Thiol Modulation of the Chloroplast ATP Synthase is Dependent on the Energization of Thylakoid Membranes

Abstract

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.

Light- and Metabolism-related Regulation of the Chloroplast ATP Synthase Has Distinct Mechanisms and Functions

“…The protein precipitates were dissolved in freshly prepared solution containing 1% SDS, 50 mM Tris-HCl (pH 8.0), and 15 mM AMS and then immigrated by SDS-PAGE using running buffer lacking reducing agent as described in Ref. 16. Redox states of the ␥ subunits in wild type and the mothra were visually detected using an antibody against the ␥ 1 subunit using Western blotting.…”

“…the ability to form the regulatory disulfide bond. The redox state of wild-type and mutant ␥ subunits was probed by modification of free sulfhydryl groups with AMS followed by separation on SDS-PAGE (16,17). In wild type, the ATP synthase ␥ subunit showed the expected redox dependence upon exposure of dark-adapted leaves for 60 min to light, with the apparent molecular weight being completely shifted from low to high molecular weight, indicating transition from oxidized to reduced forms (Fig.…”

“…Insoluble proteins were solubilized in sample buffer without addition of reducing regents, incubated in the thiol binding probe AMS, and separated by SDS-PAGE. Reduced proteins bands migrate less and appear in the upper part of the gel, whereas oxidized proteins migrate further (16,17). D, AMS gel shift assay for redox state of wild-type and mothra ␥ subunit by light and diamide using the methods described for C. As a control, wild-type and leaf discs were treated with buffer in light (100 mol photons m Ϫ2 s Ϫ1 ) or darkness, as described in Ref.…”

Role of cyclic electron transport around photosystem I in regulating proton motive force

“…On the basis of the relatively fast recovery of elevated g H + caused by high light (20 min), this phenotype in g H + observed after long dark adaptation may reflect a different physiological event. A candidate is inactivation of the Calvin cycle enzymes, which shows a slower response to light than that of ATP synthase [42]. In the pgr5 mutant, however, g H + was higher even after 15 to 16-min AL exposure, in which the Calvin cycle enzymes are activated to the steady-state levels (Fig.…”

“…This idea is supported by the fact that the activity of another thiol enzyme, NADPmalate dehydrogenase (NADP-MDH), is higher at low light intensity in the pgr5 mutant than in the WT [41]. However, the reduction of the disulfide bond in the γ subunit is more sensitive to light than is the case in other thiol enzymes, including FBPase [42]. The disulfide bond in the γ subunit is fully reduced in a few seconds at very low light intensity (4 μmol photons m − 2 s − 1 ) [43].…”