OLIGOMYCIN SENSITIVITY CONFERRING PROTEIN OF MITOCHONDRIAL ATP SYNTHASE - DELETIONS IN THE N-TERMINAL END CAUSE DEFECTS IN INTERACTIONS WITH F1, WHILE DELETIONS IN THE C-TERMINAL END CAUSE DEFECTS IN INTERACTIONS WITH F-O

The structure/function relationships of oligomycin sensitivity conferr ing protein (OSCP) of bovine mitochondrial ATP synthase were studied b y nested deletion mutagenesis. followed by analyses of the resultant O SCPs for their ability to restore partial reactions of ATP synthesis i n 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 wit hout 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 OSC P, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-P-i exchange activity, Detailed ana lyses of mutant ND-28 revealed that it was able to bind to the membran e segment (F-o) of ATP synthase and restore oligomycin-sensitive ATPas e activity in OSCP-depleted F-1-F-o complexes. However, it did not bin d to soluble segment F-1, nor did it confer cold stability to either s oluble F-1 or reconstituted F-1-F-o complex, On the other hand, studie s on nested deletions on the C-terminal end indicate that three residu es could be deleted without compromising the energy-coupling activity of OSCP, However, truncations of five or more residues caused an impai rment in the ability of resultant mutant forms to restore ATP-P-i exch ange activity in OSCP-depleted complexes, Mutant CD-10 (deletion of am ino acids 181-190) was completely ineffective as a coupling factor, De tailed analyses of this mutant revealed that the subunit was able to b ind 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 ab le to reconstitute high oligomycin sensitivity in depleted F-1-F-o com plexes, We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coup ling factor to soluble F-1 but not for coupling the energy of proton t ranslocation 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 tile coupling of the energy of D elta 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 an d 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.