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 (CFO). 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 CFO in NaBr-treated thylakoids. Although full-length [epsilon] rapidly inhibited the ATPase activity of CF1(-[epsilon]) in solution or bound to CFO, 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 CFO, it did not block the proton conductance through CFO 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 CFO.
Cruz
J. A.
JA
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2658.
Radkowski
C. A.
CA
McCarty
R. E.
RE