Mutagenesis disrupts posttranslational processing of the Na,K-ATPase catalytic subunit.
The first 5 amino acids of the catalytic alpha 1 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 alpha 1 in which an epitope tag from the c-myc oncogene 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 alpha 1 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-myc antibody. 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. The myc-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 alpha 1 isoform was prevented by mutation, yet that processing had little influence on the kinetic parameter most likely to be influenced by such changes.
Petrosian
S A
SA
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Carr
D L
DL
Guerrero
G
G
Pressley
T A
TA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D000939:Epitopes / Q000235:genetics; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000939:Epitopes; D007527:Isoenzymes; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D000254:Sodium-Potassium-Exchanging ATPase