The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.A long recognized disease of zinc metabolism is the human genetic disorder acrodermatitis enteropathica (AE) 1 (1, 2). This autosomal recessive disorder causes classic symptoms of zinc deficiency (3), such as dermatological lesions, changes in the gastric mucosa associated with digestive system problems, lack of weight gain, and immune and reproductive problems (4 -8). Remarkably, these symptoms can be ameliorated by dietary zinc supplement (4, 6, 9 -11), consistent with the finding of reduced, but not eliminated, uptake of 65 Zn by the intestine from AE patients (12, 13), and the reduced uptake and total content of zinc in AE fibroblasts (14,15). Recent genetic mapping localized the AE gene to chromosome 8q24.3 (16) and led to its identification as a member of the ZIP superfamily (17, 18). That gene was named hZIP4 (the Human Genome Organization Nomenclature Committee named this gene SLC39A4).ZIP4 was found to be expressed in enterocytes and to reside in the plasma membrane. Mutations in hZIP4 were detected in AE patients (17, 18), strongly suggesting that they cause this genetic disorder.The recently identified ZIP superfamily of metal ion uptake transporters (19,20) are found in all eukaryotes, and many of its members mediate zinc uptake. In yeast, ZRT1 encodes the high affinity zinc transporter, and ZRT2 encodes the low affinity zinc transport system. The Arabidopsis iron-regulated transporter gene (IRT1) encodes a metal transporter that has remarkable sequence similarity with the yeast ZRTs and with other Arabidopsis zinc transporters (19, 21). Thus, the acronym ZIP was adopted to reflect ZRT/IRT-related proteins. Many members of the ZIP gene superfamily have now been detected based on sequence homology with yeast and Arabidopsis ZIP genes (22, 23). The ZIP proteins typically have eight membrane-spanning domains, and spanning domain four contains fully conserved histidyl and glycyl residues in an amphipathic ␣-helix. These proteins also often have a histidine-rich intracellular loop between s...

The ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zincadequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in ␤-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4.

Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.

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 eighttransmembrane 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 is an essential trace element that is required for the catalytic activity of numerous metalloenzymes (4, 5) and can also serve a purely structural role by stabilizing the conformation of certain zinc-dependent protein domains, such as zinc fingers, zinc clusters, and RING fingers, that are commonly found in transcriptional regulatory proteins (5, 6). Deficiency of this essential metal can have devastating effects. In mammals, inadequate levels of zinc in the diet lead to dermatologic lesions, growth retardation, mental disorders, and compromised function of the immune and reproductive systems (7-9). Likewise, high levels of zinc can be cytotoxic. Thus, cells must maintain tight control over intracellular zinc levels. This control is achieved through a balance of zinc efflux, cellular zinc storage, and zinc uptake. Each of these activities is mediated by a distinct family of proteins (8).In mammals, the zinc transporter (ZnT) 1 family of proteins function in a tissue-, cell-, and organelle-specific manner to regulate intracellular zinc homeostasis. These proteins contain six predicted transmembrane domains and are thought to function as multimers. Seven members of the Znt family have been identified to date in mammals (Znt1-7), and genetic studies have confirmed the importance of many of these genes in mammalian zinc metabolism (10 -17). Recent studies demonstrated that zinc can regulate expression of the Znt1 gene (18), and dietary zinc modulates Znt1 and Znt2 mRNA levels in the kidney, intestine, and liver (19). Zinc can also regulate the intracellular localization of the ZnT4 and ZnT6 proteins (16).Intracellular zinc is bound by small cysteine-rich proteins called metallothioneins (MTs) (20). In the mouse, the MT family consists of four members. Exposure to high levels of zinc causes MT accumulation through increased gene expression, whereas dietary zinc deficiency leads to decreased MT abundance through decreased gene expression and protein destabilization (21). These proteins are thought to sequester zinc when present at high levels, protecting against heavy metal toxicity, and to provide a labile pool of zinc under limiting conditions that can be released for use by other proteins.In eukaryotes, uptake of several essential metals is mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters (22, 23). The first member of this family to be i...