A Histidine-rich Cluster Mediates the Ubiquitination and Degradation of the Human Zinc Transporter, hZIP4, and Protects against Zinc Cytotoxicity

Xiaoqing Mao,Byung-Eun Kim,Fudi Wang,David J Eide,Michael J Petris

doi:10.1074/jbc.m610552200

Abstract

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.

Highlights

The levels of mZIP4 mRNA are increased in response to zinc limitation in enterocytes and yolk sac and suppressed upon zinc repletion [5, 7]
Human and murine ZIP4 was shown to accumulate at the plasma membrane during zinc deficiency and undergo endocytosis when cells were exposed to low zinc concentrations (ϳ1 ␮M Zn)
Plasma Membrane Localization and Endocytosis Assays of hZIP4 Protein—The pool of hZIP4-HA at the plasma membrane was assessed by measuring the levels of anti-HA antibodies bound to the surface of HEK/hZIP4-HA cells as we have previously described for the murine ZIP4 protein [8]

Summary

Introduction

The levels of mZIP4 mRNA are increased in response to zinc limitation in enterocytes and yolk sac and suppressed upon zinc repletion [5, 7]. Immunoprecipitation Experiments— hZIP4-HA-expressing HEK293 cells or vector controls were transiently transfected with 10 ␮g of the 3ϫFLAG-Ubiquitin construct and incubated for the indicated times in zinc-depleted or zinc-supplemented medium. When zinc was added to Chelex-treated media there was a concentration-dependent increase in hZIP4-HA endocytosis, as determined by the marked increase in anti-HA antibody uptake during a 5-min incubation.

Results

Conclusion