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Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins

  • Takatoshi Sekiguchi
    Affiliations
    Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-Ku, Yokohama, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
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  • Keisuke Yoshida
    Affiliations
    Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-Ku, Yokohama, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
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  • Ken-Ichi Wakabayashi
    Affiliations
    Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-Ku, Yokohama, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
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  • Toru Hisabori
    Correspondence
    For correspondence: Toru Hisabori
    Affiliations
    Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-Ku, Yokohama, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
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Open AccessPublished:September 26, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102541
      Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔμH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔμH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔμH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔμH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

      Keywords

      Abbreviations:

      2CP (2-Cys peroxiredoxin), ACMA (9-amino-6-chloro-2-methoxyacridine), AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), DTTred (reduced DTT), DTTox (oxidized DTT), FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), TCA (trichloroacetic acid)
      According to the chemiosmotic theory, chloroplast FoF1-ATP synthase (CFoCF1) plays a central role in photosynthetic energy conversion in green plants (
      • Mitchell P.
      Chemiosmotic coupling in oxidative and photosynthetic phosphorylation.
      ,
      • Jagendorf A.T.
      • Uribe E.
      ATP formation caused by acid-base transition of spinach chloroplasts.
      ). When photosynthetic electron transport reactions generate the electrochemical gradient of protons across the thylakoid membrane (ΔμH+) under light conditions, the CFoCF1 embedded in this membrane synthesizes ATP using the gradient as a driving force (
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ATP synthase–a marvellous rotary engine of the cell.
      ). The regulation of CFoCF1 activity is important for two reasons. First, when the ΔμH+ is insufficient to drive ATP synthesis, CFoCF1 can catalyze the reverse reaction, ATP hydrolysis, coupled with ΔμH+ formation as a general mechanism of FoF1-ATP synthase (
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ATP synthase–a marvellous rotary engine of the cell.
      ). Second, the CFoCF1 in thylakoid membranes primarily controls proton efflux from the thylakoid lumen and is also involved in the maintenance of lumen side acidity. This acidification regulates the electron transfer activity of the cytochrome b6f complex. In addition, it is also thought to be an essential signal for initiating the nonphotochemical quenching required for photoprotection (
      • Kanazawa A.
      • Kramer D.M.
      In vivo modulation of nonphotochemical exciton quenching (NPQ) by regulation of the chloroplast ATP synthase.
      ,
      • Bosco C.D.
      • Lezhneva L.
      • Biehl A.
      • Leister D.
      • Strotmann H.
      • Wanner G.
      • et al.
      Inactivation of the chloroplast ATP synthase γ subunit results in high non-photochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana.
      ,
      • Kohzuma K.
      • Froehlich J.E.
      • Davis G.A.
      • Temple J.A.
      • Minhas D.
      • Dhingra A.
      • et al.
      The role of light-dark regulation of the chloroplast ATP synthase.
      ). Therefore, the activity of CFoCF1 seems to be controlled tightly according to the ΔμH+ level across the thylakoid membrane, that is, under both light and dark conditions.
      CFoCF1 is a molecular motor enzyme, and the c-ring portion of Fo rotates during the proton translocation from the lumen side to the stroma side in the ATP synthesis reaction. This c-ring rotation leads to rotation of the central axis portion of F1, which is composed of the γ and ε subunits, and induces conformational changes at the catalytic subunit β. In turn, a catalytic reaction occurs at three catalytic sites on each β subunit. CFoCF1 is also known as a thiol-modulated enzyme, and its rotation axis, that is, the γ subunit (CF1-γ), has a plant-specific insertion sequence containing a redox-active Cys pair (Cys199 and Cys205 in spinach CF1-γ) (
      • Mckinney D.W.
      • Buchanan B.B.
      • Wolosiuk R.A.
      Association of a thioredoxin-like protein with chloroplast coupling factor (CF1).
      ,
      • Nalin C.M.
      • McCarty R.E.
      Role of a disulfide bond in the γ subunit in activation of the ATPase of chloroplast coupling factor 1.
      ,
      • Miki J.
      • Maeda M.
      • Mukohata Y.
      • Futai M.
      The γ-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence.
      ). In an early study in the field, Junesch and Gräber investigated the influence of redox regulation on CFoCF1 activity using isolated spinach thylakoid membranes, finding that the apparent Vmax was the same for the oxidized and reduced forms of the enzymes but that the ΔμH+ threshold required to drive the reduced-form enzyme was lower than that required for the oxidized form (
      • Junesch U.
      • Graber P.
      Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis.
      ). In other studies, the process of activating CFoCF1 via reduction at the dark-to-light transition has been clarified. Specifically, the disulfide bond of the oxidized form of CF1-γ is reduced by reducing equivalents supplied via ferredoxin (Fd), Fd-thioredoxin reductase, and thioredoxin (Trx) from the photosynthetic electron transport chain (
      • Buchanan B.B.
      Role of light in the regulation of chloroplast enzymes.
      ,
      • Mills J.D.
      • Mitchell P.
      • Schurmann P.
      Modulation of coupling factor ATPase activity in intact chloroplasts, the role of the thioredoxin system.
      ,
      • Schwarz O.
      • Schurmann P.
      • Strotmann H.
      Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase.
      ). Trx is a small and ubiquitous redox-active protein that possesses a highly conserved amino acid sequence, WCGPC, at its active site and catalyzes a dithiol–disulfide exchange reaction with its target proteins (
      • Holmgren A.
      Thioredoxin.
      ,
      • Jacquot J.P.
      • Lancelin J.M.
      • Meyer Y.
      Thioredoxins: structure and function in plant cells.
      ). In green plants, Trx constitutes a gene superfamily (e.g., 20 genes in Arabidopsis thaliana), which is classified into seven major classes based on amino acid sequences and their subcellular localization (
      • Meyer Y.
      • Reichheld J.P.
      • Vignols F.
      Thioredoxins in Arabidopsis and other plants.
      ,
      • Meyer Y.
      • Riondet C.
      • Constans L.
      • Abdelgawwad M.R.
      • Reichheld J.P.
      • Vignols F.
      Evolution of redoxin genes in the green lineage.
      ). Five Trx subtypes exist in chloroplasts, namely Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z, which exhibit specific target selectivity (
      • Lemaire S.D.
      • Michelet L.
      • Zaffagnini M.
      • Massot V.
      • Issakidis-Bourguet E.
      Thioredoxins in chloroplasts.
      • Yoshida K.
      • Hara S.
      • Hisabori T.
      Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.
      ). By in vitro and in vivo analyses, we showed that both Trx-f and Trx-m are involved in reducing CF1-γ (
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ). It is also known that ΔμH+ formation across the thylakoid membrane is a prerequisite for the reduction of CF1-γ (
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ,
      • Ketcham S.R.
      • Davenport J.W.
      • Warncke K.
      • McCarty R.E.
      Role of the γ subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.
      ). Thus, CFoCF1 is active only under light conditions.
      In contrast to the light-induced CFoCF1 activation process, the oxidative deactivation process under dark conditions has not been clarified. Indeed, the oxidation process related to all Trx-regulated enzymes in chloroplasts, not only CFoCF1, is poorly understood, and the proteins involved in this oxidation have not been assigned. In the 1980s, the in vivo redox state of CF1-γ was assumed to be in equilibrium with the NADP+/NADPH pool via the Fd/Trx system or glutathione pool (
      • Quick W.P.
      • Mills J.D.
      Thiol modulation of chloroplast CFo-CF1 in isolated barley protoplasts and its significance to regulation of carbon dioxide fixation.
      • Kramer D.M.
      • Crofts A.R.
      Activation of the chloroplast ATPase measured by the electrochromic change in leaves of intact plants.
      ). However, Mills and Mitchell found that CF1-γ in lysed pea chloroplasts cannot be oxidized in vitro by Trx-f alone or by glutathione (
      • Mills J.D.
      • Mitchell P.
      Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark.
      ).
      The recent studies of the protein oxidation system in chloroplasts have provided us with the important information. With advances in analysis technology, comparative genomic studies have revealed the presence of Trx-like proteins in chloroplasts as well as the typical Trxs (
      • Cain P.
      • Hall M.
      • Schroder W.P.
      • Kieselbach T.
      • Robinson C.
      A novel extended family of stromal thioredoxins.
      ,
      • Chibani K.
      • Wingsle G.
      • Jacquot J.P.
      • Gelhaye E.
      • Rouhier N.
      Comparative genomic study of the thioredoxin family in photosynthetic organisms with emphasis on Populus trichocarpa.
      ). Among these Trx-like proteins, Trx-like 2 (TrxL2) and atypical Cys-His–rich Trx (ACHT) from Arabidopsis were found to be the oxidizing factors for several Trx target proteins. Both TrxL2 isoforms (TrxL2.1 and TrxL2.2) have been shown to oxidize the proteins involved in the Calvin–Benson cycle [Rubisco activase, fructose 1,6-bisphosphatase (FBPase), and sedoheptulose 1,7-bisphosphatase] (
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ), oxidative pentose phosphate pathway (glucose-6-phosphate dehydrogenase) (
      • Yoshida K.
      • Uchikoshi E.
      • Hara S.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade acts as oxidative activator of glucose-6-phosphate dehydrogenase in chloroplasts.
      ), and glycolytic pathway (phosphofructokinase) (
      • Yoshida K.
      • Hisabori T.
      Biochemical basis for redox regulation of chloroplast-localized phosphofructokinase from Arabidopsis thaliana.
      ). Among five ACHT isoforms, ACHT1 and ACHT2 can also oxidize FBPase (
      • Yokochi Y.
      • Sugiura K.
      • Takemura K.
      • Yoshida K.
      • Hara S.
      • Wakabayashi K.I.
      • et al.
      Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.
      ). These proteins are oxidants with the following common features: (i) possession of a Trx-like motif of CxxC instead of the typical active site sequence, (ii) a higher midpoint redox potential than that of the typical Trxs and Trx target proteins (TrxL2.1: −258 mV, TrxL2.2: −245 mV, ACHT1: −252 mV, and ACHT2: −247 mV at pH 7.5), and (iii) a higher efficiency in terms of reducing 2-Cys peroxiredoxin (2CP) (
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ,
      • Yokochi Y.
      • Sugiura K.
      • Takemura K.
      • Yoshida K.
      • Hara S.
      • Wakabayashi K.I.
      • et al.
      Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.
      ,
      • Dangoor I.
      • Peled-Zehavi H.
      • Levitan A.
      • Pasand O.
      • Danon A.
      A small family of chloroplast atypical thioredoxins.
      ). 2CP is responsible for detoxifying hydrogen peroxide (H2O2) reductively because it is the most abundant peroxiredoxin in chloroplasts (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • et al.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). Thus, the oxidation process involving thiol-modulated enzymes in chloroplasts is now thought to be due to the transfer of reducing equivalents from the reduced-form enzymes to H2O2 via Trx-like proteins and 2CP. This oxidation process is assumed to be always functional under conditions where H2O2 is generated, such as in a photosynthetic environment. However, under light conditions, the reducing power supplied by the photosynthetic electron transfer system exceeds the final oxidizing power of H2O2, resulting in the reduction of various chloroplast enzymes. In contrast, as the transition from light to dark conditions occurs, the supply of reducing power decreases, and the oxidation process of these enzymes becomes dominant (
      • Yoshida K.
      • Yokochi Y.
      • Hisabori T.
      New light on chloroplast redox regulation: molecular mechanism of protein thiol oxidation.
      ).
      In terms of the redox regulation of CFoCF1, it is known that the dynamics of reduction occurring at the dark-to-light transition and those of oxidation occurring at the light-to-dark transition differ significantly in this enzyme relative to those in other stromal redox-regulated enzymes in chloroplasts (
      • Yoshida K.
      • Matsuoka Y.
      • Hara S.
      • Konno H.
      • Hisabori T.
      Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.
      ). However, the molecular mechanisms underlying these dynamic responses are unclear. Therefore, a thorough analysis of this regulation system is needed to improve our understanding of how the ATP synthesis reaction in chloroplasts is regulated in response to fluctuations in light. In the present study, we focused on the capacity of Trx-like proteins and typical Trxs to oxidize CF1in vitro. We also examined the influence of ΔμH+ formation across the thylakoid membrane on the oxidation of CF1-γ. Our results provide important insights into the relationship between the redox regulation of CFoCF1 and ΔμH+ formation in the thylakoid membrane.

      Results

      Thylakoid membranes from leaves infiltrated with reductants show clear H+ pump activity

      We intended to prepare thylakoid membranes containing the reduced form of CFoCF1 from spinach leaves; however, CFoCF1 was oxidized entirely when the thylakoid membranes were isolated from untreated spinach leaves without light irradiation or reducing agent treatment (Fig. 1A, labeled as “Untreated”). For the reduction, spinach leaves were irradiated at 1000 to 1500 μmol photons m−2 s−1 for 10 min and infiltrated with 20 mM of reduced DTT (DTTred) under vacuum conditions. Thylakoid membranes were then prepared from the leaves, and DTTred was removed in a subsequent washing step. The redox state of CF1-γ in the thylakoid membranes was confirmed using the thiol-modifying reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (AMS). Using this method, we successfully isolated thylakoid membranes in which at least 80% of the contained CF1-γ was in the reduced form [Fig. 1A, labeled as “+ DTTred (Vac.)”]. In contrast, CF1-γ was in the fully oxidized form when the infiltration of a DTTred-free solution was used without irradiation [Fig. 1A, labeled as “− DTTred (Vac.)”].
      Figure thumbnail gr1
      Figure 1Manipulation of the redox state of CF1-γ in the thylakoid membrane. A, determination of the redox state of CF1-γ in the thylakoid membrane. Thylakoid membranes were isolated from spinach leaves using three different methods (untreated, DTTred infusion, and infusion without DTTred) as described in the Experimental procedures. After the modification of the free thiols of thylakoid proteins with AMS, proteins were subjected to nonreducing SDS-PAGE, and the redox state of CF1-γ was visualized by Western blotting. Unmodified samples dissolved in nonreducing SDS sample buffer without AMS were also loaded (labeled as “− AMS”). B, ATP-driven H+-pump activity measurements taken in the thylakoid membrane. Acidification of the thylakoid lumen was monitored using fluorescence quenching of ACMA (excitation at 410 nm, emission at 480 nm) at 25 °C. The reaction was initiated by the addition of 5 mM ATP, and the lumen acidification was dissipated by the addition of 5 μM FCCP to the thylakoid membrane. AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate; ACMA, 9-amino-6-chloro-2-methoxyacridine; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Ox, oxidized form; Red, reduced form.
      Next, we assessed the ATP-driven H+ pump activity in each thylakoid membrane preparation (Fig. 1B). When thylakoid membranes are supplemented with ATP, proton translocation into the thylakoid lumen and the ATP hydrolysis reaction should occur simultaneously. The resulting proton gradient formed across the thylakoid membrane can be detected using the ΔpH indicator 9-amino-6-chloro-2-methoxyacridine (ACMA), the fluorescence of which is quenched by protonation (
      • Huang C.S.
      • Kopacz S.J.
      • Lee C.P.
      Mechanistic differences in the energy-linked fluorescence decreases of 9-aminoacridine dyes associated with bovine heart submitochondrial membranes.
      ). As shown in Figure 1B, when ATP was added to the thylakoid membranes, two quenching phases were observed: a rapid phase immediately after ATP addition and a subsequent gradual phase. The rapid phase can be attributed to a direct interaction between ATP and ACMA, whereas the gradual phase is thought to be due to H+ pump activity caused by CFoCF1 (
      • McCarty R.E.
      ATP synthase of chloroplast thylakoid membranes: a more in depth characterization of its ATPase activity.
      ). Among the membrane preparations from leaves treated differently, only thylakoid membranes containing the reduced form of CFoCF1 exhibited high H+ pump activity and restored fluorescence intensity with the addition of an uncoupler, namely carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), implying that the H+ pump activity in thylakoid membranes depends on the CF1-γ redox state. Therefore, we used the thylakoid membranes isolated using this reduction method in the following in vitro oxidation assay.

      TrxL2 and ACHT oxidize CF1-γ efficiently under uncoupled conditions

      We reconstituted an in vitro oxidation assay system to evaluate the ability of Trx-like proteins to oxidize the CF1-γ of CFoCF1 in thylakoid membranes. The thylakoid membranes containing reduced CF1-γ was incubated with each isoform of TrxL2 and ACHT at various concentrations (0–500 nM) or for various incubation periods (0–900 s) in the presence of 50 μM of oxidized DTT (DTTox) as a final oxidation power (Figs. 2 and 3 and Fig. S1). To form the ΔμH+ across the thylakoid membrane, the reaction solution was supplemented with an artificial electron mediator, 1-methoxy-5-methylphenazinium methylsulfate, and the thylakoid membranes were irradiated at 600 to 650 μmol photons m−2 s−1 (
      • Schwarz O.
      • Schurmann P.
      • Strotmann H.
      Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase.
      ,
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ,
      • Schmid G.H.
      • Menke W.
      • Radunz A.
      • Koenig F.
      Polypeptides of thylakoid membrane and their functional characterization.
      ). To achieve control conditions under which no ΔμH+ was formed, the same experiments were performed under light conditions in the presence of FCCP or under dark conditions. We confirmed that the chemical oxidant DTTox could not oxidize CF1-γ when used alone, especially under conditions in which the ΔμH+ was formed (Fig. 2). Conversely, all Trx-like proteins examined in this study, namely TrxL2.1, TrxL2.2, ACHT1, and ACHT2, were able to oxidize CF1-γ under the appropriate conditions; however, each protein showed a different affinity for CF1-γ oxidation (Fig. 3, A and B) and a different kinetic pattern (Fig. 3, C and D). Furthermore, these oxidation patterns were affected by the ΔμH+ formation conditions. Therefore, we determined the half concentration (S1/2) and half oxidation time (t1/2) of reduction of CF1-γ by fitting the following equation to the plotted data:
      CF1γred=CF1-γred0×ekv


      where {CF1red} is the reduction level of CF1-γ, {CF1red}0 is the reduction level of CF1-γ before the assay, k is the rate constant determined by each curve fitting, and v is the variable (reaction time or concentration of the Trx-like protein). When the ΔμH+ was canceled through the addition of FCCP or not formed under dark conditions, TrxL2.2 oxidized CF1-γ rapidly; however, this rapid oxidation was suppressed by the ΔμH+ formed under light conditions. These differences were evident in the analysis of t1/2 values (Table 1). For ACHT1 and ACHT2, changes in oxidation kinetics were similar to those of TrxL2.2, but changes in S1/2 values were also observed in a ΔμH+-dependent manner, implying that the affinity between ACHT and CF1-γ is altered according to ΔμH+ formation. In contrast, the oxidation kinetics of CF1-γ by TrxL2.1 differed from those by the other three Trx-like proteins, and a ΔμH+-dependent change in the efficiency of CF1-γ oxidation was not detected due to the rapid oxidation reaction. Therefore, we measured the t1/2 values again using TrxL2.1 at a lower concentration, 50 nM (Fig. S2 and Table S1), finding similar ΔμH+-dependent oxidation of CF1-γ. These results suggest that the oxidation of CF1-γ by Trx-like proteins is strongly affected by the formation/dissipation of the ΔμH+ across the thylakoid membrane, and the affinity of Trx-like proteins to CF1-γ may also be affected by the ΔμH+.
      Figure thumbnail gr2
      Figure 2CF1-γ oxidation by DTTox in the presence and absence of the ΔμH+. A, visualization of CF1-γ oxidation by DTTox. Thylakoid membranes (50 μg Chl/ml) were incubated with 50 μM DTTox for 15 min in the presence or absence of the ΔμH+. After the modification of the free thiols of the proteins residing on the thylakoid membrane with AMS, proteins were subjected to nonreducing SDS-PAGE, and the redox state of CF1-γ was visualized by Western blotting. Unmodified samples dissolved in nonreducing SDS sample buffer without AMS were also loaded (labeled as “− AMS”). B, quantification of the redox state of CF1-γ. The CF1-γ reduction level shown in (A) was quantified as the ratio of the reduced form to the total and plotted against the reaction time. Each value represents the mean ± SD (n = 7–8). AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate; Ox, oxidized form; Red, reduced form.
      Figure thumbnail gr3
      Figure 3CF1-γ oxidation assisted by TrxL2 and ACHT in the presence and absence of the ΔμH+. A, Trx-like protein concentration-dependence of CF1-γ oxidation. Thylakoid membranes (50 μg Chl/ml) were incubated with the indicated concentrations of Trx-like proteins and 50 μM DTTox for 15 min in the presence or absence of the ΔμH+, and the redox state of CF1-γ was visualized as described in the A legend. B, quantification of the redox state of CF1-γ. The CF1-γ reduction level shown in (A) was quantified as described in the B legend. C, time course of CF1-γ oxidation by Trx-like proteins. Thylakoid membranes (50 μg Chl/ml) were incubated with 500 nM Trx-like proteins and 50 μM DTTox for the indicated time in the presence or absence of the ΔμH+, and the redox state of CF1-γ was visualized as described in the A legend. D, quantification of the CF1-γ redox state. The CF1-γ reduction level shown in (C) was quantified as described in the B legend. A and C, whole images are also shown in . C and D, each value represents the mean ± SD (n = 3). The exponential fitting curves for the mean values are also shown. Ox, oxidized form; Red, reduced form.
      Table 1Kinetic parameters of Trx-like protein-dependent oxidation in CF1
      SpeciesConditionsΔμH+S1/2 (nM)Significance by condition (p < 0.05)|R|t1/2 (s)Significance by condition (p < 0.05)|R|
      TrxL2.1Light, − FCCPFormed7.1 ± 2.5a0.9939.4 + 1.1a0.986
      Light, + FCCPNot formed7.1 ± 3.8a0.8658.4 + 1.0a0.994
      Dark, − FCCPNot formed8.9 ± 1.0a0.9669.9 + 1.9a0.964
      TrxL2.2Light, − FCCPFormed24.8 ± 10.6a0.976114.9 ± 21.9a0.955
      Light, + FCCPNot formed8.1 ± 2.0a0.87814.4 ± 3.0b0.995
      Dark, − FCCPNot formed12.5 ± 5.1a0.97617.9 ± 2.1b0.991
      ACHT1Light, − FCCPFormed110.8 ± 43.4a0.973433.2 ± 62.5a0.964
      Light, + FCCPNot formed22.7 ± 4.6b0.94188.9 ± 37.5b0.990
      Dark, − FCCPNot formed48.1 ± 8.7ab0.98762.5 ± 6.4b0.967
      ACHT2Light, − FCCPFormed272.4 ± 50.2a0.916688.9 ± 385.9a0.855
      Light, + FCCPNot formed50.1 ± 8.0b0.92070.3 ± 15.3b0.984
      Dark, − FCCPNot formed66.1 ± 6.4b0.97459.2 ± 11.3b0.974
      Data were analyzed using one-way ANOVA and Tukey’s honest significance difference test (p < 0.05). Different letters indicate significant differences among the same Trx-like proteins under different ΔμH+ formation conditions. The letters “a” and “b” mean that the values are significantly different, and “ab” means that the values are not significantly different from both “a” and “b”.

      Typical Trxs fail to oxidize CF1

      We also examined the ability of typical Trxs to oxidize CF1-γ using the same method described above (Fig. 4). First, we investigated CF1-γ oxidation by Trx-f, which is recognized as a major reducing mediator of many redox enzymes in chloroplasts, including CF1-γ (
      • Yoshida K.
      • Hara S.
      • Hisabori T.
      Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.
      ). Trx-f1 oxidized CF1-γ slightly in the absence of the ΔμH+, but the kinetic parameters of this oxidation could not be determined due to its low efficiency (Fig. 4, A–D). In addition, we examined the effects of the following Trxs: Trx-m, which is the most abundant chloroplast Trx in Arabidopsis (
      • Okegawa Y.
      • Motohashi K.
      Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo.
      ) and has been shown to reduce CF1-γ (
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ), and Trx-x and Trx-y, which are thought to be a part of the antioxidant system because they efficiently reduce 2CP and peroxiredoxin Q, respectively (
      • Yoshida K.
      • Hara S.
      • Hisabori T.
      Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.
      ,
      • Collin V.
      • Issakidis-Bourguet E.
      • Marchand C.
      • Hirasawa M.
      • Lancelin J.M.
      • Knaff D.B.
      • et al.
      The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity.
      ,
      • Collin V.
      • Lamkemeyer P.
      • Miginiac-Maslow M.
      • Hirasawa M.
      • Knaff D.B.
      • Dietz K.J.
      • et al.
      Characterization of plastidial thioredoxins from Arabidopsis belonging to the new y-type.
      ). Trx-z was not considered in the present study because it is known to function as part of an RNA polymerase complex (
      • Arsova B.
      • Hoja U.
      • Wimmelbacher M.
      • Greiner E.
      • Ustun S.
      • Melzer M.
      • et al.
      Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana.
      ). As shown in Figure 4, E and F, Trx-m1, Trx-x, and Trx-y2 did not oxidize CF1-γ effectively. Thus, we conclude that the typical Trxs present in chloroplasts do not function in CF1-γ oxidation.
      Figure thumbnail gr4
      Figure 4CF1-γ oxidation assisted by typical Trxs in the presence and absence of the ΔμH+. A, Trx-f1 concentration-dependence of CF1-γ oxidation. Thylakoid membranes (50 μg Chl/ml) were incubated with the indicated concentrations of Trx-f1 and 50 μM DTTox for 15 min in the presence or absence of the ΔμH+, and the redox state of CF1-γ was visualized as described in the A legend. B, quantification of the CF1-γ redox state. The CF1-γ reduction level shown in (A) was quantified as described in the B legend. C, time course of CF1-γ oxidation by Trx-f1. Thylakoid membranes (50 μg Chl/ml) were incubated with 500 nM Trx-f1 and 50 μM DTTox for the indicated time in the presence or absence of the ΔμH+, and the redox state of CF1-γ was visualized as described in the A legend. D, quantification of the CF1-γ redox state. The CF1-γ reduction level shown in (C) was quantified as described in the B legend. E, CF1-γ oxidation assisted by the other Trxs. Thylakoid membranes (50 μg Chl/ml) were incubated with 500 nM Trx isoforms and 50 μM DTTox for 15 min in the presence or absence of the ΔμH+, and the redox state of CF1-γ was visualized as described in the A legend. F, quantification of the CF1-γ redox state. The CF1-γ reduction level was quantified as described in the B legend. Different letters indicate significant differences (p < 0.05; one-way ANOVA and Tukey’s honest significance difference test). A, C and E, Ox, oxidized form; Red, reduced form. B, D and F, each value represents the mean ± SD [n = 3 or 4 (B); n = 3 (D and F)].

      Discussion

      Thiol-based reductive activation and oxidative deactivation of CFoCF1 are believed to play crucial roles in the efficient management of ATP production and ΔμH+ consumption during photosynthesis. However, the complete regulation mechanism has not been determined because biochemical analysis has been lacking, especially analysis of the CFoCF1 oxidation process. In the present study, we characterized the kinetics of CF1-γ oxidation by two physiological oxidants, namely the proteins TrxL2 and ACHT.
      First, we established a procedure for preparing thylakoid membranes containing CFoCF1 for which the redox state is controlled artificially. Using our thylakoid membrane preparations, we confirmed that reduced CFoCF1 generates the ΔμH+ across the thylakoid membrane via ATP hydrolysis and H+ translocation (Fig. 1). Second, we performed oxidation experiments using the thylakoid membrane preparations because light irradiation enabled the formation of a steady ΔμH+ across the thylakoid membrane. CF1-γ was oxidized efficiently by TrxL2 and ACHT, especially under uncoupled conditions (Figs. 2 and 3). The formation of the ΔμH+ across the thylakoid membrane is known to be a prerequisite for reductive activation of CFoCF1 by Trx (
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ,
      • Ketcham S.R.
      • Davenport J.W.
      • Warncke K.
      • McCarty R.E.
      Role of the γ subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.
      ); thus, the present results imply that oxidation and reduction of CFoCF1 are inversely dependent on the ΔμH+ formed across the thylakoid membrane. A similar result was reported previously in relation to the redox dynamics of CF1-γ in spinach intact chloroplasts when the chloroplasts were exposed to light, and adding an uncoupler to the chloroplasts induced the rapid oxidation of CF1-γ even under light conditions, although the reduction level of the stromal redox-regulated enzyme FBPase was not affected by adding FCCP to the chloroplasts (
      • Konno H.
      • Nakane T.
      • Yoshida M.
      • Ueoka-Nakanishi H.
      • Hara S.
      • Hisabori T.
      Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.
      ). Differences in the redox dynamics of membrane-embedded CFoCF1 and other stromal Trx target proteins in Arabidopsis plants were also observed under artificial light-controlled conditions mimicking those in the field (
      • Yoshida K.
      • Matsuoka Y.
      • Hara S.
      • Konno H.
      • Hisabori T.
      Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.
      ). Interestingly, while other stromal redox-regulated enzymes were oxidized gradually as light intensity decreased, only CF1-γ maintained an almost fully reduced state even under weak light conditions and was eventually oxidized when light was turned off. This ΔμH+-dependent oxidative deactivation of CFoCF1 also likely functions in vivo to avoid wasteful ATP hydrolysis in plants under nonphotosynthetic conditions.
      Using our in vitro assay system, we were able to estimate the apparent affinity between Trx-like proteins and CF1-γ, finding that this affinity increased markedly under uncoupled conditions (Fig. 3 and Table 1). However, among the proteins examined in the present study, TrxL2.1 did not show this tendency, and no ΔμH+-dependent change in S1/2 values was observed in association with this protein. This may have been due to the higher affinity between TrxL2.1 and CF1-γ compared with that between the other Trx-like proteins and CF1-γ. TrxL2.1 and TrxL2.2 share a common atypical Trx motif, WCRKC, but they show only about 53% identity. The difference in kinetic parameters between TrxL2 isoforms may be due to this amino acid sequence difference. We also found that none of the typical Trxs tested oxidized CF1-γ efficiently (Fig. 4), which is consistent with a previous study (
      • Mills J.D.
      • Mitchell P.
      Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark.
      ), whereas Trx-f and Trx-m have been shown to reduce CF1-γ under ΔμH+ formation conditions through physical interactions (
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ). Conversely, both Trx-f and Trx-m have been reported to oxidize their specific target proteins (
      • Yokochi Y.
      • Sugiura K.
      • Takemura K.
      • Yoshida K.
      • Hara S.
      • Wakabayashi K.I.
      • et al.
      Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.
      ,
      • Vaseghi M.J.
      • Chibani K.
      • Telman W.
      • Liebthal M.F.
      • Gerken M.
      • Schnitzer H.
      • et al.
      The chloroplast 2-cysteine peroxiredoxin functions as thioredoxin oxidase in redox regulation of chloroplast metabolism.
      ). Therefore, further studies on the conformational changes in CF1-γ that regulate the affinity with Trx-like proteins and Trxs are required. The ΔμH+ formation across the thylakoid membrane is known to induce the conformational changes in CF1-γ (
      • Hightower K.E.
      • McCarty R.E.
      Proteolytic cleavage within a regulatory region of the γ subunit of chloroplast coupling factor 1.
      ,
      • Komatsu-Takaki M.
      Energizing effects of illumination on the reactivities of lysine residues of the γ subunit of chloroplast ATP synthase.
      ,
      • Sugiyama K.
      • Hisabori T.
      Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the γ subunit.
      ). Komatsu-Takaki reported that these structural changes were achieved within about 1 to 30 s of light irradiation of the thylakoid membrane (
      • Komatsu-Takaki M.
      Energizing effects of illumination on the reactivities of lysine residues of the γ subunit of chloroplast ATP synthase.
      ). However, the atomic-level structure of CFoCF1 when energized by ΔμH+ remains to be clarified. The conformational change of CF1-γ induced by the ΔμH+ formation may alter its midpoint redox potential more positively. In contrast, cryo-EM studies have revealed structural differences between the reduced and oxidized forms of spinach CFoCF1 in the nonenergizing state (
      • Hahn A.
      • Vonck J.
      • Mills D.J.
      • Meier T.
      • Kuhlbrandt W.
      Structure, mechanism, and regulation of the chloroplast ATP synthase.
      ,
      • Yang J.H.
      • Williams D.
      • Kandiah E.
      • Fromme P.
      • Chiu P.L.
      Structural basis of redox modulation on chloroplast ATP synthase.
      ). In the structures shown in (
      • Yang J.H.
      • Williams D.
      • Kandiah E.
      • Fromme P.
      • Chiu P.L.
      Structural basis of redox modulation on chloroplast ATP synthase.
      ), the short β hairpin loop of CF1-γ is destabilized when the disulfide bond formed by two Cys at the redox switch is reduced. In addition, three negatively charged amino acids (Glu210, Asp211, and Glu212 in spinach CF1-γ) located at the end of the short β hairpin loop structure change their interactions with the surrounding amino acids depending on the redox state of the switch. The importance of these charged amino acids in redox regulation was reported in several studies (
      • Konno H.
      • Yodogawa M.
      • Stumpp M.T.
      • Kroth P.
      • Strotmann H.
      • Motohashi K.
      • et al.
      Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the γ subunit of chloroplast ATP synthase.
      ,
      • Ueoka-Nakanishi H.
      • Nakanishi Y.
      • Konno H.
      • Motohashi K.
      • Bald D.
      • Hisabori T.
      Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the γ subunit of chloroplast ATP synthase.
      ,
      • Wu G.
      • Ortiz-Flores G.
      • Ortiz-Lopez A.
      • Ort D.R.
      A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase γ-subunit regulatory disulfide above the range of thioredoxin modulation.
      ,
      • Kohzuma K.
      • Dal Bosco C.
      • Meurer J.
      • Kramer D.M.
      Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.
      ). The redox state of CF1-γ may also regulate the affinity for Trxs; however, further structural analyses are needed to reveal the underlying mechanism more clearly.
      Reverse-genetic studies in Arabidopsis have provided important insights into the oxidative regulation of CF1-γ by Trx-like proteins. Yokochi et al. generated separate Arabidopsis mutant plants deficient in TrxL2 and ACHT using the CRISPR/Cas9 system and tested the redox dynamics of various Trx target proteins in each mutant strain in response to a light-to-dark transition (
      • Yokochi Y.
      • Fukushi Y.
      • Wakabayashi K.I.
      • Yoshida K.
      • Hisabori T.
      Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.
      ). They found that TrxL2 deficiency retarded the in vivo oxidation of CF1-γ, whereas ACHT deficiency did not have this effect. These results are consistent with our finding that TrxL2 has a high affinity for CF1-γ (Fig. 3 and Table 1). However, CF1-γ was almost fully oxidized in dark-adapted TrxL2-deficient plants (
      • Yokochi Y.
      • Fukushi Y.
      • Wakabayashi K.I.
      • Yoshida K.
      • Hisabori T.
      Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.
      ), suggesting that other oxidation factors, such as ACHT, may also be involved in CF1-γ oxidation in vivo. Although TrxL2 and ACHT are about 10-fold less abundant in vivo than typical Trxs and their target proteins (
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ,
      • Yokochi Y.
      • Fukushi Y.
      • Wakabayashi K.I.
      • Yoshida K.
      • Hisabori T.
      Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.
      ), they may more efficiently oxidize CF1-γ if the affinity of CF1-γ with Trx-like proteins and typical Trxs changes due to ΔμH+ dissipation. In 2CP-knockdown mutant plants generated by T-DNA insertion, CF1-γ was found to be oxidized with a delay (
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ). Collectively, these results indicate that CF1-γ oxidation in vivo is accomplished primarily via the TrxL2/2CP cascade.
      Based on the present findings, we propose a new schematic model of the redox regulation of CFoCF1 (Fig. 5) with reference to the model proposed previously by Junesch and Gräber (
      • Junesch U.
      • Graber P.
      Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis.
      ). In the oxidation process, ΔμH+ dissipation causes a conformational change at CF1-γ, and Trx-like proteins then act to oxidize the redox switch on CF1-γ. Overall, our study provides an overview of the specific mechanism underlying the inactivation of CFoCF1 as well as insights into the energetic strategy of plants in response to fluctuations in light conditions.
      Figure thumbnail gr5
      Figure 5Scheme of the redox regulation of CFoCF1 adapted from that of Junesch and Gräber (
      • Junesch U.
      • Graber P.
      Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis.
      ). Oxidative deactivation of CFoCF1 by Trx-like proteins promoted by ΔμH+ dissipation is included in the scheme. Straight arrows indicate the direction of change in CFoCF1 activation states. Curved arrows indicate the flow of reducing power. Triangles labeled with ‘H+’ indicate the formation of ΔμH+ across the thylakoid membrane. 2CP, 2-Cys peroxiredoxin; H2O2, hydrogen peroxide.

      Experimental procedures

      Preparation of thylakoid membranes containing the reduced form of CFoCF1

      Fresh market spinach was washed well and left overnight in the dark at 4 °C. Harvested leaves (about 10 g in fresh weight) were irradiated with 1000 to 1500 μmol photons m−2 s−1 using a LED illuminator for 10 min at room temperature. The leaves were then immediately immersed in a reducing solution containing 50 mM Tricine–NaOH (pH 7.5), 0.4 M sucrose, 5 mM MgCl2, 10 mM NaCl, and 20 mM DTTred and vacuum-infiltrated for 3 min in this solution. Subsequently, the leaves were homogenized three times for 3 s in a mixer with 200 ml of the reducing solution at 4 °C. The homogenate was then filtrated through four layers of gauze and centrifuged at 3000g and 4 °C for 10 min. The pellet was suspended using the reducing solution and centrifuged at 300g and 4 °C for 1 min. The supernatant was collected and centrifuged at 3000g and 4 °C for 10 min. The abovementioned wash step was performed twice with a nonreducing wash solution containing 50 mM Tricine–NaOH (pH 7.5), 0.4 M sucrose, 5 mM MgCl2, and 10 mM NaCl at 4 °C. The resulting pellet was suspended in the nonreducing wash solution with a chlorophyll (Chl) concentration of 0.5 mg/ml. The preparation was left in the dark on ice for at least 1 h before use.

      Preparation of recombinant Arabidopsis Trx and Trx-like proteins

      All expression plasmids for Trx-like proteins and typical Trxs used in this work (TrxL2.1, At5g06690; TrxL2.2, At5g04260; ACHT1, At4g26160; ACHT2, AT4G29670.1; Trx-f1, At3g02730; Trx-m1, At1g03680; Trx-x, At1g50320; and Trx-y2, At1g43560) were constructed as described previously (
      • Yoshida K.
      • Hara S.
      • Hisabori T.
      Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.
      ,
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ,
      • Yokochi Y.
      • Sugiura K.
      • Takemura K.
      • Yoshida K.
      • Hara S.
      • Wakabayashi K.I.
      • et al.
      Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.
      ,
      • Yoshida K.
      • Hisabori T.
      Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts.
      ). Each expression plasmid was transformed into Escherichia coli strain BL21(DE3), and transformed cells were cultured at 37 °C. The desired protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside followed by a further culture at 21 °C overnight. The cells were then disrupted using sonication and centrifuged at 125,000g for 40 min, after which the resulting supernatant was used in protein purification. TrxL2.1 and Trx2.2 proteins were purified using a Ni-nitrilotriacetic acid affinity column as described previously (
      • Yoshida K.
      • Hara A.
      • Sugiura K.
      • Fukaya Y.
      • Hisabori T.
      Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts.
      ) following an additional size-exclusion chromatography step conducted using a Superdex 75 column 10/300 (GE Healthcare). ACHT1 and ACHT2 proteins were purified using a combination of Ni-nitrilotriacetic acid affinity chromatography, cation-exchange chromatography, and size-exclusion chromatography as described previously (
      • Yokochi Y.
      • Sugiura K.
      • Takemura K.
      • Yoshida K.
      • Hara S.
      • Wakabayashi K.I.
      • et al.
      Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.
      ). The other Trx proteins contained no affinity tag and were purified using a combination of anion-exchange chromatography, cation-exchange chromatography, or hydrophobic-interaction chromatography as described previously (
      • Yoshida K.
      • Hara S.
      • Hisabori T.
      Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.
      ,
      • Sekiguchi T.
      • Yoshida K.
      • Okegawa Y.
      • Motohashi K.
      • Wakabayashi K.I.
      • Hisabori T.
      Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.
      ,
      • Yoshida K.
      • Hisabori T.
      Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts.
      ). The concentrations of purified proteins were determined using a BCA protein assay (Pierce).

      Measurement of H+ pump activity in thylakoid membranes

      The ATP-driven H+ pump activity of CFoCF1 in the thylakoid membranes was measured according to the fluorescence quenching of ACMA based on a previous method with some modifications (
      • McCarty R.E.
      ATP synthase of chloroplast thylakoid membranes: a more in depth characterization of its ATPase activity.
      ,
      • Akanuma G.
      • Tagana T.
      • Sawada M.
      • Suzuki S.
      • Shimada T.
      • Tanaka K.
      • et al.
      C-terminal regulatory domain of the ε subunit of FoF1 ATP synthase enhances the ATP-dependent H+ pumping that is involved in the maintenance of cellular membrane potential in Bacillus subtilis.
      ). The emitted fluorescence of ACMA (excitation at 410 nm, emission at 480 nm) was measured using a FP-8500 spectrofluorometer (Jasco), in which the temperature in the sample chamber was 25 °C. At 180 s after the initiation of the measurement, 5 mM ATP was added to the reaction solution containing 50 mM Tricine–NaOH (pH 7.5), 0.4 M sucrose, 5 mM MgCl2, 10 mM NaCl, 0.3 μg ml−1 ACMA, and 20 μg Chl/ml of thylakoid membranes. After the measurement was continued for 15 min, 5 μM FCCP was added, and the stable fluorescence intensity following the addition of FCCP was taken as 1.0. The reaction solution was stirred continuously during the measurement.

      Determination of the CF1-γ redox state in thylakoid membranes

      To quantify the redox state ratio of CF1-γ in thylakoid membranes, a thylakoid membrane solution was mixed with an equal volume of 20% trichloroacetic acid (TCA) and left on ice for 30 min. The TCA precipitants were then washed with ice-cold acetone, and the resulting precipitated proteins were labeled with AMS using the following procedure. The precipitants were suspended in nonreducing SDS sample buffer [62.5 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 7.5% (v/v) glycerol, and 0.01% (w/v) bromophenol blue] containing 2 mM of AMS. After incubation for 30 min at room temperature, protein samples were boiled for 5 min at 95 °C. Proteins were then separated using SDS-PAGE and transferred to a PVDF membrane. Antibodies against CF1-γ (
      • Yoshida K.
      • Matsuoka Y.
      • Hara S.
      • Konno H.
      • Hisabori T.
      Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.
      ) were used to perform Western blotting. Chemiluminescence was detected using horseradish peroxidase–conjugated secondary antibodies and ECL Prime (GE Healthcare) and visualized on an LAS 3000 Mini Imaging System (Fuji Film). The resultant band intensities were quantified using ImageJ.

      In vitro assay of CFoCF1 oxidation by Trx-like proteins and Trxs

      The nonreducing wash solution used in the preparation of thylakoid membranes was degassed for 1 h at room temperature, and the following reactions were performed in this solution. Prior to the oxidation assay, 150 to 5000 nM of Trx-like proteins or Trx were incubated for 10 min at 25 °C with a DTTox mixture containing 50 mM Tricine–NaOH (pH 7.5), 0.4 M sucrose, 5 mM MgCl2, 10 mM NaCl, and 500 μM DTTox. Subsequently, 100 μl of the DTTox mixture containing Trx-like proteins or Trx was added to 900 μl of a thylakoid solution to initiate the oxidation reaction. The composition of the final reaction mixture was 50 mM Tricine–NaOH (pH 7.5), 0.4 M sucrose, 5 mM MgCl2, 10 mM NaCl, 100 μM 1-methoxy-5-methylphenazinium methylsulfate, 50 μg Chl/ml of thylakoid membranes, 50 μM DTTox, and 15 to 500 nM Trx-like proteins or Trx. The oxidation reaction was performed for a specific time (0–900 s) at 25 °C and terminated by adding 10% TCA. For the formation of a ΔμH+ across the membrane, the thylakoid solution was irradiated with 600 to 650 μmol photons m−2 s−1 using a LED illuminator while stirring 5 min before the start of the reaction. The irradiation was then continued during the reaction. The followings were examined to assess uncoupled conditions: 5 μM FCCP was added to the reaction mixture or the reaction tube was wrapped in aluminum foil and placed in a dark room.

      Data availability

      All data are contained within the article and can be shared upon request ( [email protected] ).

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We acknowledge the Open Facility Center , Tokyo Institute of Technology , for supporting DNA sequencing.

      Author contributions

      T. S., K. Y., and T. H. methodology; T. S. investigation; K. Y. resources; T. S. writing–original draft; K. Y., K.-I. W., and T. H. writing–review and editing; K.-I. W. and T. H. supervision; T. S. and T. H. conceptualization.

      Funding and additional information

      This study was supported by Grants-in-Aid for Scientific Research (Grant 21H02502 to T. H.) from the Japan Society for the Promotion of Science and partly by a Grant-in-Aid for JSPS Research Fellows (Grant 22J13334 to T. S.) as well as the Dynamic Alliance for Open Innovation Bridging Human, Environment, and Materials.

      Supporting information

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