Role of Glycosylation and Disulfide Bond Formation in the β Subunit in the Folding and Functional Expression of Na,K-ATPase

Abstract

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.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

“…9). Glycosylation of the ␤ subunit is thought to stabilize the interaction between ␣ and ␤ subunits in the endoplasmic reticulum, because deglycosylation reduces the yield of Na ϩ ,K ϩ -ATPase reaching the cell membrane, although the function of those pumps that are expressed is normal (53). Formally, the result in Fig.…”

Mutational Analysis of Cysteine-rich Domains of the Epithelium Sodium Channel (ENaC)

“…Among the transport proteins, the formation of disulfide bonds in the extracellular loop or N and C terminus of the protein is usually critical for proper co-translational folding of the protein and subsequent assembly and oligomerization in the ER compartment. For instance, mutation of conserved cysteines forming disulfide bonds in the ectodomain of the ␤ subunit of Na,K-ATPase prevents assembly with the ␣ subunit without changing the half-life of the ␤ protein (24). Likewise, cysteine bonds in the ectodomain of the low density lipoprotein receptor allow the folding of this protein domain (repeated many times) into an octahedral cage, binding of calcium molecules, required for apoE and apoB binding and receptor recycling (25).…”

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

“…The extent to which ␤ 1 -subunit N-linked glycans are processed, compared with glycans of known adhesion molecules, as well as the recognition that ␤ 2 is identical to the adhesion molecule of glia, suggests an additional role for ␤ 1 -subunit as an adhesion molecule (15,38). Although ␤ 1 -glycosylation is not necessary for sodium pump activity or transport to the PM, it is implicated as an important feature of sodium pump stability (2,22). When MDCK cells exogenously express the unglycosylated form of ␤ 1 -subunit, a reduction in adherens junctional resis-tance to detergent extraction has been reported (41).…”