A CYTOPLASMIC REGION OF THE NA,K-ATPASE ALPHA-SUBUNIT IS NECESSARY FOR SPECIFIC ALPHA/ALPHA ASSOCIATION

While most structural studies of the Na,K-ATPase support a subunit stoichiometry of one alpha subunit to one beta-subunit, the exact quaternary structure of the Na,K-ATPase and its relevance to enzyme function is the subject of much debate. Formation of a higher order enzyme complex is supported by our previous study demonstrating specific alpha/alpha interactions among the rat Na,K-ATPase isoforms (alpha 1, alpha 2, alpha 3), expressed in virally infected Sf-9 insect cells and among native alpha isoforms in rat brain (1). This detergent-resistant association was not observed in insect cells coexpressing the homologous gastric H,B-ATPase alpha-subunit, nor was it dependent on the coexpression of the beta-subunit. To delineate domains necessary for alpha/alpha assembly, a series of H,K-ATPase-Na,K-ATPase chimeras were constructed by combining the N-terminal, cytoplasmic midregion and C-terminal segments derived hom the Na,K-ATPase (N) and the H,K-ATPase (H) alpha-polypeptides (HNN, HNH, NHH, NHN, and HHN), The alpha-subunit chimeras were coexpressed with the Na,R-ATPase alpha 1-subunit in Sf-9 cells using the baculovirus expression system. Specific and detergent-stable association is observed between the Na,R-ATPase alpha-subunit and the HNN and HNH chimeras, but not with the NHH, NHN, or HHN chimeras. Consistent with the Na,K-ATPase cytoplasmic domain as being necessary for alpha/alpha interactions, the full-length alpha-subunit stably associates with an alpha N-terminal deletion mutant (Delta Gly(2)-Leu(273)), but not with an alpha cytoplasmic deletion mutant (Delta Arg(350)-Pro(785)). In addition, the naturally occurring C-terminal truncated alpha 1 isoform, alpha 1T (Delta Gly(554) to, C terminus), does not associate with the alpha 1-subunit in Sf-9 cells coexpressing both polypeptides. Thus, a cytoplasmic region in the alpha-subunit (Gly(554)-Pro(785)) is necessary for specific alpha/alpha association. The same cytoplasmic region contains a strongly hydrophobic segment that, by analogy with oligomerization of water-soluble proteins, may form the interface of the extramembranous alpha/alpha contact site.