Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter
Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter
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DOI：
10.1074/jbc.m109.007203
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发表时间：
2009-06-26
影响因子：
4.8
通讯作者：
Sekler, Israel
中科院分区：
文献类型：
--
作者：
Ohana, Ehud;Hoch, Eitan;Sekler, Israel
囊泡锌转运蛋白（ZnTs）在调节各种细胞区室中的Zn 2+稳态中起关键作用，并且与从阿尔茨海默病到糖尿病的主要疾病有关。尽管它们的重要性，ZnTs的细胞内定位提出了一个重大的挑战，建立它们的功能和身份的离子结合位点的机制。在这里，我们将基于荧光的功能分析和结构建模结合起来，旨在阐明这些功能方面。联合收割机。ZnT 5的表达之后，从细胞质中加速去除Zn 2+，并增加囊泡隔离。此外，这种锌运输的活动耦合到trans-Golgi网络的碱化。最后，基于细菌金属转运蛋白YiiP的X射线结构，ZnT 5的结构建模确定了四个可能形成ZnT 5上锌结合位点的残基。与该模型一致，用丙氨酸替换这些残基Asp（599）和His（451）足以阻断Zn 2+转运。这些研究结果表明，第一次，由哺乳动物ZnT介导的Zn 2+转运是由H+/Zn 2+交换催化的，并确定了锌转运所必需的ZnT蛋白的锌结合位点。
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, Asp(599) and His(451), 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.