At. Beggah et al., ROLE OF GLYCOSYLATION AND DISULFIDE BOND FORMATION IN THE BETA-SUBUNIT IN THE FOLDING AND FUNCTIONAL EXPRESSION OF NA,K-ATPASE, The Journal of biological chemistry, 272(15), 1997, pp. 10318-10326
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.