SYNTHESIS AND TRANSLOCATION OF NA+-K+-ATPASE ALPHA-SUBUNIT AND BETA-SUBUNIT TO PLASMA-MEMBRANE IN MDCK CELLS

Synthesis and translocation of Na+-K+-ATPase alpha-catalytic and beta-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 beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha-beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta-approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature-beta (beta(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-beta (beta(m)) became detectable after 30 min, and conversion of beta(i) to beta(m) was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta(m)-subunit in this sample, suggesting movement as alpha-beta-heterodimers.