Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.