The Zrt/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. As a highly diverse family, ZIPs exhibit very different substrate preference, but the molecular mechanism underlying the variations in substrate specificity has not been clarified. The ultimate goal of my research is to elucidate the mechanism of how ZIPs distinguish chemically similar d-block divalent metals. Here, I present an engineering method for human ZIP8, where four differentially conserved residues were rationally mutated into their counterparts in human ZIP4, creating a zinc-preferring quadruple variant (Q180H/E343H/C310A/N357H), which exhibited largely reduced transport activities towards Cd2+, Fe2+, and Mn2+ whereas increased activity toward Zn2+, “turning” human ZIP8 into a human ZIP4-like transporter. Current progress is related to a conditional selectivity filter identified in the process, which appeared to be the molecular determinants of the substrate preference of ZIPs. To better biochemically study ZIPs, I developed a non-radioactive metal uptake assay by taking advantages of inductively coupled plasma mass spectrometry (ICP-MS) and rare stable isotopes. Combined with the quantification of the cell surface expression of human ZIP4 using biotinylation or surface-bound antibody, I estimated the turnover number of human ZIP4 to be 0.08-0.2 s-1, clearly classifying human ZIP4 as a carrier. Zn efflux effect during the cell-based assay was evaluated to exclude the possibility that metal efflux may significantly affect data interpretation in the cell-based metal transport assay. The potential of converting the current approach into a high-throughput format by using laser ablation (LA) ICP-MS was explored. Taking advantage of these progress, I am applying the ICP-MS method to a further study on the conditional selectivity filter, where different amino acid compositions of residues were introduced into the selectivity filter (Q180/D318/E343) to screen the change in the substrate preference. This work led to the discovery of variants with drastically increased activity and altered substrate preference. Selected variants also exhibited novel activities against metals that are not substrates of wild-type ZIP8, including Pb2+ and the diatomic cation VO2+. I believe this research provided clearer understanding of the ZIP family and useful approaches that can be applied to the studies of metal transporters beyond the ZIP family.
Read
