Mutagenesis disrupts posttranslational processing of the Na,K-ATPase catalytic subunit

Susan A. Petrosian, Deborah L. Carr, Georgina Guerrero, Thomas A. Pressley

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

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-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 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-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 α1 isoform was prevented by mutation, yet that processing had little influence on the kinetic parameter most likely to be influenced by such changes.

Original languageEnglish
Pages (from-to)249-258
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume357
Issue number2
DOIs
StatePublished - Sep 15 1998

Keywords

  • Immunoblotting
  • Ing
  • Ion deocclusion
  • Na,K-ATPase
  • Posttranslational process
  • Site-directed mutagenesis

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