PROTEOLYTIC CLEAVAGE WITHIN A REGULATORY REGION OF THE GAMMA-SUBUNIT OF CHLOROPLAST COUPLING FACTOR-1

The gamma subunit of chloroplast coupling factor 1 (CF1) is susceptibl e to selective proteolysis when the enzyme is in solution and the epsi lon subunit is removed [CF1(-epsilon)]. In spinach thylakoid membranes , rapid cleavage of gamma is dependent on the generation of an electro chemical proton potential. The tryptic cleavage sites within the gamma of oxidized CF1 in illuminated thylakoids as well as of reduced CF1(- epsilon) in solution were determined by N-terminal amino acid sequenci ng. Two large gamma fragments of 27 000 (gamma 27) and 10 000 (gamma 1 0) molecular weight were generated by trypsin treatment of membrane-bo und CF1. The N-terminal gamma 27 contains amino acids 1-215, and the C -terminal gamma 10 contains 232-323. These polypeptides were tightly a ssociated with the trypsin-resistant core of the enzyme. In contrast, three large gamma fragments were produced by trypsinolysis of reduced CF1(-epsilon). These polypeptides, which were also tightly associated with the trypsin-resistant core, have molecular weights of 7900 (gamma 8), 14 850 (gamma 15), and 10 000 (gamma 10). These fragments contain residues 1-70, 71-204, and 232-323, respectively. The C-terminal gamm a 10 fragment generated by trypsin treatment of membrane-bound and sol uble CF1 are identical. These results suggest that the gamma subunit o f CF1 in illuminated thylakoids resembles that of CF1(-epsilon) With r espect to accessibility to proteolytic cleavage. Cleavage of gamma bet ween residues 215 and 232 is sufficient to fully activate the ATPase a ctivity of the enzyme without reduction of the gamma disulfide. In add ition, cutting within this region is responsible for loss of affinity for the inhibitory epsilon subunit.