Zn2+-stimulated Endocytosis of the mZIP4 Zinc Transporter Regulates Its Location at the Plasma Membrane

Abstract

Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis.

Zinc-stimulated Endocytosis Controls Activity of the Mouse ZIP1 and ZIP3 Zinc Uptake Transporters

“…As was found previously for mZip4 (23), increased surface levels of mZip1-HA and mZip3-HA under zinc deficiency could be the result of decreased rates of endocytosis. To test this hypothesis, we examined antibody internalization in zinc-replete and deficient cells as described in Fig.…”

“…Cells were transferred to ice to prevent further endocytosis, washed three times with ice-cold PBS, and surface-bound antibodies were removed by five washes with ice-cold acidic buffer (100 mM glycine, 20 mM magnesium acetate, 50 mM potassium chloride, pH 2.2). This treatment is sufficient to remove all detectable surface-bound antibodies (16,23). After two additional washes with ice-cold PBS, the cells were collected, lysed, and analyzed for anti-HA antibodies by immunoblotting as described above.…”

“…Construction of these plasmids is described elsewhere (15,19). Each protein was expressed from the cytomegalovirus promoter and shown previously to be functional for zinc transport (15,19,23).…”

“…1A), surface levels of mZip4-HA protein appeared to be much higher than either mZip1-HA or mZip3-HA. As an alternative approach to assess surface levels, we used an immunoblotting technique used previously to assay surface levels of mZip4 (16,23). This method uses immunoblots to assess the levels of anti-HA antibodies bound to the surface of non-permeabilized HEK/mZip-HA cells.…”

“…We have recently found that mZip4 activity is regulated at a post-translational level as well. As was the case for yeast Zrt1, zinc stimulates endocytosis of the mouse and human Zip4 protein (23). It is therefore likely that regulation at the level of mRNA abundance combined * This work was supported by National Institutes of Health Grant RO1 DK59369.…”