Biosynthesis and turnover of Na,K-ATPase in cultured rat hepatoma cells

Na,K-ATPase is an integral protein of the plasma membrane of cells in all vertebrates. The details of its biosynthesis and, to a lesser degree, its degradation (turnover) remain unclear after more than 25 years of investigation on this enzyme. I have used antibodies prepared to the catalytic (α) subunit of rat kidney Na,K-ATPase to investigate the biosynthesis and turnover of the α subunit in HTC cells, a cultured rat hepatoma line.

Na,K-ATPase was isolated in a form approximately 50% pure, as indicated by its activity and SDS-PAGE profile, by the detergent extraction of rat kidney microsomes, followed by ultracentrifugation through 50% glycerol. The pellet of purified enzyme was denatured in SDS and the α subunit collected by gel filtration. Anti-α IgG, isolated from the serum of rabbits immunized with this preparation, precipitated a major protein of 93,000 daltons from HTC cells labeled with [³⁵S]methionine. The evolutionarily conserved structure of this subunit was confirmed by the cross-reactivity of anti-rat α subunit with the enzyme from hamster, dog and human cells.

No ³⁵S-labeled glycoprotein (β) subunit was detected in the immunoprecipitates from cells lysed under a variety of nondenaturing conditions, or when chemical cross-linking preceded the detergent solubilization. A protein corresponding in size to the β subunit was observed in immunoprecipitates from cells labeled with either [³H]leucine or a mixture of [³H]fucose and [³H]mannose, suggesting that the β peptide of Na,K-ATPase in HTC cells contains little or no methionine.

The α subunit was turned over, when corrected for cell generation time (T_G), with a half-life of .45 T_G and a turnover coefficient of 1.5 T_G^-1. A 5 hour lag period was observed prior to the onset of turnover which was interpreted as the transit time of the newly-synthesized α proteins from intracellular membrane compartments to the plasma membrane. This is inconsistent with theories that α is translated as a soluble protein and inserts directly into the plasma membrane. Furthermore, immunoprecipitable (93,000 daltons) α subunit was found, after brief pulses of [³⁵S]methionine, only in cell membranes, not in the cytosol. I conclude that the α subunit of Na,K-ATPase follows the classic pathway for membrane protein biogenesis, namely translation on bound ribosomes and transport through intracellular (Golgi apparatus) membranes to the plasma membrane.