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. 2014 Jun 24;111(25):9175-80.
doi: 10.1073/pnas.1405355111. Epub 2014 Jun 9.

An Iron-Regulated and Glycosylation-Dependent Proteasomal Degradation Pathway for the Plasma Membrane Metal Transporter ZIP14

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Free PMC article

An Iron-Regulated and Glycosylation-Dependent Proteasomal Degradation Pathway for the Plasma Membrane Metal Transporter ZIP14

Ningning Zhao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins.

Keywords: SLC39A14; hereditary hemochromatosis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Iron deficiency decreases cellular levels of ZIP14, whereas iron overload increases ZIP14. HepG2-ZIP14 cells were used in AF. (A) Immunoblots of ZIP14 by using FLAG antibody after cells were treated with DFO (100 μM) or FAC (100 µg/mL) for 24 h. The intensities of ZIP14 bands were quantified by Image J and presented as the relative levels to controls (Lower). Con: control. (B) Immunoblots of ZIP14 after cells were treated for 24 h with DFO or FAC. (C) Cells were treated with DFO (100 μM) or FAC (100 µg/mL) for 24 h, and then cell-surface proteins were labeled with NHS-SS-biotin. Samples were analyzed by immunoblotting. Blots were probed for P-type Na+, K+ ATPase as a marker for plasma membrane proteins and actin as a control for cytosolic proteins. (D) Cells were treated with 100 µM DFO and 100 µM SIH or (E) 50 µM hemin to alter intracellular iron. Immunoblotting was performed to examine ZIP14 levels. (F) ZIP14 and TfR1 mRNA after DFO or FAC treatment were quantified by qRT-PCR (n = 3 independent experiments). CON: control. HEK293 cells stably expressing ZIP14-FLAG were used afterward. Clone #1 and clone #2 indicate different stable cell clones (Table S1). Cells were treated with 100 µM SIH or 25 µg/mL FAC for 24 h. (G) Immunoblots of ZIP14 and TfR1. (H) ZIP14 and TfR1 mRNA were analyzed by qRT-PCR (n = 3 independent experiments). *P < 0.05, **P < 0.001, ***P < 0.0001, compared with control.
Fig. 2.
Fig. 2.
ZIP14 degradation induced by iron deficiency is proteasome-dependent and is preceded by deglycosylation. HepG2-ZIP14 cells were used in AF. (A) Cells were incubated with 100 μM DFO, DMSO (as vehicle-treated control), and 10 μM leupeptin (Leup) individually or in combination with DFO for 12 h. ZIP14 levels were analyzed by immunoblotting. (B) Cells were incubated with DFO, DMSO, 10 μM pepstatin A (Pep A), or 10 μM E64 in combination with DFO for 24 h, and ZIP14 levels were analyzed by immunoblotting. (C) Cells were incubated with DFO, DMSO (as vehicle-treated control), and 10 μM MG132 individually or in combination with DFO for 12 h. ZIP14 levels were analyzed by immunoblotting. (D) The time-course effect of MG132 was examined. (E) Cells were treated with epoxomicin (Epoxo, 10 μM). (F) Lysates collected from DMSO-treated (Con) or MG132-treated cells were digested with PNGase F. (G) Diagram of ZIP14 glycosylation sites on its extracellular amino terminus. SP: signal peptide; TM: transmembrane domain. (H) HEK293 stable transgenic cells were treated with MG132 (12 h) before harvesting for immunoblotting. Numbers with “#” indicate different stable cell clones (Table S1). An asterisk (*) indicates unstripped signal from anti-FLAG antibody. Note that the endogenous ZIP14-3XFLAG travels with a higher MW than that of the ZIP14-FLAG-Myc construct transfected into HEK293 cells.
Fig. 3.
Fig. 3.
Plasma membrane ZIP14 is internalized before proteasomal processing. HepG2-ZIP14 cells were used. (A) Schematic illustration of experiment. Cell-surface proteins were biotinylated at 4 °C for 30 min, and cells were then treated with 10 μM MG132 in complete medium at 37 °C for 2 h. Cell lysates were collected and cell-surface proteins were isolated by streptavidin gel. (B) Cell-surface proteins were labeled with cell-membrane–impermeable NHS-SS-biotin at 4 °C and then chased for 2 h at 37 °C with or without the addition of MG132. (C) Cells were incubated with or without dynasore (80 µM) for 15 min followed by the addition of transferrin. After 30 min the cells were placed on ice, washed extensively to remove unbound surface transferrin, and fixed with paraformaldahyde. Cells stained for transferrin (red), actin (green), and nuclei (blue). (D) Immunoblots of cell-surface protein after preincubation with dynasore.
Fig. 4.
Fig. 4.
Iron prevents internalized plasma membrane ZIP14 from being extracted from the cytosol. HepG2-ZIP14 cells were used. (A) Schematic illustration of the protocol to analyze plasma membrane protein associated with cytosolic or membrane fractions. (B) Total membrane (Mem) and cytosolic (Cyto) fractions were isolated by two-step centrifugations. Biotin-labled cell-surface ZIP14 in each fraction was isolated by streptavidin gel and analyzed by immunoblotting. (C) Cells were treated with PBS (CON) or FAC for 24 h, and then the internalized ZIP14 in the cytosol and the membrane fractions were compared by immunoblotting. (D) Cells treated with BFA (10 µg/mL) for 1 h were stained for Golgi97 (red), actin (green), and nuclei (blue). (E) Cells were treated with BFA (10 µg/mL) for 12 h. Total cell lysates and cell media from control and BFA-treated cells were collected and analyzed by immunoblotting for transferrin. (F) Cell-surface proteins were labeled with NHS-SS-biotin at 4 °C. Cells were then incubated with BFA for 1 h followed by 2 h of incubation with MG132. Biotin-labled cell-surface proteins were isolated by streptavidin gel and analyzed by immunoblotting.
Fig. 5.
Fig. 5.
The iron sensitivity of ZIP14 depends on N-linked oligosaccharides, but not on the histidine-rich motif-containing intracellular loop. (A) Topology of the ZIP14 fragment between TM3 and TM4 within the endocytic compartment. (B) HEK293 stable transgenic cells expressing ZIP14 without its histidine-rich region or (C) the predicted second intracellular loop were treated with 100 µM SIH or 25 µg/mL FAC for 24 h, followed by immunoblotting. (D) Cell-surface proteins were labeled with NHS-SS-biotin. Samples were analyzed by immunoblotting. (E) Cells were labeled with NHS-SS-biotin at 4 °C, and then cells were warmed to 37 °C to induce endocytosis (under control conditions, cells were kept at 4 °C to prevent endocytosis). Subsequently, cells were incubated again at 4 °C and were treated with the cell-impermeable reducing agent MesNa to remove surface biotinylation. Cells were lysed, immunoprecipitated with streptavidin–agarose, and analyzed by immunoblotting to measure internalization. Biotinylated cell-surface NG ZIP14 was detected when cells were allowed to undergo endocytosis. Cells maintained at 4 °C were used as control. Under these conditions, no biotinylated protein was detected, indicating efficient stripping of the cell-surface biotinylation by MesNa treatment. (F) Cells stably transfected with NG ZIP14 were treated with 100 µM SIH or 25 µg/mL FAC for 24 h, followed by immunoblotting. Numbers with “#” indicate different stable cell clones (Table S1).
Fig. 6.
Fig. 6.
Glycosylation at asparagine 102 is required for the membrane extraction and iron sensitivity of ZIP14. HEK293 stable transgenic cells expressing WT or NG ZIP14 were used in AD. (A) Immunoblotting analysis by Licor ODYSSEY scanner and quantification of the blots. n = 3 independent experiments. (B) The expression levels of the WT and the NG ZIP14 mRNA were quantified and normalized to β-actin. n = 3 independent experiments. (C) The HEK293 WT ZIP14 and HEK293 NG ZIP14 cells were incubated with cycloheximide (CHX, 100 µg/mL) for 0, 1, 2, and 4 h, followed by immunoblotting analysis. (D) Comparison of the internalized plasma membrane proteins in the membrane (Mem) and cytosolic (Cyto) fractions from the WT and NG ZIP14 cell lysates. (E) HEK293 stably transfected cells expressing single Asn-to-Ala mutant ZIP14 were treated with SIH (100µM) and analyzed by immunoblotting. (F) Comparison of the internalized plasma membrane proteins in the Mem and Cyto fractions from the WT and N102A ZIP14 cell lysates. Numbers with “#” indicate different stable cell clones (Table S1). (G) Cells were treated with CHX, and then the stability of the N102A ZIP14 was compared with that of the WT ZIP14 by immunoblotting.

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