MODIFICATIONS OF THE GAMMA-SUBUNIT OF CHLOROPLAST COUPLING FACTOR-I ALTER INTERACTIONS WITH THE INHIBITORY EPSILON-SUBUNIT

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma-subunit. Reduction of the gamma-subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (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 epsilon-subunit inhibits the, Ca2+-ATPase activity of both forms of the diluted CF1, suggesting that epsilon-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 epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon-subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma-subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon-subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.