利用体外类囊体实验评估硫氧还蛋白介导的 CFoCF1 还原

Takatoshi Sekiguchi; Keisuke Yoshida; Toru Hisabori

doi:10.21769/BioProtoc.5706

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Peer-reviewed

Evaluating Thioredoxin-Mediated CF_oCF₁ Reduction Using an In Vitro Thylakoid Assay

利用体外类囊体实验评估硫氧还蛋白介导的 CFoCF1 还原

TS Takatoshi Sekiguchi

KY Keisuke Yoshida

TH Toru Hisabori email

发布: 2026年06月05日第16卷第11期 DOI: 10.21769/BioProtoc.5706 浏览次数: 255

评审: Felix BuchertAnonymous reviewer(s)

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Cover of The Journal of Biological Chemistry, featuring study using the protocol.

Sep 2024

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Abstract

The activity of chloroplast ATP synthase (CF_oCF₁) is precisely regulated through a thioredoxin (Trx)-mediated dithiol/disulfide reaction in response to varying light conditions. This regulatory mechanism is further controlled by ΔpH formation across the thylakoid membrane. To better understand this complicating regulatory function of CF_oCF₁, a method is required to evaluate the extent of CF_oCF₁ reduction by Trx under controlled ΔpH conditions and to directly evaluate the redox state of CF_oCF₁. In this study, we present a simple in vitro procedure to assess the CF_oCF₁ reduction system using spinach thylakoids. The method consists of three key steps: (A) simple preparation of intact thylakoids from spinach leaves; (B) reduction of CF_oCF₁ on the thylakoid membrane using recombinant Trx under light irradiation; and (C) in situ determination of the redox state of CF_oCF₁ by labeling thiol groups with a maleimide reagent followed by protein detection using western blotting. The redox state of CF_oCF₁ was determined by mobility shifts on non-reducing SDS-PAGE. This protocol provides a refined strategy for elucidating the regulatory mechanism controlling energy conversion by CF_oCF₁ under fluctuating photosynthetic conditions.

Key features

• A simple isolation method for intact thylakoids from Spinacia oleracea that enables the evaluation of the reduction of chloroplast ATP synthase by thioredoxin.

• The combination of LED light irradiation and electron mediators allows controlled adjustment of the proton electrochemical gradient across thylakoid membranes.

• The redox state of chloroplast ATP synthase can be distinguished by labeling free thiols with maleimide reagents and quantitative detection by western blotting.

Keywords: Chloroplast ATP synthase (叶绿体 ATP 合酶)

Thioredoxin (硫氧还蛋白)

Thylakoid membrane (类囊体膜)

Redox regulation (氧化还原调控)

Thiol labeling (巯基标记)

Western blotting (Western blot)

Spinacia oleracea (菠菜)

Graphical overview

Background

Chloroplast ATP synthase (CF_oCF₁) is a pivotal enzyme in photosynthetic energy conversion in green plants. When photosynthetic electron transport generates a proton electrochemical gradient (ΔμH⁺) across the thylakoid membrane under light conditions, this gradient is primarily consumed by CF_oCF₁ to drive ATP synthesis [1–3]. Therefore, CF_oCF₁ activity is strongly regulated to maintain efficient energy conversion under varying light conditions. One representative regulatory mechanism is redox control [4,5]. The γ subunit (CF₁-γ), which rotates within the enzyme complex during catalysis, harbors a redox-active cysteine pair [6,7]. These cysteines are targets of thioredoxin (Trx), which mediates the reducing power from the photosynthetic electron transport chain [8]. Furthermore, CF₁-γ is reduced by Trx only when ΔpH is formed across the thylakoid membrane under photosynthetic conditions [9–11]. Thus, CF_oCF₁ is regulated through a multistep control mechanism involving Trx-mediated redox reactions, closely linked to ΔpH formation, reflecting changes in light environment.

Over the past half-century of research on CF₁, including by our research group, methods for examining the redox state of CF₁-γ have been established. Jagendorf first demonstrated ATP synthesis driven by ΔpH across thylakoid membranes in isolated chloroplasts using the so-called acid–base transition experiment, in which chloroplasts were sequentially incubated in buffers of different pHs [12]. Subsequently, Gräber et al. applied the acid–base transition method to isolated thylakoids or liposomes containing isolated CF_oCF₁ in the presence of excess reducing agents, enabling kinetic analysis of the relationship between the redox state of CF₁-γ and CF_oCF₁ enzymatic activity [13,14]. In another approach, Schwarz et al. investigated the selectivity of Trx toward CF₁-γ by measuring ATP hydrolysis activity under defined pH gradients across thylakoid membranes in the presence of different Trx isoforms [15]. However, these studies focused primarily on the enzymatic activity of CF_oCF₁ rather than directly determining the exact redox state of CF₁-γ. Because the extent of ΔpH affects both ATP synthesis and hydrolysis activities of CF_oCF₁, a system capable of directly monitoring the redox state of CF₁-γ under ΔpH-forming conditions, independent of enzyme activity measurements, is required. In previous work, our laboratory successfully determined the redox state of CF₁-γ by directly applying a maleimide reagent to isolated spinach chloroplasts under light conditions [16]. Using this approach, we observed that CF₁-γ is rapidly reduced upon light irradiation, and that reduction is completely inhibited in the presence of an uncoupler in the chloroplast solutions.

In this study, we refine this conventional approach and provide a simple protocol for quantitative reduction analysis of CF₁-γ using isolated spinach thylakoids combined with an artificial electron transfer mediator, which is required for membrane potential formation in the light [17], and recombinant Trx. Using this method, we successfully demonstrated the selectivity of major Trx family proteins toward CF₁-γ [18], oxidation of CF₁-γ by Trx-like proteins associated with ΔpH dissipation [19], and the distinct contributions of ΔpH and membrane potential to CF₁-γ reduction [20], supporting the sensitivity and accuracy of this method.

Materials and reagents

Biological materials

1. Fresh bunched spinach (Spinacia oleracea) (purchased from the market)

Reagents

1. Distilled water

2. Tricine (Wako, catalog number: 347-02844)

3. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Nacalai Tesque, catalog number: 20909-55)

4. Sodium chloride (NaCl) (Nacalai Tesque, catalog number: 31320-05)

5. Potassium chloride (KCl) (Nacalai Tesque, catalog number: 28513-85)

6. Sodium hydroxide (NaOH) (Nacalai Tesque, catalog number: 31511-05)

7. Sucrose (Nacalai Tesque, catalog number: 30404-45)

8. Dithiothreitol (DTT) (Wako, catalog number: 049-08972)

9. 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS) (Sigma-Aldrich, catalog number: M8640)

10. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920)

11. Ethanol (Nacalai Tesque, catalog number: 14713-24)

12. Sodium lauryl sulfate (SDS) (Nacalai Tesque, catalog number: 31607-65)

13. Glycerol (Nacalai Tesque, catalog number: 17018-83)

14. Bromophenol blue (BPB) (Wako, catalog number: 029-02912)

15. 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonate (AMS) (Invitrogen, catalog number: A485)

16. Tris(hydroxymethyl)aminomethane (Tris) (Nacalai Tesque, catalog number: 35434-21)

17. Glycine (Nacalai Tesque, catalog number: 17141-95)

18. Polyoxyethylene (20) sorbitan monolaurate (Tween 20) (Wako, catalog number: 167-11515)

19. Methanol (Nacalai Tesque, catalog number: 21914-74)

20. Disodium hydrogen phosphate (Nacalai Tesque, catalog number: 31801-05)

21. Potassium dihydrogen phosphate (Nacalai Tesque, catalog number: 28721-55)

22. SDS-PAGE protein marker (Bio-Rad, catalog number: 161-0373)

23. ECL Prime western blotting detection reagent (GE Healthcare, catalog number: RPN2232)

24. Primary antibody for the protein of interest

25. Secondary antibody for the protein of interest

26. Skim milk (Morinaga Nyugyo, catalog number: 0652842)

27. 100% (w/v) trichloroacetic acid (TCA) (Wako, catalog number: 200-08085)

28. Acetone (Wako, catalog number: 019-00353)

Solutions

1. 4× stock solution for grinding buffer (see Recipes)

2. Grinding buffer (see Recipes)

3. 25× DTT stock solution (see Recipes)

4. 50× PMS stock solution (see Recipes)

5. FCCP stock solution (see Recipes)

6. Thiol-labeling solution (see Recipes)

7. Electrophoresis buffer for SDS-PAGE (see Recipes)

8. Transfer buffer for western blotting (see Recipes)

9. PBS buffer (see Recipes)

Recipes

1. 4× stock solution for grinding buffer

Reagent	Final concentration	Quantity or volume
Tricine	200 mM	35.8 g
MgCl₂·6H₂O	20 mM	4.06 g
NaCl	40 mM	2.34 g
KCl	200 mM	14.9 g
Distilled water	N/A	Up to 1 L
Total	N/A	1 L

Adjust pH to 7.5 using NaOH. Prepare in a media bottle and store at 4 °C in a refrigerator; it is stable for several months.

2. Grinding buffer

Reagent	Final concentration	Quantity or volume
4× stock solution for grinding buffer	N/A	100 mL
Sucrose	0.4 M	54.8 g
Distilled water	N/A	Up to 400 mL
Total	N/A	400 mL

Prepare in a media bottle. Always use a freshly prepared grinding buffer for each experiment and maintain it at low temperature until just before use.

3. 25× DTT stock solution

Reagent	Final concentration	Quantity or volume
DTT	500 mM	77.1 mg
Distilled water	N/A	1 mL

Prepare in a microcentrifuge tube and store at -20 °C in a freezer; stable for several months. Dilute the 25× DTT stock solution to 1× (2 mM DTT) with grinding buffer before use.

4. 50× PMS stock solution

Reagent	Final concentration	Quantity or volume
1-Methoxy PMS	10 mM	3.36 mg
Distilled water	N/A	1 mL

Prepare in a microcentrifuge tube and store in a storage box away from light at room temperature; stable for several months. Dilute the 50× PMS stock solution to 1× (200 μM 1-Methoxy PMS) with grinding buffer before use.

5. FCCP solution

Reagent	Final concentration	Quantity or volume
FCCP	2 mM	1.02 mg
Ethanol	N/A	2 mL

Prepare in a microcentrifuge tube and store at -20 °C in a freezer; stable for several months.

6. Thiol-labeling solution

Reagent	Final concentration	Quantity or volume
Tris	62.5 mM	75.7 mg
SDS	2% (w/v)	200 mg
Glycerol	7.5% (w/v)	750 mg
BPB	0.01% (w/v)	1 mg
AMS	2 mM	10 mg
Distilled water	N/A	Up to 10 mL
Total	N/A	10 mL

Adjust pH to 6.8 using HCl. Prepare in a conical tube. Always use a freshly prepared thiol-labeling solution for each experiment. Store in a storage box away from light at room temperature until just before use.

7. Electrophoresis buffer for SDS-PAGE

Reagent	Final concentration	Quantity or volume
Tris	25 mM	30.25 g
Glycine	192 mM	144.1 g
SDS	0.1% (w/v)	10 g
Distilled water	N/A	Up to 10 L
Total	N/A	10 L

Prepare in a solution tank and store at room temperature; stable for several months.

8. Transfer buffer for western blotting

Reagent	Final concentration	Quantity or volume
Tris	40 mM	24.2 g
Glycine	242 mM	90.7 g
SDS	0.1% (w/v)	5 g
Ethanol	20% (v/v)	1 L
Distilled water	N/A	Up to 5 L
Total	N/A	5 L

Prepare in a solution tank and store at room temperature; stable for several months.

9. PBS buffer

Reagent	Final concentration	Quantity or volume
NaCl	137 mM	80 g
KCl	2.69 mM	2 g
Na₂HPO₄	7.75 mM	11 g
KH₂PO₄	1.47 mM	2 g
Tween 20	0.4% (v/v)	20 mL
Distilled water	N/A	Up to 10 L
Total	N/A	10 L

Prepare in a solution tank and store at room temperature; stable for several months.

Laboratory supplies

1. Newspaper

2. 1.5 mL microcentrifuge tube (WATSON, catalog number: 131-7155C)

3. 10 μL pipette tips (WATSON, catalog number: 110-201C)

4. 200 μL pipette tips (WATSON, catalog number: 110-705C)

5. 1,000 μL pipette tips (WATSON, catalog number: 110-804C)

6. Gauze (Iwatsuki, catalog number: 002-21540)

7. Absorbent paper (ATTO, catalog number: CB-09A)

8. Immun-Blot PVDF membrane (Bio-Rad, catalog number: 1620177)

Equipment

1. 10 μL pipette (Gilson, model: P10, catalog number: F144802)

2. 20 μL pipette (Gilson, model: P20, catalog number: F123600)

3. 200 μL pipette (Gilson, model: P200, catalog number: F123601)

4. 1,000 μL pipette (Gilson, model: P1000, catalog number: F123602)

5. Blender (Kuvings, model: KPB-351SP)

6. High-speed refrigerated centrifuge (Himac, model: CR20GIII)

7. Refrigerated microcentrifuge (TOMY, model: MX-307)

8. Equipment for SDS-PAGE (NIHON EIDO, model: NA-1012)

9. Equipment for western blotting (BIO CRAFT, model: BE-320)

10. Luminescence image analyzer (Fujifilm, model: LAS-3000 mini)

11. Spectrofluorometer (Jasco, model: FP-6500)

12. Quartz cell

13. Paintbrush

14. LED light (CCS Inc., model: ISLM-150150-HWHR)

15. Grass tube (AS ONE, catalog number: 14101005B)

16. Conical beaker (AS ONE, catalog number: 1-7117-03)

17. Funnel (AS ONE, catalog number: 6-319-06)

18. Magnetic stirrer (TOMY, model: FS-210)

Software and datasets

1. ImageJ, http://imagej.nih.gov/ij//

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Mar 4, 2026

接收日期: Apr 22, 2026

在线发布日期: May 9, 2026

出版日期: Jun 5, 2026

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Sekiguchi, T., Yoshida, K. and Hisabori, T. (2026). Evaluating Thioredoxin-Mediated CF_oCF₁ Reduction Using an In Vitro Thylakoid Assay. Bio-protocol 16(11): e5706. DOI: 10.21769/BioProtoc.5706.
Sekiguchi, T., Yoshida, K., Wakabayashi, K. I. and Hisabori, T. (2024). Proton gradient across the chloroplast thylakoid membrane governs the redox regulatory function of ATP synthase. J Biol Chem. 300(9): 107659. https://doi.org/10.1016/j.jbc.2024.107659