Abstract. Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of a and [3 subunits of Na,K-ATPase. Xenopus oocytes injected with c~ and/or 13 cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or a-[3 complexes in the ER. Only ot subunits that are associated with 13 subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with 13 subunits is necessary and probably sufficient for the catalytic et subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand-dependent conformations and to hydrolyze ATP in an Na ÷-and K÷-dependent, ouabaininhibitable fashion. Not only the a but also the [3 subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NHz-terminally modified 13 subunits revealed a K+-dependent interaction of the cytoplasmic NH2 terminus of the [3 subunit with the subunit, which is likely to be involved in the modulation of the K÷-activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.M ANY plasma membrane and secretory proteins are oligomeric. The subunits of these proteins are synthesized independenly of each other and are inserted into the ER membrane or the lumen during their synthesis. They are subjected to cotranslational modifications, fold, and then oligomerize. Once correctly assembled, the proteins leave the ER and are targeted to their final cellular site of action. Misfolded or unassembled subunits are retained in the ER and are degraded (15). Although it is increasingly clear that the oligomeric state controls the proper function of the protein, little is known about the nature of interactions that are involved in subunit assembly and about the structural and functional consequences of subunit oligomerization. In this study, we investigate several aspects of this question by analyzing the oligomerization and the functional maturation of Na,KATPase.The ubiquitous Na,K-ATPase is responsible for the maintenance of the sodium and potassium gradients between the intra-and extracellular milieu. The enzyme is composed of two heterologous subunits. The ct subunit is a

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

Initial folding is a prerequisite for subunit assembly in oligomeric proteins. In this study, we have compared the role of co-translational modifications in the acquisition of an assembly-competent conformation of the beta subunit, the assembly of which is required for the structural and functional maturation of the catalytic Na,K-ATPase alpha subunit. Cysteine or asparagine residues implicated in disulfide bond formation or N-glycosylation, respectively, in the Xenopus beta1 subunit were eliminated by site-directed mutagenesis, and the assembly efficiency of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes. Our results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term assembly of the beta subunit. On the other hand, lack of the most N-terminal disulfide bonds allows the expression of a small number of functional Na+,K+ pumps at the cell surface. Elimination of all three but not of one or two glycosylation sites produces beta subunits that remain stably expressed in the endoplasmic reticulum, in association with binding protein but not as irreversible aggregates. The assembly efficiency of nonglycosylated beta subunits is decreased but a reduced number of functional Na+,K+ pumps is expressed at the cell surface. The lack of sugars does not influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Thus, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial folding of oligomeric protein subunits. Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent.

The active Na+/K' pump is composed of an a and a (3 (1 subunit and is preferentially expressed in the nervous system (4). This isoform is nearly identical with the adhesion molecule on glia cloned from mouse brain (5). The (32 isoform has been copurified with the a2 subunit of rat brain Na+/K+-ATPase (6). More recently, Good et al. (7)