Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.
Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.
Mukhopadhyay
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University of Health Sciences, Chicago Medical School, Department of Biological Chemistry, Illinois 60064.
Zhou
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XQ
Uh
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Mueller
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DM
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009840:Oligomycins / Q000494:pharmacology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D013367:Submitochondrial Particles / Q000378:metabolism*
D002352:Carrier Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D009840:Oligomycins; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein