The α and β subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Abstract

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated α and β subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus- infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase α or β subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate α and β polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase α subunit and a H,K-ATPase β subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase α subunit are fused to cells coexpressing the Na,K-ATPase β subunit and the H,K-ATPase β subunit, the Na,K-ATPase α subunit selectively assembles with the Na,K-ATPase β subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase α subunit assembles with both β subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase α and β subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.