ROLE OF GLYCOSYLATION AND DISULFIDE BOND FORMATION IN THE BETA-SUBUNIT IN THE FOLDING AND FUNCTIONAL EXPRESSION OF NA,K-ATPASE

Initial folding is a prerequisite for subunit assembly in oligomeric p roteins, 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 struct ural and functional maturation of the catalytic Na,K-ATPase alpha subu nit. Cysteine or asparagine residues implicated in disulfide bond form ation or N-glycosylation, respectively, in the Xenopus beta 1 subunit were eliminated by site-directed mutagenesis, and the assembly efficie ncy of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes, Our results show that lac k of each one of the two most C-terminal disulfide bonds indeed permit s short term but completely abolishes long term assembly of the beta s ubunit, On tile other hand, lack of the most N-terminal disulfide bond s allows the expression of a small number of functional Na+,K+ pumps a t the cell surface, Elimination of all three but not of one or two gly cosylation sites produces beta subunits that remain stably expressed i n the endoplasmic reticulum, in association with binding protein but n ot as irreversible aggregates, The assembly efficiency of nonglycosyla ted beta subunits is decreased but a reduced number of functional Na+, K+ pumps is expressed at the cell surface, The lack of sugars does nob influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Th us, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial fol ding of oligomeric protein subunits, Moreover, the results suggest tha t an endoplasmic reticulum degradation pathway exists, which is glycos ylation-dependent.