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Structural interactions of the oligomycin sensitivity-conferring protein in the yeast ATP synthase
1Department of Biological Chemistry, The Chicago Medical School, North Chicago, Illinois 60064, USA.
Abstract:
The structure/function relationship of oligomycin sensitivity-conferring protein (OSCP), subunit 5 of the mitochondrial ATP synthase, from yeast Saccharomyces cerevisiae has been studied by a combination of genetic and biochemical methods. OSCP was studied by deletion mutagenesis of the N- and C-terminal regions by modifying the gene coding for OSCP. Two deletion mutations were made immediately downstream of the leader peptide of OSCP and five were made at the C-terminus. OSCP was functional with deletions of amino acids 3 to 17 (ND15) and of the last 8 amino acids (CD8), while deletion of amino acids 3 to 31 (ND29) and the last 9 amino acids (CD9) inactivated the ATP synthase, as determined by in vivo analysis. The deletion mutants were expressed in Escherichia coli, purified, and studied by in vitro reconstitution studies. Circular dichroism studies suggested that the mutant proteins, with the possible exception of ND29, were folded in a similar fashion as wild-type OSCP. Mutants ND15 and CD8 were able to reconstitute an oligomycin-sensitive ATPase complex, although not as well as wild-type OSCP, while ND29 and CD9 were completely ineffective. Binding studies of ND29 and CD9 indicate that these mutants in OSCP were unable to bind to the membrane portion of the ATP synthase, F0, and these results were supported by competition binding studies. These results support the hypothesis that the N- and C-terminal regions of subunit 5 interact with F0 and suggest that the central region interacts with F1.
Insights
Investigating the yeast mitochondrial ATP synthase subunit 5 (OSCP), this study reveals that its N- and C-terminal regions are crucial for binding to the F0 component, impacting oligomycin sensitivity.
Area of Science:
- Mitochondrial biogenesis and function
- Protein structure-function relationships
- Enzyme kinetics and regulation
Background:
- Mitochondrial ATP synthase (also known as Complex V) is essential for cellular energy production.
- Oligomycin sensitivity-conferring protein (OSCP), subunit 5, plays a critical role in coupling the F0 and F1 components of ATP synthase.
- Understanding OSCP's structure is key to elucidating ATP synthase assembly and function.
Purpose of the Study:
- To investigate the structure/function relationship of yeast Saccharomyces cerevisiae OSCP.
- To determine the roles of OSCP's N- and C-terminal regions in ATP synthase assembly and activity.
- To explore the interaction of OSCP with the F0 and F1 subunits.
Main Methods:
- Deletion mutagenesis of the OSCP gene to create N- and C-terminal deletion mutants (ND15, ND29, CD8, CD9).
- In vivo analysis of ATP synthase activity in yeast strains expressing mutant OSCP.
- In vitro reconstitution studies, circular dichroism spectroscopy, and binding assays using purified mutant proteins.
Main Results:
- Mutants ND15 (amino acids 3-17 deleted) and CD8 (last 8 amino acids deleted) retained partial function.
- Mutants ND29 (amino acids 3-31 deleted) and CD9 (last 9 amino acids deleted) completely inactivated ATP synthase.
- ND29 and CD9 mutants failed to bind to the F0 component, indicating impaired interaction with the membrane sector.
Conclusions:
- The N- and C-terminal regions of OSCP are essential for its interaction with the F0 subunit of ATP synthase.
- Proper interaction with F0 is critical for oligomycin sensitivity and overall ATP synthase function.
- The central region of OSCP likely interacts with the F1 component, mediating energy transduction.