Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates.
Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein consisting of an alpha-catalytic and a beta-glycoprotein subunit. Because these two subunits are derived from two separate genes, they may not be synthesized with stoichiometric equivalence. The aim of this study was to estimate relative rates of synthesis and degradation of nascent and mature Na(+)-K(+)-ATPase alpha- and beta-subunits to determine whether either of the nascent subunits accumulates in excess and, if so, the fate of the excess subunits. We studied a pig kidney cell line (LLC-PK1/Cl4) that expresses only alpha 1- and beta 1-subunits. Relative synthesis and degradation rates of nascent subunits were first estimated by pulsing cells for 10 min with [35S]methionine followed by chase periods of up to 120 min and by immunoprecipitation. We found that directly after labeling, beta-subunits were present in threefold excess over alpha-subunits and that nearly 50% of this beta-subunit pool was degraded by 60 min. Nascent alpha-subunits were not degraded during the chase period. In a second strategy to examine relative rates of nascent alpha- vs. beta-subunit accumulation, cells were pulsed for 5-60 min and immunoprecipitated directly (without chase). The rate of accumulation of labeled alpha was greater than that of beta between 5 and 60 min, consistent with the results of the pulse-chase strategy, demonstrating a significant component of degradation of beta during this period. Despite the very different degradation rates of newly synthesized alpha- vs. beta-subunits, the degradation rates of alpha- and beta-subunits beyond 4 h after synthesis were indistinguishable (t0.5 = 10-12 h).(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
L
L
Lescale-Matys
D S
DS
Putnam
A A
AA
McDonough
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007687:Kidney Tubules, Proximal / Q000166:cytology; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011233:Precipitin Tests; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D013460:Sulfur Isotopes; D013552:Swine; D013997:Time Factors
D046911:Macromolecular Substances; D013460:Sulfur Isotopes; D008715:Methionine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium