Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits 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.
Mircheff
A K
AK
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Bowen
J W
JW
Yiu
S C
SC
McDonough
A A
AA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D011233:Precipitin Tests; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014178:Translocation, Genetic
D000254:Sodium-Potassium-Exchanging ATPase