Zrt/Irt-like Protein Research Articles

Response ofSchizosaccharomyces pombeto Zinc Deficiency

A component of the cellular response to zinc deficiency operates via control of transcript abundance. Therefore, microarray analysis was employed to identify Schizosaccharomyces pombe genes whose mRNA levels are regulated by intracellular zinc status. A set of 57 genes whose mRNA levels were substantially reduced in response to zinc deficiency was identified, while the mRNA levels of 63 genes were increased by this condition. In order to investigate the mechanisms that control these responses, a genetic screen was employed to identify mutants with defective zinc-responsive gene expression. Two strains (II-1 and V7) that were identified by this screen harbor mutations that are linked to zrt1+, which encodes a putative Zrt/IRT-like protein (ZIP) zinc uptake transporter. Importantly, zrt1+ mRNA levels are increased in response to zinc deprivation, and cells lacking functional Zrt1 are highly impaired in their ability to proliferate at limiting zinc concentrations. Furthermore, zrt1 null cells were found to have severely reduced zinc contents, indicating that Zrt1 functions as a key regulator of intracellular zinc levels in fission yeast. The deletion of fet4+, another zinc-responsive gene encoding a putative metal ion transporter, exacerbated the phenotypes associated with the loss of Zrt1, suggesting that Fet4 also plays a role in zinc uptake under limiting conditions.

Cloning of ZIP family metal transporter genes from the manganese hyperaccumulator plant Chengiopanax sciadophylloides, and its metal transport and resistance abilities in yeast

Manganese (Mn) specific hyperaccumulation in the tree Chengiopanax sciadophylloides in soil not polluted by metals has received attention as a distinctive character of this plant, but its mechanism has not yet been clarified. Based on the speculation that ZIP (Zrt-, Irt-like proteins) transporters of high Mn affinity are involved in Mn-specific hyperaccumulation in this plant, we isolated ZIP family transporter gene homologs and investigated their metal transport characteristics in yeast cells. An isolated Zip gene homolog, designated as CsZIP1, coded a protein of 475 amino acids in length and showed 65% or lower sequence identity with ZIPs of other herbal plants. Expression of this gene was induced in the callus of Mn-deficient C. sciadophylloides. However, its expression did not rescue the Mn transport ability of yeast lacking the Mn transporter gene smf1. CsZIP1 did not show Zn and Fe transport, but its expression increased Cd sensitivity and Ni tolerance of wild-type yeast.

Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.)

Two ZIP (Zrt, Irt-like Protein) cDNAs were isolated from rice (Oryza sativa L.) by RT-PCR approach, and named as OsZIP7a and OsZIP8 respectively. The predicted proteins of OsZIP7a and OsZIP8 consist of 384 and 390 amino acid residues respectively, and display high similarity to other plant ZIP proteins. Each protein contains eight transmembrane (TM) domains and a highly conserved ZIP signature motif, with a histidine-rich region in the variable region between TM domains III and IV. By semi-quantitative RT-PCR approach, it was found that the expression of OsZIP7a was significantly induced in rice roots by iron-deficiency, while that of OsZIP8 induced in both rice roots and shoots by zinc-deficiency. When expressed in yeast cells, OsZIP7a and OsZIP8 could complement an iron-uptake-deficient yeast mutant and a zinc-uptake-deficient yeast mutant respectively. It suggested that the OsZIP7a and OsZIP8 might encode an iron and a zinc transporter protein in rice respectively.

Fear of intimacy - a close LIV-1 acquaintancy?

A recent paper in Development by Mathews and colleagues demonstrates the essential role of Drosophila Fear of Intimacy (FOI) in gonad morphogenesis (Mathews et al.,2006) that is linked to its ability to transport zinc. FOI is a member of the LIV-1 subfamily of the Zrt, IRT-like protein (ZIP) family of ion transporters. Given the increasing interest in this family of zinc transporters, we wish to highlight certain points of nomenclature in the Mathews et al. paper, which do not reflect on its conclusions.The ZIP family of ion transporters divides into four subfamilies that belong to solute carrier family 39 (SLC39A)(Eide, 2003). LIV-1 is designated SLC39A6 or ZIP6. FOI belongs to the LIV-1 subfamily(Taylor and Nicholson, 2003),which contains nine human sequences in total.We question the designation of FOI as dSLC39A6/dZIP6, which suggests that it is the Drosophila equivalent of LIV-1, which is already known as SLC39A6 (Eide, 2003). There appears to be no evidence to justify this, and an examination of the phylogenetic tree of over 110 members of the SLC39A family(Taylor and Nicholson, 2003)clarifies this view. There are four Drosophila sequences in the LIV-1 subfamily termed Dm1-4. Dm4 represents FOI.The section of the tree that includes both FOI and LIV-1 also contains another Drosophila molecule and five human ones. The two Drosophila sequences, Dm3 and Dm4 (FOI), are present in a different branch to that containing the human sequences: ZIP10/SLC39A10, ZIP5/SLC39A5,LIV-1/ZIP6/SLC39A6, ZIP14/SLC39A14 and ZIP8/SLC39A8. Additionally, when the LIV-1 subfamily sequences alone are aligned(Taylor et al., 2004), Dm3 and FOI (Dm4) again locate in a separate branch, whereas LIV-1, ZIP10/SLC39A10 and ZIP5/SLC39A5 locate in another. Furthermore, an alignment of the family sequences across transmembrane domains IV and V(Taylor and Nicholson, 2003),containing the zinc transport active site, shows the FOI sequence to be closest to ZIP5/SLC39A5 and ZIP10/SLC39A10, although there is also good sequence similarity with LIV-1, ZIP12/SLC39A12, ZIP8/SLC39A8 and ZIP14/SLC39A14. In light of this, it is our opinion that it is inappropriate to assign FOI to any individual SLC39A family member, especially LIV-1, until further research clarifies the situation.

Characterization of Gene Expression Profiles of Metallothionein-Null Cadmium-Resistant Cells

To understand metallothionein (MT)-independent mechanisms for cadmium (Cd) resistance in mammalian cells, we previously established MT-null Cd-resistant cells from embryonic fibroblast cells of MT-I and -II knockout mice. The Cd resistance of these cells was conferred primarily by a marked reduction of Cd accumulation. To identify genes responsible for Cd resistance as well as Cd transport, we carried out several DNA microarray analyses using cDNAs obtained from two clones of Cd-resistant cells and parental cells. A competitive hybridization of Cy3- and Cy5-probed cDNAs on a DNA chip was carried out with dye-swapping. After a careful examination of the reproducibility and reliability of the data obtained using five different chips, it was found that the expression of 78 genes was enhanced and that of 48 genes was reduced in Cd-resistant cells compared with those in parental cells. These genes include those involved in signal transduction, ubiquitin pathway, and cell-to-cell interactions. Several genes for transporters including solute carrier family transporters and ATP-binding cassette transporters were up- or down-regulated. The examination of mRNA levels using quantitative real-time PCR revealed that the expression of Slc39a14 encoding ZIP14, a member of the zinc transporter ZIP (ZRT-, IRT-like protein) family, was markedly down-regulated in both clones of Cd-resistant cells. Although it is not yet clear whether ZIP14 has the ability to transport Cd, these results suggest that the lowered expression of ZIP14 may be involved in Cd resistance in MT-null cells.

Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis

Testicular necrosis is a sensitive endpoint for cadmium (Cd(2+), Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.

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.

Structure–function analysis of a novel member of the LIV-1 subfamily of zinc transporters, ZIP14

Here, we report the first investigation of a novel member of the LZT (LIV-1 subfamily of ZIP zinc Transporters) subfamily of zinc influx transporters. LZT subfamily sequences all contain a unique and highly conserved metalloprotease motif (HEXPHEXGD) in transmembrane domain V with both histidine residues essential for zinc transport by ZIP (Zrt-, Irt-like Proteins) transporters. We investigate here whether ZIP14 (SLC39A14), lacking the initial histidine in this motif, is still able to transport zinc. We demonstrate that this plasma membrane located glycosylated protein functions as a zinc influx transporter in a temperature-dependant manner.

Intestinal and placental zinc transport pathways.

Mammalian members of the cation diffusion facilitator (CDF) and zrt-, irt-like protein (ZIP) families of Zn transporters, initially identified in Saccharomyces cerevisiae and Arabidopsis thalania spp., have been cloned during the last 8 years and have been classified as families SLC30 and SLC39 respectively. The cloning of human Zn transporters ZnT-like transporter 1 (hZTL1)/ZnT5 (SLC30A5) and hZIP4 (SLC39A4) were major advances in the understanding of the molecular mechanisms of dietary Zn absorption. Both transporters are localised at the enterocyte apical membrane and are, therefore, potentially of fundamental importance in dietary Zn uptake. hZTL1 mediates Zn uptake when expressed in Xenopus laevis oocytes and hZIP4 is mutated in most cases of the inherited Zn deficiency disease acrodermatitis enteropathica. Localisation of hZTL1/ZnT5 at the apical membrane of the placental syncytiotrophoblast indicates a fundamental role in the transfer of Slc30 Zn to the foetus. Observations in rodent models indicate that in the intestine increased Zn availability increases expression of Zn transporters. Human intestinal Caco-2 cells show a similar response to increasing the Zn2+ concentration of the nutrient medium in relation to the expression of mRNA corresponding to several Zn transporters and that of ZnT1 (SLC30A1) and hZTL1/ZnT5 proteins. In the human placental cell line JAR, however, expression at the mRNA level of a number of Zn transporters is not modified by Zn availability, whilst ZnT1 and hZTL1/ZnT5 proteins are reduced under Zn-supplemented conditions. These differences between Caco-2 and JAR cells in Zn transporter gene responses to Zn supply may reflect the different extracellular Zn concentrations encountered by the corresponding cell types in vitro.

Structure-function analysis of HKE4, a member of the new LIV-1 subfamily of zinc transporters.

The KE4 proteins are an emerging group of proteins with little known functional data. In the present study, we report the first characterization of the recombinant human KE4 protein in mammalian cells. The KE4 sequences are included in the subfamily of ZIP (Zrt-, Irt-like Proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc Transporters). All these LZT sequences contain similarities to ZIP transporters, including the consensus sequence in transmembrane domain IV, which is essential for zinc transport. However, the new LZT subfamily can be separated from other ZIP transporters by the presence of a highly conserved potential metalloprotease motif (HEXPHEXGD) in transmembrane domain V. Here we report the location of HKE4 on intracellular membranes, including the endoplasmic reticulum, and its ability to increase the intracellular free zinc as measured with the zinc-specific fluorescent dye, Newport Green, in a time-, temperature- and concentration-dependent manner. This is in contrast with the zinc influx ability of another LZT protein, LIV-1, which was due to its plasma membrane location. Therefore we have added to the functionality of LZT proteins by reporting their ability to increase intracellular-free zinc, whether they are located on the plasma membrane or on intracellular membranes. This result, in combination with the crucial role that zinc plays in cell growth, emphasizes the importance of this new LZT subfamily, including the KE4 sequences, in the control of intracellular zinc homoeostasis, aberrations of which can lead to diseases such as cancer, immunological disorders and neurological dysfunction.

Overview of mammalian zinc transporters.

In recent years, a number of mammalian zinc transporters have been identified, and candidate genes are rapidly growing. These transporters are classified into two families: ZIP (ZRT, IRT-like protein) and CDF (cation diffusion facilitator). ZIP members facilitate zinc influx into the cytosol, while CDF members facilitate its efflux from the cytosol. Molecular characterization of the transporters has brought about major advances in our understanding of their physiological functions. Zinc metabolism is regulated primarily through zinc-dependent control of transcription, translation, and intracellular trafficking of transporters. Analyses of mice whose zinc transporter genes have been genetically disrupted and of the naturally occurring mutant mice with symptoms related to abnormal zinc metabolism have provided compelling evidence that some zinc transporters play critical roles in zinc homeostasis. In this review, we review the literature of mammalian zinc transporters with emphasis on very recent findings and elicit integrative knowledge of zinc homeostasis.

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.

Structure-function analysis of LIV-1, the breast cancer-associated protein that belongs to a new subfamily of zinc transporters.

The LIV-1 gene has been previously associated with oestrogen-positive breast cancer and its metastatic spread to the regional lymph nodes. We have investigated the protein product of this gene as a marker for disease progression of breast cancer. The protein sequence contains a potential metalloprotease motif (HEX P H E XGD), which fits the consensus sequence for the catalytic zinc-binding site motif of the zincin metalloproteases. This motif has identified a new subfamily of ZIP (Zrt-, Irt-like proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc transporters). Expression of recombinant LIV-1 in Chinese-hamster ovary cells confirmed the prediction that LZT proteins can act as zinc-influx transporters. Zinc is essential for growth and zinc transporters have an important role in maintaining intracellular zinc homoeostasis, aberrations of which could lead to diseases such as cancer. This is the first report of the expression of a recombinant human LZT protein in mammalian cells. Recombinant LIV-1 locates to the plasma membrane, concentrated in lamellipodiae, similar to membrane-type metalloproteases. Examination of LIV-1 tissue expression located it mainly to hormonally controlled tissues with widespread expression in the brain. Interestingly, the LIV-1 sequence contains a strong PEST site and other potential degradation motifs, which, combined with our evidence that recombinant LIV-1 associates with ubiquitin, may explain the low-level expression of LIV-1. Combining the crucial role that zinc plays in cell growth and the proven role of metalloproteases in metastasis presents an exciting indication of how LIV-1 plays a role in breast cancer progression.

Characterisation of zinc uptake into rat cultured cerebrocortical oligodendrocyte progenitor cells

In the mammalian brain, extracellular Zn 2+ is reported to play a neuromodulatory role and, during acute CNS injury, increased Zn 2+ release may be neurotoxic. Although several recent studies have examined possible mechanisms of neuronal Zn 2+ accumulation, little is known about oligodendroglial Zn 2+ uptake, the focus of the present study. 65 Zn 2+ uptake was time-dependent and saturable (K m =3.2±1.0 μM, V max =697.2±67.3 pmoles mg protein −1 15 min −1 ). Neither kainate (an AMPA/kainate receptor agonist) nor nicardipine (an L-type Ca 2+ channel inhibitor) influenced 65 Zn 2+ uptake, in contrast with pyrithione (a Zn 2+ ionophore). Either increasing extracellular H + concentration (pH 5.5) or co-application of either 100 μM Co 2+ or 100 μM Cu 2+ reduced 65 Zn 2+ uptake. However, 100 μM Fe 2+ failed to influence 65 Zn 2+ uptake and 100 μM La 3+ increased 65 Zn 2+ accumulation. These data are consistent with oligodendrocyte progenitor cells possessing a high-affinity Zn 2+ uptake mechanism similar to that described for the Zrt, Irt-like protein (ZIP) transporter family.

Characterisation of zinc uptake into rat cultured cerebrocortical oligodendrocyte progenitor cells

In the mammalian brain, extracellular Zn 2+ is reported to play a neuromodulatory role and, during acute CNS injury, increased Zn 2+ release may be neurotoxic. Although several recent studies have examined possible mechanisms of neuronal Zn 2+ accumulation, little is known about oligodendroglial Zn 2+ uptake, the focus of the present study. 65Zn 2+ uptake was time-dependent and saturable (K m=3.2±1.0 μM, V max=697.2±67.3 pmoles mg protein −1 15 min −1). Neither kainate (an AMPA/kainate receptor agonist) nor nicardipine (an L-type Ca 2+ channel inhibitor) influenced 65Zn 2+ uptake, in contrast with pyrithione (a Zn 2+ ionophore). Either increasing extracellular H + concentration (pH 5.5) or co-application of either 100 μM Co 2+ or 100 μM Cu 2+ reduced 65Zn 2+ uptake. However, 100 μM Fe 2+ failed to influence 65Zn 2+ uptake and 100 μM La 3+ increased 65Zn 2+ accumulation. These data are consistent with oligodendrocyte progenitor cells possessing a high-affinity Zn 2+ uptake mechanism similar to that described for the Zrt, Irt-like protein (ZIP) transporter family.

Eukaryotic zinc transporters and their regulation.

The last ten years have witnessed major advances in our understanding of zinc transporters and their regulation in eukaryotic organisms. Two families of transporters, the ZIP (Zrt-, Irt-like Protein) and CDF (Cation Diffusion Facilitator) families, have been found to play a number of important roles in zinc transport. These are ancient gene families that span all phylogenetic levels. The characterized members of each group have been implicated in the transport of metal ions, frequently zinc, across lipid bilayer membranes. This remarkable conservation of function suggests that other, as yet uncharacterized members of the family, will also be involved in metal ion transport. Many of the ZIP family transporters are involved in cellular zinc uptake and at least one member, the Zrt3 transporter of S. cerevisiae, transports stored zinc out of an intracellular compartment during adaptation to zinc deficiency. In contrast, CDF family members mediate zinc efflux out of cells or facilitate zinc transport into intracellular compartments for detoxification and/or storage. The activity of many of these transporters is regulated in response to zinc through transcriptional and post-transcriptional mechanisms to maintain zinc homeostasis at both the cellular and organismal levels.