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.     

A role for dZIP89B in Drosophila dietary zinc uptake reveals additional complexity in the zinc absorption process

Dietary zinc is the principal source of zinc in eukaryotes, with its uptake and distribution controlled by a complex network of numerous membrane-spanning transport proteins. Dietary absorption is achieved by members of the SLC39A (ZIP) gene family, which encode proteins that are generally responsible for the movement of zinc into the cytosol. ZIP4 is thought to be the primary mammalian zinc uptake gene in the small intestine, with mutations in this gene causing the zinc deficiency disease Acrodermatitis enteropathica. In Drosophila, dual knockdown of the major dietary zinc uptake genes dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) results in a severe sensitivity to zinc-deficient media. However, the symptoms associated with ZIP4 loss can be reversed by zinc supplementation and dZIP42C.1 and 2 knockdown has minimal effect under normal dietary conditions, suggesting that additional pathways for zinc absorption exist in both mammals and flies. This study provides evidence that dZIP89B is an ideal candidate for this role in Drosophila, encoding a low-affinity zinc uptake transporter active in the posterior midgut. Flies lacking dZIP89B, while viable and apparently healthy, show indications of low midgut zinc levels, including reduced metallothionein B expression and compensatory up-regulation of dZIP42C.1 and 2. Furthermore dZIP89B mutants display a dramatic resistance to toxic dietary zinc levels which is abrogated by midgut-specific restoration of dZIP89B activity. We postulate that dZIP89B works in concert with the closely related dZIP42C.1 and 2 to ensure optimal zinc absorption under a range of dietary conditions.     

Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis

Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multicopper oxidases, MCO1–4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. Here we show Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. We speculate the likely existence of a similar iron efflux apparatus as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway.     

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.     

Regulation of Iron Homeostasis in Arabidopsis thaliana by the Clock Regulator Time for Coffee

In plants, iron homeostasis is tightly regulated to supply sufficient amounts of this metal for an optimal growth while preventing excess accumulation to avoid oxidative stress. To identify new regulators of iron homeostasis, a luciferase-based genetic screen using the Arabidopsis AtFer1 ferritin promoter as a target was developed. This screen identified TIME FOR COFFEE (TIC) as a regulator of AtFer1 gene expression. TIC was previously described as a nuclear regulator of the circadian clock. Mutants in the TIC gene exhibited a chlorotic phenotype rescued by exogenous iron addition and are hypersensitive to iron during the early stages of development. We showed that iron overload-responsive genes are regulated by TIC and by the central oscillator of the circadian clock. TIC represses their expression under low iron conditions, and its activity requires light and light/dark cycles. Regarding AtFer1, this repression is independent of the previously characterized cis-acting element iron-dependent regulatory sequence, known to be involved in AtFer1 repression. These results showed that the regulation of iron homeostasis in plants is a major output of the TIC- and central oscillator-dependent signaling pathways.     

Sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters

ZIP （ZRT/IRT-like Protein） and CDF （Cation Diffusion Facilitator） are two large metal transporter families mainly transporting zinc into and out of the cytosol. Several ZIP and CDF transporters have been characterized in mammals and various model organisms, such as yeast, nematode, fruit fly, and zebrafish, and many candidate genes have been identified by genome projects. Unexpected functions of ZIP and CDF transporters have been recently reported in some model organisms, leading to major advances in our understanding of the functions of mammalian counterparts. Here, we review the recent information on the sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters obtained from the representative model organisms.     

Hepatocyte-specific deletion of peroxisomal protein PEX13 results in disrupted iron homeostasis

Peroxisomes are organelles, abundant in the liver, involved in a variety of cellular functions, including fatty acid metabolism, plasmalogen synthesis and metabolism of reactive oxygen species. Several inherited disorders are associated with peroxisomal dysfunction; increasingly many are associated with hepatic pathologies. The liver plays a principal role in regulation of iron metabolism. In this study we examined the possibility of a relationship between iron homeostasis and peroxisomal integrity.We examined the effect of deleting Pex13 in mouse liver on systemic iron homeostasis. We also used siRNA-mediated knock-down of PEX13 in a human hepatoma cell line (HepG2/C3A) to elucidate the mechanisms of PEX13-mediated regulation of hepcidin.We demonstrate that transgenic mice lacking hepatocyte Pex13 have defects in systemic iron homeostasis. The ablation of Pex13 expression in hepatocytes leads to a significant reduction in hepatic hepcidin levels. Our results also demonstrate that a deficiency of PEX13 gene expression in HepG2/C3A cells leads to decreased hepcidin expression, which is mediated through an increase in the signalling protein SMAD7, and endoplasmic reticulum (ER) stress.This study identifies a novel role for a protein involved in maintaining peroxisomal integrity and function in iron homeostasis. Loss of Pex13, a protein important for peroxisomal function, in hepatocytes leads to a significant increase in ER stress, which if unresolved, can affect liver function. The results from this study have implications for the management of patients with peroxisomal disorders and the liver-related complications they may develop.     

真核生物锌转运体及其活性的调控

真核生物的锌内稳态是由其众多特异转运体协同转运来实现的。有两个锌转运体家族ZIP和CDF被相继发现。ZIP家族的主要功能是摄取锌,而CDF家族成员主要参与锌的外排及锌在细胞内的区室化以达到解毒或贮存的目的。锌可在转录水平和翻译水平调控两类转运体的活性以维持锌在细胞和生物体水平的内稳态。