An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells

Chul-Jin Lee,H. Jerome Coyne,Michael J. Petris,Tamara Korolnek,Byung-Eun Kim,Haarin Chun,Dennis R. Winge

doi:10.1074/jbc.ra118.005203

Abstract

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

Highlights

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and plays a structural role in numerous proteins
A simple hypothesis to explain this phenomenon is that zinc may bind to extracellular region(s) of the mouse ZIP4 (mZIP4) protein, which triggers conformational changes of mZIP4 resulting in increased endocytosis, possibly by stimulating interactions between an endocytic targeting motif of mZIP4 and the cellular trafficking machinery
The N-terminal extracellular domains of both human ZIP4 (hZIP4) and mZIP4 are rich in clustered histidine residues that may bind to zinc [20, 29]

Summary

ARTICLE cro

We used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells. We found that mZIP4 exists in homodimer form at the plasma membrane based on co-immunoprecipitation and chemical cross-linking experiments These findings identify a key extracellular motif required for high sensitivity zinc-induced endocytosis of mZIP4 necessary for fine-tuned cellular zinc homeostasis

Results

Discussion

Experimental procedures

Immunofluorescence microscopy

Sequence alignments