Transmembrane topology of the mammalian Zip14 metal‐ion transporter

Abstract

Zip14 belongs to the SLC39A family of metal‐ion transporters. Similar to general topology models of SLC39A proteins, Zip14 has been predicted to have 8 transmembrane domains with extracellular N‐ and C‐termini. In the present study, we used epitope tagging to investigate the topology of mouse Zip14. We inserted into Zip14 small antigenic FLAG peptides in the N‐terminus, C‐terminus, as well as the large extramembrane domain containing a histidine‐rich motif. The tagged proteins were expressed in HEK293 cells, and the polarity of the FLAG tags with respect to the plasma membrane were determined by immunofluorescence analysis of intact and permeabilized cells. Furthermore, glycosylation sites were identified by mutating each of the 5 potential N‐linked glycosylation sites, followed by Western blotting. Based on our analyses, combined with computer‐derived models, we conclude that mouse Zip14 has 7 transmembrane domains, an intracellular histidine‐rich region and c‐terminus, and an extracellular N‐terminus that is glycosylated at asparagines 52, 75, 85 and 100. The empirically determined structure of Zip14 will help to guide structure‐function analyses of this zinc/iron transporter.Grant Funding Source: national institute of health