Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase
T A Pressley1, J C Allen, C H Clarke
1Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
The American Journal of Physiology
|September 1, 1996
Summary
The amino terminus of the sodium-potassium pump alpha 1-subunit is modified in mammals but not in all expression systems. This study developed a specific antibody to track this processing, revealing differences across species and cellular environments.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Mammalian Na(+)-K(+)-ATPase alpha 1-subunit's N-terminus differs from cDNA predictions, indicating post-translational modification.
- Understanding this cleavage is crucial for evaluating protein structure and function.
Purpose of the Study:
- To develop a site-directed antibody (anti-VGR) for detecting the N-terminal sequence of nascent rat alpha 1-subunit.
- To investigate the co- or posttranslational cleavage of the alpha 1-subunit in mammalian systems and heterologous expression systems.
Main Methods:
- Site-directed antibody production (anti-VGR) specific to the first nine residues of rat alpha 1-subunit.
- Immunoblotting of in vitro translated polypeptides and total protein from rat organs.
- Analysis of alpha 1-subunit processing in heterologous systems: E. coli, insect cells, and COS-1 cells.
Main Results:
- Anti-VGR recognized the alpha 1-subunit in vitro but showed minimal binding in various rat organs, indicating extensive in vivo modification.
- Alpha 1-subunit processing differed in heterologous systems: E. coli and insect cells showed anti-VGR binding, suggesting absent or altered modification.
- COS-1 cells transfected with alpha 1 exhibited minimal anti-VGR binding, similar to mammalian systems, indicating appropriate processing.
Conclusions:
- The N-terminal structure of the Na(+)-K(+)-ATPase alpha 1-subunit varies significantly across different expression systems.
- Efficient N-terminal processing of the alpha 1-subunit is conserved within rat organs but requires specific enzymes not present in distant phyla.
Related Concept Videos
01:48ATP Synthase: Mechanism
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...
01:18ATP Synthase: Structure
ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...
01:19Translocation of Proteins into the Mitochondria
Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:37Energy to Drive Translocation
Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct...
Generally, polypeptides are unfolded by two distinct...
01:45Tail-anchoring of Proteins in the ER Membrane
Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
01:19Allosteric Proteins-ATCase
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...