Abstract
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.
Highlights
Zinc (Zn2+) plays an important role in many key biological processes, such as immune function, redox signaling, and cell death (Vallee and Falchuk, 1993)
Our results indicate that full-length and a truncated form of Human zinc transporter 1 (hZnT1) can be produced in yeast and that protein extraction in the presence of detergent incorporated with cholesteryl hemisuccinate generates active protein
We developed methods to reproducibly express and purify full-length human ZnT1, a C-terminal truncated variant, and its soluble C-terminal domain (CTD)
Summary
Zinc (Zn2+) plays an important role in many key biological processes, such as immune function, redox signaling, and cell death (Vallee and Falchuk, 1993). Zn2+ has been associated with other medical conditions, including diabetes, Alzheimer’s disease and transient neonatal zinc deficiency (TNZD) (Lovell et al, 2005; Chowanadisai et al, 2006; Sladek et al, 2007). High levels of Zn2+ are toxic and tight regulation of intracellular Zn2+ concentrations is essential to maintain good health (Huang and Tepaamorndech, 2013). MTs bind free Zn2+ ions directly (Kimura and Kambe, 2016), whereas CDF and ZIP proteins regulate intracellular Zn2+ levels by sequestration and recruitment from organelles or the extracellular environment (Huang and Tepaamorndech, 2013)
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