Abstract
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.
Highlights
Yeast Saccharomyces cereuisiae oligomycin sensi- to be essential in bovine ATP synthase (12, 131, has not yet tivity conferring proteins (OSCP)have been expressed in Escherichia coli
Two deletion mutants have been made thatdissect OSCP. Studies with these deletion mutant proteins and antibodies directed against themprovide evidence that OSCPserves a functional role, as well as the structuralrole, in the ATP synthase
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
Summary
The experimentswere performed in variance products,indicated by the arrows In this experiment, decreasingmolecularweights),with induction with IPTG. Since OSCP and NB contain a single cysteine residue, whereas BBcontainsmethionine (Fig. 6B, lanes 4-6, [:"SI Cys) These experiments confirm the absence of the methionine in OSCP afterexpression in E. coli and provide radiolabeled OSCP which was used for monitoring the binding of OSCP toF1and ETPin equilibrium binding studies. Effect of immune serum on OSCP actiuity activity was measured in the absence and presence of oligomycin, as Oligomycin (0g)-sensitive ATPasewas reconstituted ATPase activity was measured in the absence and presence of oligomycin, as indicated These data represent the average of two experiments
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