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. 2010 Oct 15;285(42):32141-50.
doi: 10.1074/jbc.M110.143248. Epub 2010 Aug 3.

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron From Transferrin

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Free PMC article

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron From Transferrin

Ningning Zhao et al. J Biol Chem. .
Free PMC article

Abstract

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism.

Figures

FIGURE 1.
FIGURE 1.
Overexpression of ZIP14 increases the cellular assimilation of iron from TF. A, HEK 293T cells transfected with pCMVSport2 (control) or pCMVSport6/mouse ZIP14 were incubated with 100 nm 59Fe-TF for the times indicated. Cells were harvested, and cell-associated radioactivity was determined by γ-counting. The amount of 59Fe assimilated by the cells is expressed as cpm/mg of protein. *, different from respective control, p < 0.05. The data shown represent the mean ± S.E. (error bars) of three independent experiments. B, lysates of cells incubated with 100 nm 59Fe-TF for 4 h were analyzed by Western blotting for ZIP14, TFR1, and DMT1. To indicate protein loading among lanes, the blot was stripped and reprobed for actin. Data are representative of one of three experiments. C, HEK 293T cells transfected with pCMVSport2 (control) or pCMVSport6/mouse ZIP14 were incubated with 100 nm biotin-labeled holo-TF for 2, 10, or 30 min. Cell lysates were analyzed by Western blotting for ZIP14, TF, and tubulin as a lane-loading control. Data are representative of one of two experiments.
FIGURE 2.
FIGURE 2.
pH dependence of ZIP14-mediated iron transport. HEK 293T cells transfected with pCMVSport2 (control) or pCMVSport6/mouse ZIP14 were incubated with 2 μm 59Fe-ferric citrate for 1 h in uptake buffer at pH 7.5, 6.5, and 5.5. The amount of 59Fe taken up by cells is expressed as cpm/mg of protein. Data represent the mean ± S.E. (error bars) of three independent experiments.
FIGURE 3.
FIGURE 3.
Comparison of ZIP14 and DMT1 mRNA levels in HepG2 and HEK 293T cells. Total RNA was isolated from untreated HepG2 and HEK 293T cells, and transcript copy numbers were determined by using quantitative RT-PCR. Data represent the mean ± S.E. (error bars) of three independent experiments of triplicate samples.
FIGURE 4.
FIGURE 4.
Targeted knock-in of 3×FLAG into the ZIP14 locus of HepG2 cells. A, diagrams of the wild-type (WT) ZIP14 allele, the FLAG + NeoR allele after homologous recombination with the targeting vector, and the FLAG allele after excising the neomycin cassette with Cre recombinase are shown. Clones were screened by using primers P1–P6 designed to target the indicated positions. B, PCR of genomic DNA identifies clones with the FLAG + NeoR allele and FLAG allele. C, Western blot analysis of HepG2 cells expressing FLAG-tagged ZIP14 and knockdown of endogenous ZIP14 in HepG2 cells using siRNA targeting ZIP14 are shown. To indicate protein loading among lanes, the blot was stripped and reprobed for actin.
FIGURE 5.
FIGURE 5.
ZIP14 partially co-localizes with internalized TF in HepG2 cells. Cells were incubated for 30 min with Alexa Fluor 488-labeled human holo-TF prior to fixation and permeabilization. Endogenous ZIP14–3×FLAG was detected by using anti-FLAG antibody followed by rhodamine-conjugated secondary antibody. Merged image shows co-localization of ZIP14 and internalized holo-TF. Nuclei were stained with DAPI. All images were obtained by using a Leica TCS SP5 laser-scanning confocal microscope. Areas of co-localization (designated by white) were determined by using the co-localization tool provided with the Leica SP5 software.
FIGURE 6.
FIGURE 6.
Confocal microscopic analysis of the subcellular localization of ZIP14 in HepG2 cells. Fixed and permeabilized cells were analyzed for endogenous ZIP14–3×FLAG by using anti-FLAG antibody followed by rhodamine-conjugated secondary antibody. A–D, co-localization of ZIP14 with EEA1 (A), LAMP1 (B), TFR1 (C), and Rab11 (D) was determined by using respective primary antibodies followed by Alexa Fluor 488-labeled secondary antibody. Merged images show co-localization of ZIP14, with nuclei stained by DAPI. E, as a control, TF was co-localized with TFR1 by first incubating cells for 30 min with Alexa Fluor 488-labeled human holo-TF prior to fixation and permeabilization of cells. TFR1 was determined by using anti-TFR1 antibody followed by rhodamine-conjugated secondary antibody. All images were obtained by using a Leica TCS SP5 laser-scanning confocal microscope. Areas of co-localization (designated by white) were determined by using the co-localization tool provided with the Leica SP5 software.
FIGURE 7.
FIGURE 7.
Knockdown of ZIP14 in HepG2 cells decreases the assimilation of iron from TF. A, HepG2 cells were transfected with negative control (NC) or ZIP14 siRNA for 72 h prior to Western blot analysis for ZIP14, TFR1, TFR2, and actin as a lane loading control. B, HepG2 cells transfected with negative control or ZIP14 siRNA for 72 h were incubated with 100 nm 59Fe-TF for 4 h. Cells were harvested, and cell-associated radioactivity was determined by γ-counting. The amount of 59Fe assimilated by the cells is expressed as cpm/mg of protein. Data represent the mean ± S.E. (error bars) of three independent experiments. C, HepG2 cells transfected with negative control or ZIP14 siRNA for 72 h were incubated with or without 100 nm biotin-labeled holo-TF. After 4 h, cell lysates were analyzed by Western blotting for ZIP14, TF, and actin as a lane loading control. Data are representative of one of two independent experiments. D, HepG2 cells transfected with negative control or ZIP14 siRNA for 72 h were incubated with 100 nm biotin-labeled holo-TF. After 2, 10, and 30 min, cell lysates were obtained and analyzed by Western blotting for ZIP14, TF, and tubulin as a lane-loading control. Data are representative of one of two independent experiments.
FIGURE 8.
FIGURE 8.
Model of ZIP14 function in iron assimilation by hepatocytes. Binding of holo-TF (Fe3+-TF) to TFR1 results in endocytosis of the TF·TFR1 complex. Acidification of early endosomes causes TF to release its Fe3+ iron, which is reduced to Fe2+ prior to transport into the cytosol via endosomal ZIP14. The TF·TFR1 complex recycles to the plasma membrane where apo-TF dissociates from TFR1. ZIP14 is detectable in EEA1- and LAMP1-positive structures, but not Rab11-positive structures, suggesting that ZIP14 does not recycle to the plasma membrane. ZIP14 at the cell surface mediates the uptake of NTBI.

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