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

Abstract

The ε subunit of the chloroplast ATP synthase functions in part to prevent wasteful ATP hydrolysis by the enzyme. In addition, ε 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 (CF(o)). Mutant ε subunits of the spinach (Spinacia oleracea) chloroplast ATP synthase that lack 5, 11, or 20 amino acids from their N termini (ε-Δ5N, ε-Δ11N, and ε-Δ20N, respectively), were overexpressed as inclusion bodies. Using a procedure that resulted in the folding of full-length, recombinant ε in a biologically active form, none of these truncated forms resulted in ε that inhibited the ATPase activity of CF1 deficient in ε, CF1(-ε). Yet, the ε-Δ5N and ε-Δ11N peptides significantly inhibited the ATPase activity of CF1(-ε) bound to CF0 in NaBr-treated thylakoids. Although full-length ε rapidly inhibited the ATPase activity of CF1(-ε) in solution or bound to CF0, an extended period was required for the truncated forms to inhibit membrane-bound CF1(-ε). Despite the fact that ε-Δ5N significantly inhibited the ATPase activity of CF1(-ε) bound to CF(o), it did not block the proton conductance through CF(o) in NaBr-treated thylakoids reconstituted with CF1(-ε). 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 ε is important in its binding to CF1, (b) that CF(o) stabilizes ε binding to the entire ATP synthase, and (c) that the N terminus may play some role in the regulation of proton flux through CF(o).