{'fraction_non_alphanumeric': 0.02660016625103907, 'fraction_numerical': 0.029925187032418952, 'mean_word_length': 5.174358974358975, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification of residues required for iron transport activity of the transmembrane protein Zip14

Zip14 is a member of the ZIP (Zrt‐, Irt‐like Protein) family of metal‐ion transporters. Recent studies have shown that Zip14 can mediate the uptake of non‐transferrin‐bound iron (NTBI) into cells. In the present study, we used site‐directed mutagenesis to identify amino acid residues essential for iron transport activity of mouse Zip14. As our initial targets for point mutation, we chose the most highly conserved Asp, Glu, and His residues located in or near putative transmembrane domains IV and V, which are believed to form a metal‐ion translocation pore. NTBI uptake in the form of ferric (59Fe) citrate was measured in HEK293T cells transiently transfected with wild type or mutant versions of Zip14. We found that Asp348Ala and Glu378Ala abolished iron transport activity of Zip14. In addition, Asp381Ala resulted in 35% lower iron uptake, whereas His344Ala and His377Ala had no effect. Western blot analysis revealed similar expression levels of all mutants. These findings suggest that negatively charged Asp and Glu residues are critical for iron transport activity of Zip14. Funded by NIH.