Zn2+-stimulated endocytosis of the mZIP4 zinc transporter regulates its location at the plasma membrane

Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis.     

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.     

A cytosolic domain of the yeast Zrt1 zinc transporter is required for its post-translational inactivation in response to zinc and cadmium

Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells. Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition. Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals. We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis. During the transition from zinc-limiting to zinc-replete growth conditions (i.e. zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole. To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane. This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking. Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells.     

A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.     

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.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.     

Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters

K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.     

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.     

Reciprocal Regulation of Endocytosis and Metabolism

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe³⁺, K⁺) or efflux (e.g., Na⁺) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.Interactions between the cell and its environment obligatorily involve events at the plasma membrane. Cell-surface proteins mediate nutrient uptake, product release, and the sensing of environmental changes, including signals from other cells. Appropriate sensing and response to extracellular cues is essential for the individual cell’s survival and for the coordinated cellular behavior in multicellular organisms. Accordingly, maintenance and dynamics of membrane proteins are fundamental mechanisms of cellular homeostasis and survival.Most plasma membrane proteins are in defined equilibria with intracellular endosomal compartments, such that the amount of a given protein at the plasma membrane is determined by the balance of its endocytosis and its recycling back to the cell surface from endosomes and other intracellular compartments (Fig. 1). Changes in the kinetics of membrane protein traffic acutely affect the levels of individual proteins at the cell surface and thereby impact how cells intake nutrients, sense the environment, and respond to external cues.Open in a separate windowFigure 1.Dynamic regulation of the cell-surface content of membrane proteins. Integral membrane proteins found at the cell surface are dynamically localized to the plasma membrane. The amount of any of these proteins at the cell surface is the result of the balance of exocytosis or recycling of vesicles containing that protein from intracellular membrane compartments and the endocytosis of the protein from the cell surface. Regulation of either the rate of exocytosis or endocytosis results in alteration of the cell-surface content of a given protein.Selective molecular mechanisms trigger traffic of plasma membrane proteins through endomembranes. Among them, ubiquitination and phosphorylation stand out as they can directly target the cargo proteins. Ubiquitination is the covalent attachment of the 76-amino acid polypeptide ubiquitin to the ε-amino group of specific lysine residues (reviewed by Miranda and Sorkin 2007; and see also Piper et al. 2014). Ubiquitination of cell-surface proteins is the principal mechanism of control of endocytosis in yeast (MacGurn et al. 2012), whereas in mammals, additional molecular mechanisms regulate the endocytosis of cell-surface proteins, including alterations in conformation that impact interaction with other proteins, and as mentioned, phosphorylation. Each of these modifications can either enhance or reduce the rates internalization, recycling, or degradation of specific proteins, highlighting the complexity of the regulation of endomembrane traffic. The intricate mechanisms that underlie the reciprocal regulation of endocytosis and metabolism are beginning to be understood. Here we discuss the endocytosis mechanisms in the regulation of cellular intake or efflux of iron, cholesterol, Na⁺, and glucose, and in the regulation of receptor signaling relevant to metabolism.     

Focus Issue on Roots: Plant Nutrition: Root Transporters on the Move

Nutrient and water uptake from the soil is essential for plant growth and development. In the root, absorption and radial transport of nutrients and water toward the vascular tissues is achieved by a battery of specialized transporters and channels. Modulating the amount and the localization of these membrane transport proteins appears as a way to drive their activity and is essential to maintain nutrient homeostasis in plants. This control first involves the delivery of newly synthesized proteins to the plasma membrane by establishing check points along the secretory pathway, especially during the export from the endoplasmic reticulum. Plasma membrane-localized transport proteins are internalized through endocytosis followed by recycling to the cell surface or targeting to the vacuole for degradation, hence constituting another layer of control. These intricate mechanisms are often regulated by nutrient availability, stresses, and endogenous cues, allowing plants to rapidly adjust to their environment and adapt their development.Plants take up nutrients and water from the soil and transport them to the leaves to support photosynthesis and plant growth. However, most soils around the world do not provide optimal conditions for plant colonization. Consequently, plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients and water supply. Membrane transport proteins, including channels and transporters, play crucial roles in the uptake of nutrients and water from the soil and in their radial transport to the root vasculature. Newly synthesized membrane transport proteins have to be properly targeted to a defined compartment, usually the plasma membrane, to efficiently ensure their function. The trafficking of membrane transport proteins along the secretory pathway is tightly controlled and involves the recognition of exit signals by gatekeeper protein complexes. After reaching the plasma membrane, membrane transport proteins can be endocytosed and subsequently recycled to the cell surface or targeted to the vacuole for degradation. Because the subcellular localization of proteins directly influences their activity, modulating the localization of membrane transport proteins constitutes a powerful way to control nutrient and water uptake in plants. This review discusses the fundamental mechanisms at stake in membrane protein secretion and endocytosis, with a specific focus on membrane transport proteins, and how endogenous and exogenous cues affect their dynamics to integrate uptake of nutrients and water to plant growth conditions.     

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2′-deoxycytidine (AZA) increased intracellular (after 24 and 48 h) and total cellular zinc levels (after 48 h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48 h. MT mRNA was significantly enhanced after 24 h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.     

Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn²⁺ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae . We identified the first Zn²⁺-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1 Δ cells were unable to grow at low Zn²⁺ and showed reduced ⁶⁵Zn²⁺ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd²⁺ ions inhibited uptake but Fe²⁺ or Mn²⁺ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn²⁺ concentrations. Optimal growth is restricted to a narrow range of Zn²⁺ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.     

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.     

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.     

Bacterial zinc transporters and regulators

Zn2+ homeostasis in bacteria is achieved by export systems and uptake systems which are separately regulated by their own regulators. Three types of Zn2+ export systems that protect cells from high toxic concentrations of Zn2+ have been identified: RND multi-drug efflux transporters, P-type ATPases, and cation-diffusion facilitators. The RND type exporters for Zn2+ are only found in a few gram-negative bacteria; they allow a very efficient export across the cytoplasmic membrane and the outer membrane of the cell. P-type ATPases and cation-diffusion facilitators belong to protein families that are also found in eukaryotes. The exporters are regulated in bacteria by MerR-like repressor/activators or by ArsR-like repressors. For the high-affinity uptake of Zn2+, several binding-protein-dependent ABC transporters belonging to one class have been identified in different bacteria. Zn2+ ABC transporters are regulated by Zur repressors, which belong to the Fur protein family of iron regulators. Little is known about low-affinity Zn2+ uptake under zinc-replete conditions. One known example is the phosphate uptake system Pit, which may cotransport Zn2+ in Escherichia coli. Similarly, the citrate-metal cotransporter CitM in Bacillus subtilis may help to supply Zn2+.