TY - JOUR
T1 - Identification of the Zn2+ binding site and mode of operation of a mammalian Zn2+ transporter
AU - Ohana, Ehud
AU - Hoch, Eitan
AU - Keasar, Chen
AU - Kambe, Taiho
AU - Yifrach, Ofer
AU - Hershfinkel, Michal
AU - Sekler, Israel
PY - 2009/6/26
Y1 - 2009/6/26
N2 - 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 a imedate lucidating 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.
AB - 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 a imedate lucidating 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.
UR - http://www.scopus.com/inward/record.url?scp=67650540770&partnerID=8YFLogxK
U2 - 10.1074/jbc.M109.007203
DO - 10.1074/jbc.M109.007203
M3 - Article
C2 - 19366695
AN - SCOPUS:67650540770
SN - 0021-9258
VL - 284
SP - 17677
EP - 17686
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -