Abstract
Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure–function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae-based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.
Highlights
As the selection of an adequate expression host is a bottleneck in protein production, we decided to investigate whether our established S. cerevisiaebased platform will be applicable in manufacturing Human ZIPs (hZIPs)
Based on sequence analysis of the 14 hZIPs, we selected four members possessing the shortest N-termini (Figure 2A), i.e., hZIP1, 2, 11, and 13 with the assumption that the long tails may restrict overall protein rigidity and introduce an additional level of disorder that could be detrimental for the production, stability, and subsequent crystallization-based structure determination approaches
Characterization, i.e., the production of prime-quality samples required for their biophysical characterization
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Zinc is one of the most crucial minerals for both plants and animals, and constitutes the second most abundant transition metal in humans. Up to 10% of the human proteome associates with zinc, and this metal exerts essential structural and/or functional roles, enabling correct activity of many proteins [1]. Zinc is critical for many transcription factors and serves as a cofactor in all enzyme classes. It acts as a signaling molecule, and abnormalities of intracellular levels of zinc affect pathways controlling cell development, growth, differentiation, and death [2]
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