predictions confirmed that these cyanobacterial transporters belong to the SLC39 subfamily of metal transporters. Quantitative real-time (QRT)-PCR analyses suggested that the enzymes encoded by zip11 and zip63 have a broad allocrite range that includes zinc as well as cadmium, cobalt, copper, manganese and nickel. Inactivation of either zip11 or zip63 via insertional mutagenesis in N. punctiforme resulted in reduced expression of both genes, highlighting a possible co-regulation mechanism. Uptake experiments using ⁶⁵Zn demonstrated that both zip mutants had diminished zinc uptake capacity, with the deletion of zip11 resulting in the greatest overall reduction in ⁶⁵Zn uptake. Over-expression of Zip11 and Zip63 in an E. coli mutant strain (ZupT736::kan) restored divalent metal cation uptake, providing further evidence that these transporters are involved in Zn uptake in N. punctiforme. Our findings show the functional role of these twin metal uptake transporters in N. punctiforme, which are independently expressed in the presence of an array of metals. Both Zip11 and Zip63 are required for the maintenance of homeostatic levels of intracellular zinc N. punctiforme, although Zip11 appears to be the primary zinc transporter in this cyanobacterium, both ZIP’s may be part of a larger metal uptake system with shared regulatory elements.