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

Abstract

The mouse mZip1 and mZip3 zinc transporters have been implicated in zinc acquisition by the cells of many tissues. This hypothesis raised the question of whether activity of these proteins is regulated to maintain zinc homeostasis. Neither mZIP1 nor mZIP3 mRNA levels are highly regulated by zinc status. Therefore, we investigated whether zinc controls the activity of these proteins post-translationally by altering their subcellular distribution. When expressed in transfected cells grown in zinc-replete medium, both mZip1 and mZip3 were largely present in intracellular organelles. However, these proteins were found to rapidly transit between the plasma membrane and intracellular compartments in zinc-replete cells. Zinc deficiency increased plasma membrane levels of mZip1 and mZip3 by decreasing their rates of endocytosis. Greater zinc deficiency was required to alter mZip3 distribution than was needed to affect mZip1. Increased surface levels correlated with increased zinc uptake activity. Taken together, these results suggest that post-translational control of mZip1 and mZip3 localization plays a role in zinc homeostasis. Moreover, our results indicate that zinc-responsive endocytosis is a conserved mechanism controlling activity of many mammalian zinc uptake transporters.

The Mammalian Zip5 Protein Is a Zinc Transporter That Localizes to the Basolateral Surface of Polarized Cells

“…Nonetheless, the subcellular distribution of mZip4-HA and mZip5-HA was assessed by immunofluorescence confocal microscopy to provide the foundation for studies of zinc-stimulated endocytosis in HEK293 cells. This same approach was used previously to show zinc-stimulated endocytosis of mZip1, mZip3, mZip4, and hZip4 (23,28). Cells were grown in a zinc-replete basal medium and then fixed, permeabilized, incubated with anti-HA antibody, and subsequently incubated with a fluorophore-conjugated secondary antibody.…”

“…For example, we have shown that several mammalian ZIP proteins are down-regulated by zinc post-translationally. Endocytosis of mZip1, mZip3, and mZip4 is stimulated by zinc thereby decreasing plasma membrane protein levels and reducing zinc uptake activity (23,28). To assess whether mZip5 is similarly regulated by zinc-stimulated endocytosis, we first examined mZip5-HA distribution in stably transfected HEK293 cells.…”

“…A substantial amount of this protein was located on the plasma membrane. This was shown using an immunoblotting assay to assess the levels of anti-HA antibodies bound to the surface of non-permeabilized HEK/mZip-HA cells (23,28). The carboxylterminal epitope tag of each protein is predicted to be extracellular (Fig.…”

“…First, among the mutations in SLC39A4 gene associated with AE were several mutations that are likely to completely abolish the transport function of the Zip4 protein (20,21,24,25) yet the symptoms of AE can be ameliorated by increased levels of dietary zinc. Furthermore, some AE mutations fail to map to the same chromosomal region as SLC39A4 (20).…”

Prolactin (PRL)-stimulated Ubiquitination of ZnT2 Mediates a Transient Increase in Zinc Secretion Followed by ZnT2 Degradation in Mammary Epithelial Cells

“…For example, zinc exposure ubiquitinates and degrades the yeast zinc uptake transporter ZRT1 (23). Similarly, ZIP4 is quickly ubiquitinated and degraded in response to high zinc levels (47), suggesting that ubiquitination and degradation of zinc transporters may be a critical regulatory mechanism controlling zinc homeostasis. To our knowledge, our study is the first to identify a ubiquitin-mediated mechanism that is responsible for transiently increasing zinc efflux, contributing to our general understanding of zinc regulation.…”