Modifications of the γ subunit of chloroplast coupling factor 1 alter interactions with the inhibitory ε subunit

The treatment of chloroplast coupling factor 1 (CF₁) with dithiothreitol or with trypsin modifies the γ subunit. Reduction of the γ subunit disulfide bond in CF₁ in solution with dithiothreitol enhances the dissociation of ε (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca²⁺-ATPase activity of either oxidized or reduced CF₁ increases as the enzyme is diluted. Added ε subunit inhibits the Ca²⁺-ATPase activity of both forms of the diluted CF₁, suggesting that ε dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF₁, indicating that reduction decreases the affinity for ε about 20-fold. Immunoblotting techniques show that there is only one ε subunit/CF₁ in intact chloroplasts, in thylakoid membranes, and in solution. No ε is released from CF₁ in thylakoids under conditions of ATP synthesis. The γ subunit of CF₁ in illuminated thylakoids is specifically cleaved by trypsin. CF₁ purified from thylakoids treated with trypsin in the light is deficient in ε subunit, and has a high rate of ATP hydrolysis. Added ε neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.