Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability.
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.
Shanbaky
N M
NM
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77030.
Pressley
T A
TA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002470:Cell Survival; D002522:Chlorocebus aethiops; D006706:Homeostasis; D007158:Immunologic Techniques; D007477:Ions; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D011499:Protein Processing, Post-Translational; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000502:physiology*
D007477:Ions; D015345:Oligonucleotide Probes; D000254:Sodium-Potassium-Exchanging ATPase