The ␤-subunit of the Na,K-ATPase is required to deliver functional ␣␤؊heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the ␤-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the ␣-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of ␤-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by ␤-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated ␤-subunits and some disulfide bridge substitution mutants assemble with the ␣-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.

The higher order oligomeric state of the Na,K-ATPase ␣␤ heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with ␣-subunits bearing either His 6 or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase ␣␤ heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding ␣-His/␤ and ␣-FLAG/␤ Na,K-ATPases. Co-immunoprecipitation of the Histagged ␣-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,KATPase is present in cell membranes in an oligomeric state of at least (␣␤) 2 composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the ␣-subunit results in the deletion ␣-4,5-loop-less (␣-4,5LL), which associates with ␤ but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive ␣-4,5LL/␤ heterodimer is co-expressed with wild-type ␣␤, oligomers of wild-type ␣␤ and ␣-4,5LL/␤ form in the ER, but the ␣-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric ␣␤ heterodimer in native cell membranes.