Ahern Meghan E, Bafaro Elizabeth M, Cowan Ann, Dempski Robert E
Department of Chemistry and Biochemistry , Worcester Polytechnic Institute , Worcester , Massachusetts 01609-2247 , United States.
Department of Molecular Biology and Biophysics and Center for Cell Analysis and Modeling , University of Connecticut Health Center , Farmington , Connecticut 06030 , United States.
Biochemistry. 2019 Apr 2;58(13):1705-1708. doi: 10.1021/acs.biochem.9b00131. Epub 2019 Mar 20.
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 (ε) 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.
人类（h）锌转运蛋白ZIP4在质膜上表达，其功能是提高胞质锌水平。hZIP4的突变会导致肠病性肢端皮炎。在包括胰腺癌和前列腺癌在内的实体组织癌症中，也已表明hZIP4的调节功能存在异常。尽管细胞外结构域的结构研究和膜结构域的计算模型表明hZIP4以二聚体形式存在，但hZIP4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了在HEK293细胞中表达的hZIP4的寡聚状态。hZIP4用增强绿色荧光蛋白（eGFP）标记，通过将单体和串联eGFP构建体的亮度值（ε）与hZIP4/eGFP的亮度值进行比较，我们发现hZIP4是二聚体。确定hZIP4是二聚体是了解该蛋白质功能和加工过程的重要一步，这可以为深入了解受hZIP4影响的疾病如何发生以及如何进行治疗提供更多线索。
