Novel proteolytic processing of the ectodomain of the zinc transporter ZIP4 (SLC39A4) during zinc deficiency is inhibited by acrodermatitis enteropathica mutations.

Zinc deficiency during pregnancy impairs embryonic, fetal, and postnatal development, leading to growth retardation, abnormal morphogenesis, immune system dysfunction, skin lesions, and neurological disorders in mammals (reviewed in references 8 and 22). Therefore, the ability to acquire zinc from the diet via the intestine and transfer it to the embryonic environment via the visceral yolk sac ([VYS] in mice) plays a critical role in the growth and morphogenesis of the embryo and subsequent health status of offspring. The zinc transporter SLC39A4 (ZIP4) is an essential component for the acquisition of zinc. Mutations in the human SLC39A4 gene cause a rare autosomal recessive genetic disorder of zinc deficiency called acrodermatitis enteropathica (AE) (10, 32); in mice the Slc39a4 gene is essential during early embryogenesis, and homozygous embryos die soon after implantation (5). Furthermore, heterozygous Slc39a4 knockout mice are significantly underrepresented after birth and are hypersensitive to dietary zinc deficiency (5). Recent studies reveal that the expression of Slc39a4 is regulated at multiple posttranscriptional levels in response to changes in zinc availability (2, 9, 15, 33). For example, during zinc deficiency this mRNA is stabilized, leading to increased accumulation of Slc39a4 mRNA and ZIP4 protein and the localization of ZIP4 at the apical surfaces of enterocytes and visceral endoderm cells (4, 33). In contrast, repletion of zinc to normal levels causes the rapid endocytosis and degradation of ZIP4 and destabilization of Slc39a4 mRNA (33). Similar results were obtained with in vitro transfection studies of recombinant ZIP4, which demonstrated that ZIP4 was degraded via a process that requires both the proteasomal and lysosomal compartments (9, 15). Thus, dynamic posttranscriptional control of ZIP4 in response to zinc plays an important role in regulating zinc homeostasis. Previous studies from our laboratory revealed that during prolonged zinc deficiency, apparently full-length ZIP4 and its glycosylated forms (∼75 kDa and larger) are detectable in membrane preparations from the intestine and VYS, but by far the major immunoreactive ZIP4 peptide detected by Western blotting was ∼37 kDa in apparent molecular mass, or about half the predicted size of full-length ZIP4 (2, 7, 33). This observation was explored further herein, and our results demonstrate that the ∼37-kDa peptide represents ZIP4 lacking the N-terminal extracellular domain or ectodomain. This novel processing of ZIP4 occurs in response to zinc deficiency in polarized epithelial cells like MDCK and CaCo2 as well as in mouse Hepa cells, mimicking the results obtained in mice. The evidence suggests that the ectodomain of ZIP4 accumulates as a peripheral membrane protein, whereas the remainder of the processed protein is apparently recycled back to the apical membrane. Overexpression of processed ZIP4 or ZIP4 with ectodomain truncations correlated with hypersensitivity to zinc, as shown by a dramatic reduction in the dose response for induction of Mt1 (metallothionein 1) gene expression. Furthermore, AE mutations near the predicted cleavage site of the ectodomain block processing of ZIP4. Thus, this novel regulation of ZIP4 may be an additional and important regulatory mechanism controlling zinc transport or other activities of this critical zinc transporter.