title: Oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase. The carboxyl-terminal region of OSCP is essential for the reconstitution of oligomycin-sensitive H(+)-ATPase.
abstract: Studies to establish the structure/function relationships of oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase were carried out using genetic engineering and biochemical approaches. A full-length cDNA clone encoding OSCP was isolated from a bovine heart cDNA library, and the mature form of OSCP was expressed in Escherichia coli using plasmid expression vector pKP1500. Recombinant OSCP was found to accumulate in the cytoplasmic inclusion bodies, by virtue of which the recombinant protein could be purified to greater than 85% purity by simple low speed centrifugation of cell lysates. Recombinant OSCP was found to be indistinguishable from OSCP isolated from mitochondria with respect to (i) apparent molecular mass on sodium dodecyl sulfate gel electrophoresis, (ii) immunological reactivity to anti-OSCP serum, (iii) biological activity in restoring oligomycin-sensitive ATPase and Pi-ATP exchange activities to OSCP-depleted ATP synthase complexes, and (iv) insensitivity of the biological activity to sulfhydryl-directed alkylating reagents. The amino-terminal sequence of the recombinant protein revealed that the initiating methionine was not removed by E. coli, although that apparently did not affect protein folding or its biological activity. Data on nested deletion mutations starting from the carboxyl terminus in OSCP demonstrated that, in each instance, the mutant form was expressed and the protein product was sequestered in cytoplasmic inclusion bodies, similar to the wild-type form. However, none of the variants, including the one in which only the last 10 residues were deleted, was able to restore cold-stable oligomycin-sensitive ATPase or Pi-ATP exchange activity in OSCP-depleted complexes. Taken together, these data suggest that amino acid residues 181-190 (or some of the residues in this region) in the OSCP sequence may be important for OSCP-F1 interactions.
authors.affiliation: Department of Cell and Molecular Biology, Boston Biomedical Research Institute, Massachusetts 02114.
authors.forename: S
authors.initials: S
authors.lastname: Joshi
authors.forename: A A
authors.initials: AA
authors.lastname: Javed
authors.forename: L C
authors.initials: LC
authors.lastname: Gibbs
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D009154:Mutation; D009840:Oligomycins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D009840:Oligomycins; D010710:Phosphates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein