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

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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.