Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization

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.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

“…6 A , top). This likely is a dimer; experiments with recombinant ZIP proteins have demonstrated that both monomer and dimer forms are detected in denaturing gels like those used here ( 40 ).

Figure 6 CaCl 2 treatment increases cell surface residence of ZIP8 and ZIP14 in hBMVECs.

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“…6A, top). This likely is a dimer; experiments with recombinant ZIP proteins have demonstrated that both monomer and dimer forms are detected in denaturing gels like those used here (40).…”

“…The observation that intracellular Ca 2+ contributes to ZIP8 and ZIP14 trafficking underscores the complexity of the posttranslational regulation of these divalent metal-ion transporters. In addition to posttranslational modification, a variety of evidence suggests that along with other SLC39 family members, ZIP8 and ZIP14 may form heterodimers ( 40 , 46 , 47 , 48 ). Our Western blot analyses did give evidence of dimeric species; however, our experimental method did not explicitly interrogate whether these were homodimers or heterodimers.…”

Application of Metabolomics in the Study of Starvation-Induced Autophagy in Saccharomyces cerevisiae: A Scoping Review

“…We also hoped that the application of not just a single metabolomics platform, but cross omics technologies coupled with big-data analytics would help explain these gaps and providing new and vital targets in the finetune of autophagy processes following nutritional deprivations. The discovery of new metabolic (by)products, and by extension the pathways that are being regulated, provides potential intervention targets as predictive roles for studies such as cancer [30] as well as neurodegenerative diseases [52] of which autophagy is implicated.…”

“…Baker's yeast Saccharomyces cerevisiae has been used as research models for diseases such as neurodegenerative disorders [22], aging [23], lifespan [24] oxidative stress [25], and autophagy [26]. The genes in S. cerevisiae are highly homologous to those in humans [27,28], and the simplicity of manipulating the genes [14,29,30] and nutrition [31,32] of this shortlived yeast make it a favorable eukaryotic representative. Autophagy defective mutant, atg1 was first discovered in S. cerevisiae, and through this led to the discoveries of other ATG related genes that are involved in the autophagy following starvation [33].…”