Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Abstract

Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNAinjected oocytes stimulated the uptake of 55 Fe in the presence of L-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe 2ϩ ) over ferric ion (Fe 3ϩ uptake also was saturable (K0.5 Ϸ 2 M) but, notably, the metal-ion inhibition profile and Ca 2ϩ dependence of Zn 2ϩ transport differed from those of Fe 2ϩ transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions. cadmium transport; hereditary hemochromatosis; iron; homeostasis iron transport; Xenopus laevis oocyte; SLC39A14; thalassemia; zinc transport IRON-OVERLOAD CONDITIONS (e.g., thalassemia, hereditary hemochromatosis) are characterized by the appearance in plasma of nontransferrin-bound iron (NTBI) and result in cardiomyopathy, diabetes, hepatic cancer, and cirrhosis. Identification of the routes of cellular NTBI uptake will therefore provide novel targets for therapeutics.Zrt-and Irt-like protein-14 (Zip14) is a member of a large family of mammalian metal-ion transporters, the SLC39 gene family (6,11,12,22,26). Zip14 (synonyms SLC39A14, KIAA0062) is strongly expressed in the intestine (25, 35) but its subcellular localization there is not yet clear. Notably, Zip14 is abundantly expressed in the liver, heart, and pancreas (25, 35, 42), the major sites of organ damage in iron overload. Our previous data identified Zip14 as a candidate route for NTBI uptake since overexpression of Zip14 in human embryonic kidney (HEK)293, SF9, or HeLa cell lines stimulated NTBI uptake (14, 25), whereas small interfering RNA (siRNA) suppression of endogenous Zip14 in AML12 mouse hepatocytes decreased NTBI uptake (25).We have expressed mouse Zip14 in RNA-injected Xenopus oocytes, an efficient heterologous expression system ideal for direct assays of membrane transport and tolerant of broad manipulation of experimental conditions. We used radiotracer assays to test the hypothesis that Zip14 transports free iron and to examine the functional properties and metal-ion substrate profile of Zip14. MATERIALS AND METHODSReagents. Restriction enzymes were obtained from New England Biolabs (Ipswich, MA). All other reagents were obtained from SigmaAldrich (St. Louis, MO) or Research Products International (Prospect, IL) unless otherwise indicated.Expression of mouse Zip14 and human DMT1 in Xenopus oocytes. We performed laparotomy and ovariectomy on adult female Xenopus laevis frogs (Nasco, Fort Atkinson, WI) under 3-aminoethylbenzoate methanesulfonate anesthesia...

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

“…Although K m or normalized V max values between differing expression systems may vary, it is likely that trends, such as mutations that result in higher/lower affinities or changes in normalized V max , will be consistent between overexpression systems. Furthermore, considering that both micromolar and nanomolar K m values have been observed upon heterologous expression in mammalian cells as well as X. laevis oocytes, this suggests that either both affinities are present in vivo or that overexpression in both cell types gives equivalent artifacts (3,4,46). Therefore, analysis of hZIP4 transport experiments performed in X. laevis oocytes is a viable and valuable method to examine transport properties for this class of proteins.…”

“…Although mutations to these proteins result in cellular zinc and/or iron deficiency, analysis of in situ experiments have demonstrated that ZIP transporters can also transport Ni 2ϩ , Cu 2ϩ , and/or Cd 2ϩ (3,4). ZIP member proteins have eight transmembrane domains and can be further classified into subfamilies (ZIPI, ZIPII, gufA, and LIV-1) based on sequence alignments (5).…”

Iron transport proteins: Gateways of cellular and systemic iron homeostasis

“…NTBI is taken up by the liver via ZRT/IRT-like protein-14 (ZIP14/SLC39A14), a transmembrane metal-ion transporter located on the sinusoidal membrane of hepatocytes (72). ZIP14 was originally identified as a zinc transporter, but subsequent studies showed that it could also transport iron (73,74) and that its protein levels in liver increase in response to iron loading (72). Slc39a14-null mice display markedly impaired uptake of intravenously administered 59 Fe-NTBI and fail to load iron in hepatocytes when the mice are fed an iron-loaded diet or crossed with mouse models of hereditary hemochromatosis (75).…”

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress

“…2D and 5D) supports the notion that GRP94 is a critical factor that supports recovery of ER homeostasis. Although ZIP14 can contribute to uptake of manganese and non-transferrinbound iron under certain circumstances (49,50), the hepatic concentration of these metals after TM administration was comparable between WT and Zip14 KO mice (Fig. S1 E and F).…”