Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4

Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog.     

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.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H(158)WHD(161). To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.     

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.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H₁₅₈WHD₁₆₁. To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.     

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.     

Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity

The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters.     

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.     

Phylogenetic,Structural and Functional Characteristics of the Na-K-Cl Cotransporter Family

Summary Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane α-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggests that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom. Received: 27 July 1995/Revised: 8 November 1995     

Involvement of histidine-rich domain of ZIP family transporter TjZNT1 in metal ion specificity.

The Zrt/Irt-like protein (ZIP) family generally contributes to metal homeostasis by regulating cation transport into the cytoplasm. Most ZIP members have a long variable loop between transmembrane domains III and IV, and these loops are predicted to be located in the cytoplasm. The loops contain a histidine-rich domain (HRD) postulated to serve as a metal ion binding site; however, its role has not yet been determined. We previously determined that deletion of the HRD did not affect the Ni tolerance ability of TjZNT1-a ZIP transporter that confers high Ni tolerance to yeast. In this study, we investigated the effect of HRD deletion on the ion transport ability of TjZNT1. The deletion of HRD increased the specificity for Zn2+, but not for Cd2+. In addition, we confirmed subcellular localizations of TjZNT1 and HRD-deleted mutants by green fluorescence protein (GFP)-fused proteins, indicating that the deletion of HRD did not affect the localization of TjZNT1. From these results, we propose that the HRD could be involved in the ion specificity of TjZNT1.     

Neuronal zinc regulation and the prion protein

Zinc, the most abundant trace metal in the brain, has numerous functions in health and disease. It is released into the synaptic cleft alongside glutamate and this connection between zinc and glutamatergic neurotransmission allows the ion to modulate overall excitability of the brain and influence synaptic plasticity. To maintain healthy synapses, extracellular zinc levels need to be tightly regulated. We recently reported that the cellular prion protein (PrP^C) can directly influence neuronal zinc concentrations by promoting zinc uptake via AMPA receptors. The octapeptide repeat region of PrP^C is involved in zinc sensing or scavenging and the AMPA receptor provides the channel for transport of the metal across the membrane, facilitated by a direct interaction between the N-terminal polybasic region of PrP^C and AMPA receptors. PrP^C has been evolutionarily linked to the Zrt/Irt-like protein (ZIP) metal ion transport family with the C-terminus of PrP^C sharing sequence similarities with the N-terminal extracellular domains of ZIP 5, 6 and 10. By incorporating the properties of ZIP transporters (both zinc sensing and zinc transport) into two existing neuronal proteins, (PrP^C as zinc sensor, AMPA receptor as zinc transporter), neuronal cells are enhancing their biological efficiency and functionality.     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

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.     

Changes in Sodium or Glucose Filtration Rate Modulate Expression of Glucose Transporters in Renal Proximal Tubular Cells of Rat

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na⁺ are potential candidates. In this study we examined the role of glucose and Na⁺ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na⁺-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by ∼36%, SGLT1 by ∼20%, and GLUT2 by ∼100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by ∼100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by ∼150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na⁺ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule. Received: 14 July 2000/Revised: 8 March 2001     

The LZT proteins; the LIV-1 subfamily of zinc transporters

Zinc is an essential ion for cells with a vital role to play in controlling the cellular processes of the cell, such as growth, development and differentiation. Specialist proteins called zinc transporters control the level of intracellular zinc in cells. In mammals, the ZIP family of zinc transporters has a pivotal role in maintaining the correct level of intracellular zinc by their ability to transport zinc into cells from outside, although they may also transport metal ions other than zinc. There are now recognised to be four subfamilies of the ZIP transporters, including the recently discovered LIV-1 subfamily which has similarity to the oestrogen-regulated gene LIV-1, previously implicated in metastatic breast cancer. We call this new subfamily LZT, for LIV-1 subfamily of ZIP zinc Transporters. Here we document current knowledge of this previously uncharacterised group of proteins, which includes the KE4 proteins. LZT proteins are similar to ZIP transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes. However, LZT proteins have a unique motif (HEXPHEXGD) with conserved proline and glutamic acid residues, unprecedented in other zinc transporters. The localisation of LZT proteins to lamellipodiae mirrors cellular location of the membrane-type matrix metalloproteases. These differences to other zinc transporters may be consistent with an alternative role for LZT proteins in cells, particularly in diseases such as cancer.     

Phytoextraction of Zinc: Physiological and Molecular Mechanism

Zinc is an essential trace element, necessary for plants, animals, and microorganisms. Zn is required for many enzymes as a catalytic cofactor, for photosynthetic CO₂ fixation, and in maintaining the integrity of bio-membranes. However, Zn is potentially toxic when accumulated beyond cellular needs. Phytoextraction technique, which is a part of phytoremediation, has opened new avenues for remediation of Zn-contaminated places. Hyperaccumulators like Thlaspi caerulescens and Arabidopsis halleri have been identified, which can accumulate up to 40,000 mg kg^?1 Zn in the aerial parts of the plant body. Carboxylic acids, primarily malate, citrate, and oxalate, and amino acids are found to play an important role in Zn hyperaccumulation. Transmembrane metal transporters are assumed to play a key role in Zn metal uptake, xylem loading, and vacuolar sequestration. Members of CDF (cation diffusion facilitator) and ZIP (zinc-regulated transporter, iron-regulated transporter like protein) family have been implicated in Zn-metal-tolerance mechanisms. A potential metal-binding motif, containing multiple histidine residues, is found in the variable regions of almost all of the ZIP family, including ZIP1, ZIP2, ZIP4, ZRT1, and ZRT2. Overexpression of some Zn metal transporter genes like TcZNT1 (Thlaspi caerulescens Zn transporter1), TcHMA4 (Thlaspi caerulescens heavy metal ATPase) in Thlaspi caerulescens, AhMTP1;3 (Arabidopsis halleri metal transporter1;3) in Arabidopsis halleri, and PtdMTP1(Poplar metal transporter1) from a hybrid poplar confer Zn hypertolerance in Thlaspi, Arabidopsis, and Poplar plant species.