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

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.