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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 (Zn(2+)) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn(2+) toxicity. Through homology with other CDF...

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Detalles Bibliográficos
Autores principales: Cotrim, Camila A., Jarrott, Russell J., Whitten, Andrew E., Choudhury, Hassanul G., Drew, David, Martin, Jennifer L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120272/
https://www.ncbi.nlm.nih.gov/pubmed/33996761
http://dx.doi.org/10.3389/fchem.2021.667803
Descripción
Sumario:Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn(2+)) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn(2+) 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 Zn(2+)/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.