The structure/function relationships of oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase were studied by nested deletion mutagenesis followed by analyses of the resultant OSCPs for their ability to restore partial reactions of ATP synthesis in OSCP-depleted F-1-F-o complexes. Our results indicate that, from the N-terminus of OSCP, up to 13 amino acid residues could be deleted without any effect on OSCP coupling activity. However, deletion of 16 or more residues led to a slow decline in the ability of resultant mutant forms to restore ATP synthesis, Compared to the wild-type form of OSCP, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-P-i exchange activity. Detailed analyses of mutant ND-28 revealed that it was able to bind to the membrane segment (F-o) of ATP synthase and restore oligomycin-sensitive ATPase activity in OSCP-depleted F-1-F-o complexes. However, it did not bind to soluble segment F-1, nor did it confer cold stability to either soluble F-1 or reconstituted F-1-F-o complex. On the other hand, studies on nested deletions on the C-terminal end indicate that three residues could be deleted without compromising the energy-coupling activity of OSCP. However, truncations of five or more residues caused an impairment in the ability of resultant mutant forms to restore ATP-P-i exchange activity in OSCP-depleted complexes, Mutant CD-10 (deletion of amino acids 181-190) was completely ineffective as a coupling factor. Detailed analyses of this mutant revealed that the subunit was able to bind to soluble F-1 segment and confer cold stability to the enzyme but tvas neither able to associate with the membrane segment (F-o) nor able to reconstitute high oligomycin sensitivity in depleted F-1-F-o complexes. We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coupling factor to soluble F-1 but not for coupling the energy of proton translocation to the synthesis of ATP; an the other hand, the carboxyl-terminal end of OSCP containing amino acids K181-M186 is important for F-o-OSCP interactions as well as for the coupling of the energy of Delta mu H+ during the synthesis of ATP. These results suggest a model for OSCP in which the N-terminus is associated with the F-1 segment and the C-terminus is associated with the F-o segment, while the central part of the polypeptide forms three or more helices constituting the stalk in the intact F1Fo enzyme.
