AJP - Cell Physiology, Vol 267, Issue 2 C590-C597, Copyright © 1994 by American Physiological Society

ARTICLES

Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability

N. M. Shanbaky and T. A. Pressley
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77030.

The amino terminus of the catalytic alpha-subunit from Na(+)-K(+)-adenosinetriphosphatase (ATPase) is not conserved among the various isoforms and species sequenced to date, yet it always includes a lysine-rich motif. To investigate the functional role played by this highly charged region, we altered the amino terminus of rat alpha 1 and evaluated the ability of the mutant to sustain cell viability. Nucleotide sequence encoding a 10-amino acid epitope from the human c-myc-oncogene product was substituted for the wild-type sequence encoding the first 31 amino acids of alpha 1. The chimeric cDNA containing the myc substitution was then introduced into ouabain-sensitive monkey kidney cells. Selection in ouabain produced viable colonies, suggesting that the introduced mutant was functional and conferred the ouabain-resistant phenotype of rats, despite the removal of the highly charged region from its amino terminus. Subsequent enzymatic analysis confirmed the presence of low-affinity binding sites for ouabain in the recipient colonies, and immunoblotting revealed the myc epitope on the expressed polypeptides in a membrane fraction. These results suggest that the first 31 amino acids are not required for function of alpha 1 and that the posttranslational cleavage associated with the amino terminus is unnecessary.

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