The transmembrane domains mediate oligomerization of the human ZIP4 transporter in vivo.
The transmembrane domains mediate oligomerization of the human ZIP4 transporter in vivo.
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DOI：
10.1038/s41598-022-24782-6
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发表时间：
2022-12-06
影响因子：
4.6
通讯作者：
Dempski RE
中科院分区：
文献类型：
--
作者：
Liu Y;Bafaro EM;Cowan AE;Dempski RE
人(h) ZIP4是一种质膜转运蛋白，其功能是增加胞质锌水平。hZIP4编码8个跨膜结构域和一个大的细胞外结构域（ECD）。当细胞缺锌时，这种ECD从全息转运体上断裂。同时，ECD突变可导致缺锌性肠病性肢端皮炎。先前的研究表明，hZIP4的ECD由两个结构独立的子结构域组成，其中ECD单体单元之间的接触集中在PAL基序上。这些结果导致ZIP4-ECD对全息转运体的二聚化至关重要的假设。为了验证这一假设，我们使用荧光相关光谱（FCS）来量化全长hZIP4和缺乏ECD结构域的hZIP4的低聚状态，每个hZIP4都标记有eGFP。实验结果表明，全长和截断的hZIP4在HEK293细胞中表达时都是二聚体。并行功能实验表明，截断和全长hZIP4/eGFP的Km和Vmax相似。确定截断的hZIP4/eGFP形成二聚体是理解hZIP4- ecd功能的关键一步，这为了解与hZIP4蛋白相关的疾病提供了更多的见解。
The human (h) ZIP4 is a plasma membrane transporter that functions to increase cytosolic zinc levels. hZIP4 encodes eight transmembrane domains and a large extracellular domain (ECD). This ECD is cleaved from the holo-transporter when cells are zinc-deficient. At the same time, mutations in the ECD can result in the zinc-deficiency disease Acrodermatitis enteropathica. Previously, it was shown that hZIP4’s ECD is comprised of two structurally independent subdomains where contacts between the ECD monomeric units are centered at the PAL motif. These results lead to the hypothesis that ZIP4-ECD is essential to the dimerization of the holo-transporter. To test this hypothesis, we used Fluorescence Correlation Spectroscopy (FCS) to quantify the oligomeric state of full-length hZIP4 and hZIP4 lacking the ECD domain, each tagged with eGFP. Inspection of our experimental results demonstrate that both the full-length and truncated hZIP4 is a dimer when expressed in HEK293 cells. Parallel functional experiments demonstrate that the Km and Vmax for truncated and full-length hZIP4/eGFP are similar. Determining that truncated hZIP4/eGFP forms a dimer is a crucial step for understanding the function of the hZIP4-ECD, which provides more insight into how the diseases related to hZIP4 protein.
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