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Mutagenesis disrupts posttranslational processing of the Na,K-ATPase catalytic subunit
S A Petrosian1, D L Carr, G Guerrero
1Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Archives of Biochemistry and Biophysics
|September 15, 1998
Summary
The Na,K-ATPase alpha 1 isoform undergoes N-terminal cleavage. Tagging the N-terminus prevents this processing, but downstream deletions do not, indicating specific structural requirements for cleavage.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The catalytic alpha 1 isoform of Na,K-ATPase undergoes enzymatic cleavage of its initial 5 amino acids during or after translation.
- Understanding the structural requirements for this post-translational modification is crucial for elucidating enzyme function.
Purpose of the Study:
- To investigate the structural elements within the N-terminus of the Na,K-ATPase alpha 1 isoform that are essential for its proteolytic processing.
- To determine the impact of N-terminal modifications on the enzyme's kinetic properties, particularly its response to potassium.
Main Methods:
- Construction and expression of N-terminal epitope-tagged (c-myc) and deletion mutants of the Na,K-ATPase alpha 1 isoform in monkey kidney cells.
- Immunoblot analysis using antibodies specific to the N-terminus and the epitope tag to assess proteolytic processing.
- Enzyme activity assays to evaluate the kinetic response of wild-type and mutant enzymes to varying potassium concentrations.
Main Results:
- An epitope tag at the N-terminus prevented the normal cleavage of the alpha 1 isoform, as evidenced by antibody binding to the nascent protein.
- Deletion mutants lacking residues 10-24 or 10-31 were cleaved normally, suggesting that amino acids beyond the first 9 are not required for proteolysis.
- While N-terminal processing was altered in some mutants, their sensitivity to potassium activation was largely similar to the wild-type enzyme, except for a mutant lacking the first 40 residues.
Conclusions:
- The N-terminal processing of the Na,K-ATPase alpha 1 isoform is dependent on specific structural features within the first 9 amino acids.
- Preventing N-terminal cleavage through N-terminal tagging does not significantly alter the enzyme's kinetic response to potassium, suggesting this processing step may not be critical for this specific kinetic parameter.
- A mutant lacking the first 40 residues showed altered potassium activation, hinting at a role for this region in modulating enzyme kinetics.