N-Glycan-Dependent Quality Control of the Na,K-ATPase β2 Subunit

The endoplasmic reticulum (ER) lumen contains high concentrations of molecular chaperones and folding enzymes that allow newly synthesized proteins to acquire their native conformation. Nevertheless, a large fraction of ER-synthesized proteins fail to fold properly on the first attempt. Cells overcome this problem by employing an ER-resident protein quality control system that includes a variety of proteins and operates on levels of protein synthesis, folding, and assembly. By preventing the premature exit of non-native conformers and incompletely assembled proteins from the ER, the quality control system extends exposure of substrates to the folding machinery in the ER lumen and hence improves the chances of correct maturation. If proper folding or full assembly is not achieved even after repeated attempts, proteins are then moved from the ER to the cytosol and destroyed via the ER-associated degradation (ERAD)1 pathway (1–3). Hence, the ER quality control system ensures that only properly folded and assembled proteins are exported to the Golgi to begin their travel to their final destinations. The importance of the ER quality control system in maintaining the fidelity of cellular functions is evidenced by many diseases, including cystic fibrosis, antitrypsin deficiency, Alzheimer’s disease, and Huntington’s disease, that are linked to defects in this system (3, 4). There are two major groups of ER chaperones, heat shock protein homologues (e.g., BiP, Hsp40, and Hsp90) and lectins (e.g., calnexin, calreticulin, and EDEM-1) (5–9). Correct folding of nonglycosylated proteins is assisted by the ER heat shock protein homologues that, in conjunction with luminal thiol oxidoreductases, mediate disulfide bond formation. These non-lectin chaperones also mediate quality control of nonglycosylated proteins by retarding the premature exit of incompletely folded or unassembled proteins and by targeting of misfolded proteins to ERAD (5). In addition to these non-lectin chaperones, N-glycosylated proteins, which represent the majority of ER-synthesized proteins, also utilize the lectin chaperone system to help mediate folding and quality control (5–9). Lectin chaperones use the N-glycans of glycoproteins as molecular recognition sites to guide the newly synthesized proteins through highly ordered steps of maturation and quality control. Every lectin-assisted maturation step is coupled with a particular modification of the N-glycan structure that is catalyzed by ER-resident enzymes. In eukaryotic cells, glycoproteins acquire the N-linked glycans during the process of translocation and elongation of the polypeptide chain. First, a 14-saccharide core is transferred from the dolichol phosphate precursor to the asparagine (Asn) residue within the N-glycosylation site, a consensus Asn-X-Thr/Ser (X is any amino acid residue except Pro) sequence of a nascent protein. Immediately after covalent linkage of this core oligosaccharide, its terminal glucose residues are sequentially trimmed by ER glucosidases. Removal of two of the three glucose residues enables N-glycan binding to a membrane-bound lectin, calnexin, or its soluble homologue, calreticulin. Binding to calnexin (calreticulin) can occur cotranslationally while the protein is still associated with the translocon. Calnexin forms a complex with the oxidoreductase, ERp57, which catalyzes formation of disulfide bonds in the protein (5, 6). When the newly synthesized protein is released from calnexin, it meets one of three possible fates. If it is folded correctly, is exported to the Golgi, or assembles with its partner subunits, if any, and then is exported. If the protein is terminally misfolded, it is often recognized by EDEMS and/or by a non-lectin chaperone, BiP, that facilitates translocation of the protein to the cytoplasm and proteasome-mediated degradation (5). If the protein is slightly misfolded, it is reglucosylated by UGGT1 and binds again to calnexin for refolding. This cycle can be repeated several times (5, 6). Thus,UGGT1 acts as a conformation sensor. Proteomic analysis of calnexin-interacting proteins in embryonic fibroblasts from UGGT1-null mice shows that many proteins do not require UGGT1 for maturation and complete their folding program in one calnexin binding event (10). Other proteins exhibit accelerated dissociation from calnexin and impaired folding in UGGT1-null fibroblasts, indicating that these proteins normally undergo several cycles of interaction with calnexin, mediated by UGGT1(10). Some of these proteins must be essential for mouse viability since disruption of the UGGT1 gene results in embryonic lethality (11). Here, we present data showing that one of these essential proteins that requires post-translational calnexin binding for proper folding is the β2 subunit of the Na,K-ATPase. The Na,K-ATPase is a vital transport enzyme expressed in all animal tissues where it generates ion gradients responsible for membrane potential and solute symport or antiport, as well as mediating intercellular junctions and signal transduction (12–18). The Na,K-ATPase consists of an α subunit that is responsible for catalysis of ion transport and an N-glycosylated 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) (19, 20). Assembly of the α and β subunits that occurs in the ER soon after their biosynthesis is essential for formation of the functional enzyme (21) and for the export of both subunits from the ER to the Golgi (22–25). Expression in Xenopus oocytes showed that α1, β1, and β3 subunits of the Na,K-ATPase interact with BiP and this interaction is important for retention of unassembled α1, β1, and β3 subunits in the ER (26), implying the involvement of this non-lectin chaperone system in the ER quality control of these particular isoforms of Na,K-ATPase. The presence of three, eight, and two N-glycans on the Na,K-ATPase β1, β2, and β3 subunits, respectively, also suggests the possible involvement of an ER lectin chaperone system in the folding of these subunits, their assembly with the α subunit, and quality control of the α–β complex. However, prevention of N-glycosylation of the β1 subunit by tunicamycin or by mutations of all three N-glycosylation sites only slightly impairs assembly of the α1–β1 complex (26, 27), trafficking of the pump to the plasma membrane (16, 28), or Na,K-ATPase activity (26, 28–30). Similarly, prevention of N-glycosylation of the β3 subunit by tunicamycin or by mutations of both N-glycosylation sites does not impair trafficking of this subunit to the plasma membrane (31). These results suggest that N-glycans and hence glycan–lectin interactions are not critical for folding of the β1 and β3 subunits. On the other hand, native N-glycosylation has been shown to be essential for normal trafficking of the homologous H,K-ATPase β subunit from the ER (32). The Na,K-ATPase β2 subunit is more homologous to the H,K-ATPase β subunit than to the Na,K-ATPase β1 and β3 subunits. Similar to the H,K-ATPase β subunit, the Na,K-ATPase β2 subunit has multiple N-glycosylation sites. However, the contribution of glycan–lectin interactions to folding, maturation, and quality control of the Na,K-ATPase β2 subunit has not been investigated. In this study, canine renal MDCK cells were used as the expression system for YFP-linked β1 and β2 subunit isoforms of the Na,K-ATPase and their N-glycosylation-deficient mutants to test whether the ER lectin quality control system is involved in maturation of the proteins. The results indicate that at least two of the eight N-glycans of the β2 subunit as well as the hydrophobic amino acid cluster downstream to the seventh N-glycan are critical both for calnexin binding and for normal maturation and ER exit of the subunit, suggesting the involvement of UGGT1-induced post-translational calnexin binding in folding and quality control of this subunit. In contrast, maturation of the β1 subunit does not require post-translational calnexin binding.