The transmembrane domains mediate oligomerization of the human ZIP4 transporter in vivo.

The human (h) ZIP4 is a plasma membrane transporter that functions to increase cytosolic zinc levels. hZIP4 encodes eight transmembrane domains and a large extracellular domain (ECD). This ECD is cleaved from the holo-transporter when cells are zinc-deficient. At the same time, mutations in the ECD can result in the zinc-deficiency disease Acrodermatitis enteropathica. Previously, it was shown that hZIP4's ECD is comprised of two structurally independent subdomains where contacts between the ECD monomeric units are centered at the PAL motif. These results lead to the hypothesis that ZIP4-ECD is essential to the dimerization of the holo-transporter. To test this hypothesis, we used Fluorescence Correlation Spectroscopy (FCS) to quantify the oligomeric state of full-length hZIP4 and hZIP4 lacking the ECD domain, each tagged with eGFP. Inspection of our experimental results demonstrate that both the full-length and truncated hZIP4 is a dimer when expressed in HEK293 cells. Parallel functional experiments demonstrate that the Km and Vmax for truncated and full-length hZIP4/eGFP are similar. Determining that truncated hZIP4/eGFP forms a dimer is a crucial step for understanding the function of the hZIP4-ECD, which provides more insight into how the diseases related to hZIP4 protein.