Structural interactions of the oligomycin sensitivity-conferring protein in the yeast ATP synthase.
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.
Mao
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Department of Biological Chemistry, The Chicago Medical School, North Chicago, Illinois 60064, USA.
Mueller
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DM
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D002352:Carrier Proteins; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry*; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D009840:Oligomycins / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014475:Uncoupling Agents / Q000494:pharmacology
D002352:Carrier Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009840:Oligomycins; D014475:Uncoupling Agents; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein