Synthesis and translocation of Na⁺-K⁺-ATPase α-and β-subunits to plasma membrane in MDCK cells

Synthesis and translocation of Na⁺-K⁺-ATPase α-catalytic and β- glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into β-subunit was equal to that incorporated into α-subunit after 15 min of labeling. Because the ratio of total methionines in α- vs. β-subunit is ~5:1, these results suggest that β-subunit is synthesized in molar excess over α-subunit. Half of the newly synthesized β-subunit, likely unassembled units, were degraded by 60 min after labeling, while α-subunits were stable through 120 min after synthesis, suggesting α may be limiting for αβ-assembly. By 120 min the ratio of counts incorporated into α vs. β approached 5, which is predicted by a 1:1 ratio of α to β. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature β (β(i)) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature β (β(m)) became detectable after 30 min, and conversion of β(i) to β(m) was 90% complete at 120 min. A peak of labeled α-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled β(m)-subunit in this sample, suggesting movement as αβ-heterodimers.