Assembly with the Na,K-ATPase α1 Subunit Is Required for Export of β1 and β2 Subunits from the Endoplasmic Reticulum

The sodium pump is expressed in all animal tissues where it establishes concentration gradients for Na+ and K+ by pumping three Na+ from the cytoplasm in exchange for two extracellular K+. The ion gradients generated by the Na,K-ATPase are used in many essential cellular processes, such as osmoregulation, generation of plasma membrane potential and maintenance of intracellular pH and Ca2+ concentration, vectorial transport of many solutes, and excitability in muscle fibers and neurons. Further, the Na,K-ATPase serves as a functional signal transducer (1, 2). The pump is a heterodimer, consisting of an α subunit that is responsible for ion transport and a β subunit that is implicated in maturation and membrane targeting of the enzyme. There are four isoforms of the Na,K-ATPase α subunit (α1, α2, α3, and α4) and three isoforms of the Na,K-ATPase β subunit (β1, β2, and β3) (3, 4). Additional tissue-specific regulatory subunits of the Na,K-ATPase that belong to the FXYD family of small membrane proteins have also been identified (5). Most data have shown that α and β subunits are always present in equimolar amounts in the isolated Na,K-ATPase (6–9). Recent data claiming that the α–β ratio can be less (10) or more (11) than one are difficult to reconcile with the 3D high-resolution structure of the enzyme that shows two specific regions of interaction between the α and β subunits at a 1:1 stoichiometry (12, 13). Given these sites of interaction, stable recruitment of additional β subunits to the α subunit or vice versa is unlikely. ER assembly of the α with the β subunit is necessary for export of the α subunit of the Na,K-ATPase from the ER to the Golgi and formation of functional enzyme (14), suggesting that the β subunit acts as a molecular chaperone that facilitates maturation of the enzyme (15, 16). In addition, both β1 and β2 subunits have important non-enzymatic roles such as formation and maintenance of intercellular junctions and regulation of cell migration (16–23). It is not known whether the β subunits perform these functions only as components of the α–β complexes or perhaps as individual plasma membrane proteins. Results of studies addressing the question as to whether unassembled β subunits are present in the plasma membrane are ambiguous. A consecutive series of immunoprecipitation using the antibody against the α1 subunit resulted in depletion of MDCK cell lysates of the mature but not immature β1 subunits, indicating that the mature β1 subunits are all bound to the α1 subunit (10). Along with these data, overexpressed Xenopus oocyte Na,K-ATPase β1 and β3 subunits are retained in the ER in the absence of the α subunit in Xenopus oocytes (24–26). By contrast, the Torpedo californica β1 subunit expressed in Xenopus oocytes without the α subunit shows the mature N-glycosylation pattern, implying that it is able to traffic from the ER to the Golgi (27). Whether the unassembled Na, K-ATPase β2 subunit isoform is able to reach the plasma membrane is unknown. However, the H,K-ATPase β subunit that has more homology with the Na,K-ATPase β2 subunit than the other Na,K-ATPase β subunit isoforms is detected in the plasma membrane when expressed without the H,K-ATPase α subunit in various cell types (28–32). In this work, analysis of the nature of the N-linked glycans of endogenous and expressed β subunits of the Na,K-ATPase enables determination of their localization either in the ER or in the post-ER compartments. The presence of high-mannose-type N-glycans shows ER location of the β subunits, while the presence of hybrid- or complex-type N-glycans in the β subunits that can only be generated by Golgi-located glycosyltransferases shows successful export of the β subunits from the ER. Renal MDCK cells were used as an expression system for YFP-linked β1 and β2 subunit isoforms and β subunit mutants in order to determine whether assembly with the α subunit is essential for ER exit of either β1 or β2 subunits. Confocal microscopy, immunohistochemistry, immunoprecipitation, surface-selective biotinylation, and Western blot analysis were employed to evaluate trafficking and molecular interactions of endogenous and exogenous Na,K-ATPase subunits. The results show that (i) association with the α subunit at 1:1 stoichiometry is a prerequisite for the export of both the β1 and β2 subunits from the ER and (ii) the amount of the α subunit in the ER is a limiting factor for α–β assembly.