Analysis of the Transport and Localization of Na, K-ATPase

Na，K-ATP酶由α和β亚基组成，化学计量比为1：1。α亚基含有哇巴因的结合位点以及催化位点。β亚基是一种功能未知的糖基化膜蛋白，近年来的研究表明β亚基在酶的成熟和/或转运过程中起着重要的作用。众所周知，该酶仅分布在基底外侧质膜上。酶在质膜上的定位应该存在一些机制。在此基础上，我们试图阐明该酶的转运和定位机制，并获得了以下结果。(1)我们在HeLa细胞中表达了大鼠脑Na，K-ATP酶β亚基N端的一半，观察到表达的片段蛋白定位于内质网，并抑制内源性α和β亚基的组装。这些结果表明，β亚基的N-末端部分干扰了ER中发生的内源性复合物的组装。(2)用免疫电镜定量分析了该酶在大鼠耳蜗导管、睫状体上皮细胞和视网膜色素上皮细胞中的分布。(3)我们分离并区分了两种类型的网格蛋白包覆的囊泡，分别来自神经元和神经胶质细胞。神经元细胞网格蛋白包被的囊泡主要含有神经元细胞特有的α +亚基。

Na, K-ATPase consists of the alpha and the beta subunit, with the stoichiometry of one to one. The alpha subunit contains the binding site for ouabain as well as the catalytic site. The beta subunit is a glycosylated membrane protein with unknown function.Recently, several studies have suggested the functional roles of the beta subunit in the maturation and/or transport processes of the enzyme. It is well known that the enzyme exclusively distributes on the basolateral plasma membrane. Some mechanisms should exist for the localization of the enzyme on the plasma membrane. Furthermore, it comes to be evident that the enzyme is endocytosed from the plasma membrane and affects the internal pH of the endosome.On the bases of these findings, we tried to elucidate the mechanisms of the transport and localization of the enzyme, and obtained the following results.(1) We expressed the N-terminal half of the beta sububit of rat brain Na, K-ATPase in HeLa cells, and observed that the expressed fragment protein was localized in the ER and inhibited the assembly of the endogenous alpha and beta subunits. These results suggests that the N-terminal portion of the beta subunit interferes with the assembly of the endogenous complex which takes place in the ER.(2) We quantitatively analyzed the distribution of the enzyme by using immunoelectron microscpy in rat cochlear duct, ciliary epithelial cells and retinal pigment epithelial cells.(3) we isolated and discriminated two types of clathrin-coatrd vesicles originated from neuronal and glial cells, respectively. The clathrin-coated vesicles from neuronal cells dominantly contained alpha+ subunit which is characteristic for neuronal cells.