Quantifying the Oligomeric State of hZIP4 on the Surface of Cells
Quantifying the Oligomeric State of hZIP4 on the Surface of Cells
复制标题
DOI：
10.1021/acs.biochem.9b00131
复制
发表时间：
2019-04-02
期刊：
影响因子：
2.9
通讯作者：
Dempski, Robert E.
中科院分区：
文献类型：
--
作者：
Ahern, Meghan E.;Bafaro, Elizabeth M.;Dempski, Robert E.
人（h）锌转运蛋白ZIP 4在质膜上表达，并起到增加细胞溶质锌水平的作用。hZIP 4的突变导致肠病性肢端皮炎。hZIP 4的调节功能障碍也已在实体组织癌（包括胰腺癌和前列腺癌）中被指出。尽管细胞外结构域的结构研究和膜结构域的计算建模表明hZIP 4作为二聚体存在，但hZIP 4在哺乳动物细胞质膜中的寡聚化状态尚未在体内直接定量。在此，使用荧光相关光谱法定量HEK 293细胞中表达的hZIP 4的寡聚状态。hZIP 4用eGFP标记，通过将单体和串联eGFP构建体的亮度值（epsilon）与hZIP 4/eGFP的亮度值（epsilon）进行比较，我们表明hZIP 4是二聚体。确定hZIP 4是一种二聚体是了解蛋白质功能和加工的重要一步，这可以更深入地了解受hZIP 4影响的疾病如何发生和管理。
The human (h) zinc transporter ZIP4 is expressed on the plasma membrane and functions to increase cytosolic zinc levels. Mutations in hZIP4 cause the disease acrodermatitis enteropathica. Dysfunction in the regulation of hZIP4 has also been indicated in solid tissue cancers, including pancreatic and prostate cancer. Although structural studies of the extracellular domain and computational modeling of the membrane domain suggest hZIP4 exists as a dimer, the oligomerization status of hZIP4 in the plasma membrane of mammalian cells has not been directly quantified in vivo. Here, the oligomeric state of hZIP4 expressed in HEK293 cells was quantified using fluorescence correlation spectroscopy. hZIP4 was tagged with eGFP, and by comparing brightness values (epsilon) of monomer and tandem eGFP constructs to that of an hZIP4/eGFP, we show that hZIP4 is a dimer. Determining that hZIP4 is a dimer is an important step toward understanding the function and processing of the protein, which can provide more insight into how diseases affected by hZIP4 occur and can be managed.