Purified subunit δ of chloroplast coupling factor CF₁ reconstitutes photophosphorylation in partially CF₁‐depleted membranes

Abstract

The ATP synthase of chloroplasts consists of the proton channel, CFo, and the catalytic part, CF1, which carries nucleotide-binding sites on subunits c1 and 8. The still poorly understood interaction between CFo and the catalytic sites on CF1 is mediated by the smaller subunits y, 6 and E of CF1. We investigated the ability of purified 6 to block proton leakage through CFo channels after their exposure by removal of the CF1 counterpart.Thylakoids were partially depleted of CF1 by EDTA treatment. This increased their proton permeability and thereby reduced the rate of photophosphorylation. Subunit 6 was isolated and purified by FPLC FEBS Lett. 219, 321 -3251. Addition of 6 to EDTA-treated thylakoids reconstituted high rates of phenazine-methosulfate-mediated photophosphorylation. Since 6 does not interact with nucleotides by itself, the reconstitution was due to a reduction of the proton leakage through open CFo channels. The molar ratio of purified 6 over exposed CFo, which started to elicit this effect, was 3 : 1. However, if 6 was added together with purified CF1 lacking 6, in a 1 : 1 molar ratio, the relative amount over exposed CFo was as low as 0.06. This corroborated our previous conclusion [Lill, H., Engelbrecht, S., Schonknecht, G. and Junge, W. (1986) Eur. J . Biochem. 160, 627 -6341 that only a very small fraction of exposed CFo was actually proton-conducting but with a very high unit conductance. CF1 including 6 was apparently rebound preferentially to open CFo channels.Although the ability of 6 to control proton conduction through CFo was evident, it remains to be established whether 6 acts as a gated proton valve or as a conformational transducer in the integral CFoCFl ATPase.The ATP synthase in thylakoid membranes (CFoCFl) couples proton flux to ATP synthesis. It belongs to a class of ATP synthases which also occur in the inner mitochondrial membrane (MFoMFl) and in micro-organisms, e.g. Escherichia coli (EFoEFl) and thermophilic bacteria (TFoTF1). For recent reviews see [l -51. The chloroplast ATP synthase consists of nine different subunits [6, 71. Four of them probably form the membrane-embedded proton channel CFo. Their relative stoichiometry and their quaternary structure are still under debate. The other five subunits form the extrinsic catalytic part, CF1, comprising a (56-58 kda), j (53-56kDa), y (37-39kDa), 6 (19-21 kDa), and E (1 5 kDa). Their stoichiometry is C(&Y61-3~. Electron-microscopic investigations of isolated F1 showed an alternating arrangement of the a and @ subunits in a nearly hexagonal conformation ('cyclohexane chair') [8,9]. A center mass, probably composed of subunits y, 6 and E is slightly shifted out of Correspondence to S. Engelbrecht, Biophysik, Fachbereich Biologie/Chemie, Universitat Osnabriick, BarbarastraDe 1 1, D-4500 Osnabruck, Federal Republic of Germany Abbreviations. CFoCF1, chloroplast ATP synthase; EFoEF1, E. coli ATP synthase; MFoMF1, mitochondrial ATP synthase; CF1, integral chloroplast ATPase; CFo, chloroplast proton channel; CFl(-6), CF1 lacking the 6 ...

Cross‐Linking of Chloroplast F0F1‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

“…F,(-e) was substituted with a 20-fold molar excess of TFPAM-3-modified c. NaBr-treated spinach chloroplasts (1471, 300 pg chlorophyll) were reconstituted with 300 pg of F,(-E) + e in the presence of 10 mM MgC1,. After washing to remove unbound protein, the samples were illuminated for 10 min at 312 nm on a transilluminator followed by extraction of the reconstituted Measurement of Ca'+-ATPase and Mg'+-ATPase activities of soluble F,, phosphate assays [42,49,531, and protein determination [54] were performed according to published procedures. Protein determinations [54] overestimated concentrations of t' by 18% (as determined by amino acid composition) and were corrected accordingly.…”

Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase

“…The E. coli 8 subunit has been shown to be involved in the attachment of F1 to Fo in reconstitution studies (19). Additionally, in chloroplasts, the 8 subunit has been shown to block the proton channel and to restore photophosphorylation to partially stripped thylakoid membranes (5,6,13 (14).…”

Cross‐reconstitution of the FOF1‐ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit δ

“…a) When freshly prepared chloroplast 6 was added to EDTA-treated spinach thylakoids, it restored photophosphorylation. This ability was rapidly lost upon storage of 6 and the loss of activity seemed to involve conformational transitions of isolated 6 [31]. Most of the chloroplast 6 that was used in this work had been stored at -20" C or -196" C and did not restore photophosphorylation by itself.…”

“…Our previous work on chloroplast CFoCFl has demonstrated the ability of purified subunit 6 to restore phenazinemethosulfate-mediated cyclic photophosphorylation in partially CF,-depleted vesicles from spinach thylakoids [31]. This action was due to the fact that 6 plugged the protonic leak conductance of a small fraction of 'exposed' CFo which were in a high conductance form [8, 27, 331 (see below).…”

“…Only for EFoEFl has this conclusion survived the test of time; for CFoCFl it was clearly demonstrated that 6 is not required for rebinding of CFl to CF, [25]. By various approaches it was shown that chloroplast 6 controls proton flow: when isolated and added back to partially CF1-depleted membranes, it can keep the proton pore through CFo closed [26-331. Thus at least chloroplast 6 fulfills a functional and not merely structural role [31,321. The structural role of E. coli 6 in the association of EFl with EFo does not exclude an additional functional role though, and this has been suggested [34, 351. It was interesting to compare E. coli 6 and chloroplast 6 directly in mixed reconstitution experiments, adding CF1 (-6)+E.…”