The Proton Adenosinetriphosphatase Complex of Rat Liver Mitochondria. Temperature-Dependent Dissociation-Reassociation of the F₁-ATPase Subunits

The soluble F₁ moiety of the rat liver mitochondrial proton ATPase dissociates into two easily separable fractions when cold treated and then warmed. One fraction is soluble in potassium phosphate buffer, pH 7.4, whereas the other is insoluble. Neither of these two fractions alone can catalyze ATP hydrolysis under assay conditions optimal for the native F₁-ATPase. The insoluble fraction when resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis is shown to be composed of only α and γ subunits. When this fraction is chromatographed on Sephadex G-75, it is resolved into an αγ complex and into the α subunit alone. The soluble fraction when resolved in the same electrophoretic system is shown to contain the remaining subunits, β, δ, ∊, and some γ. This fraction is resolved into two major components by chromatography on Sepharose CL-6B, a βγ complex and β subunit alone. The cold-dissociated enzyme can be readily associated when the temperature is raised to 20 °C. In the presence of either ATP or MgATP the enzyme completely regains its original ATPase specific activity. In contrast, Mg²⁺ is only about 15% effective in restoring ATPase activity. The results presented here define conditions for the dissociation and reassociation of the major subunits comprising the F₁-ATPase of rat liver and thus provide a unique system among mammalian enzymes for testing the function of individual subunits. In addition, they strongly indicate that neither the α nor β subunits, nor complexes of these subunits with the γ subunit, are capable of catalyzing ATP hydrolysis. Finally, they provide evidence that the γ subunit interacts with both the α and β subunits. As the most likely subunit stoichiometry of the rat liver F₁ is α₃β₃γδ∊ [Catterall, W. A., Coty, W. A., & Pedersen, P. L. (1973) J. Biol. Chem. 248, 7427-7431], these results suggest that the 7 subunit may “tag” one out of three αβ pairs in the molecule. The possible significance of this unusual type of subunit interaction to the function of the mitochondrial proton ATPase is discussed.