Cross-Linking of the Endogenous Inhibitor Protein (IF 1 ) with Rotor (γ,ε) and Stator (α) Subunits of the Mitochondrial ATP Synthase

The location of the endogenous inhibitor protein ( IF 1 ) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F 1 I and intact F 1 F 0 I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of α–IF 1 , IF 1 –IF 1 , γ–IF 1 , and ε–IF 1 cross-linkages in soluble F 1 I and in native F 1 F 0 I complexes. Cross-linking blocked the release of IF 1 from its inhibitory site and therefore the activation of F 1 I and F 1 F 0 I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF 1 is at a distance ≤12 Å,to γ and ε subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF 1 inhibits conformational changes of the catalytic β subunits, the inhibitory mechanism of IF 1 may involve the interference with rotation of the central stalk.