Mutagenesis Disrupts Posttranslational Processing of the Na,K-ATPase Catalytic Subunit

The first 5 amino acids of the catalytic α1 isoform from Na,K-ATPase are cleaved enzymatically during or after translation. To evaluate the structural requirements for that cleavage, we constructed amino-terminal mutants of α1 in which an epitope tag from the c-myconcogene product was added. Immunoblots of isolated membranes from transfected monkey kidney cells revealed binding of an antibody specific for the first 9 residues of the α1 nascent protein. Because this antibody does not recognize the shorter sequence corresponding to the processed polypeptide, these results indicate that the epitope tag prevented normal processing, a conclusion confirmed by the observed binding of an anti-mycantibody. In contrast, membranes from cells expressing deletion mutants that lack residues 10–24 and 10–31 of the nascent chain failed to bind the amino-terminal-directed antibody, suggesting that the mutants were cleaved normally and that amino acids downstream of the first 9 are not required for proteolysis. Amino-terminal mutants produced in other laboratories have shown an anomalous stimulation of ATPase activity by K+when measured in low ATP concentrations. Themyc-tagged and downstream deletion mutants were sensitive to K+in the range from 0.05 to 5 mM, similar to wild-type enzyme, despite the differences in posttranslational processing. A mutant missing the first 40 residues of the nascent chain, however, displayed an activation by K+. These results suggest that amino-terminal processing of the α1 isoform was prevented by mutation, yet that processing had little influence on the kinetic parameter most likely to be influenced by such changes.