- Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2+ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn2+ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp599 and His451, with alanine was sufficient to block Zn2+ transport. These findings indicate, for the first time, that Zn2+ transport mediated by a mammalian ZnT is catalyzed by H+/Zn2+ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.