Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain

Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn2+) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn2+ toxicity. Through homology with other CDF family members, ZnT1 is predicted to contain a transmembrane region and a soluble C-terminal domain though little is known about its biochemistry. Here, we demonstrate that human ZnT1 and a variant can be produced by heterologous expression in Saccharomyces cerevisiae cells and purified in the presence of detergent and cholesteryl hemisuccinate. We show that the purified hZnT1 variant has Zn2+/H+ antiporter activity. Furthermore, we expressed, purified and characterized the soluble C-terminal domain of hZnT1 (hZnT1-CTD) in a bacterial expression system. We found that the hZnT1-CTD melting temperature increases at acidic pH, thus, we used an acetate buffer at pH 4.5 for purifications and concentration of the protein up to 12 mg/mL. Small-angle X-ray scattering analysis of hZnT1-CTD is consistent with the formation of a dimer in solution with a V-shaped core.

Automated summary

In this study, it was demonstrated that human ZnT1 and its variant can be expressed and purified in yeast cells, with the purified hZnT1 variant showing Zn2+/H+ antiporter activity. Additionally, the soluble C-terminal domain of hZnT1 was successfully expressed, purified, and characterized in a bacterial expression system, showing an increase in melting temperature at acidic pH and forming a dimer in solution based on small-angle X-ray scattering analysis.