Functional consequences of deletions of the N terminus of the epsilon subunit of the chloroplast ATP synthase

The epsilon subunit of the chloroplast ATP synthase functions in part to prevent wasteful ATP hydrolysis by the enzyme. In addition, epsilon together with the remainder of the catalytic portion of the synthase (CF1) is required to block the nonproductive leak of protons through the membrane-embedded component of the synthase (CF0). Mutant epsilon subunits of the spinach (Spinacia oleracea) chloroplast ATP synthase that lack 5, 11, or 20 amino acids from their N termini (epsilon-Delta 5N, epsilon-Delta 11N, and epsilon-Delta 20N, respectively), were overexpressed as inclusion bodies. Using a procedure that resulted in the folding of full-length, recombinant epsilon in a biologically active form, none of these truncated forms resulted in epsilon that inhibited the ATPase activity of CF1 deficient in epsilon, CF1(-epsilon). Yet, the epsilon-Delta 5N and epsilon-Delta 11N peptides significantly inhibited the ATPase activity of CF1(-epsilon) bound to CF0 in NaBr-treated thylakoids. Although full-length epsilon rapidly inhibited the ATPase activity of CF1(-epsilon) in solution or bound to CF0, an extended period was required for the truncated forms to inhibit membrane-bound CF1(-epsilon). Despite the fact that epsilon-Delta 5N significantly inhibited the ATPase activity of CF1(-epsilon) bound to CF0, it did not block the proton conductance through CF0 in NaBr-treated thylakoids reconstituted with CF1(-epsilon). Based on selective proteolysis and the binding of 8-anilino-1-naphthalene sulfonic acid, each of the truncated peptides gained significant secondary structure after folding. These results strongly suggest (a) that the N terminus of epsilon is important in its binding to CF1, (b) that CF0 stabilizes epsilon binding to the entire ATP synthase, and (c) that the N terminus may play some role in the regulation of proton flux through CF0.