TY  - JOUR
TI  - Evaluating Thioredoxin-Mediated CFoCF1 Reduction Using an In Vitro Thylakoid Assay
AU  - Sekiguchi, Takatoshi 
AU  - Yoshida, Keisuke 
AU  - Hisabori, Toru 
VL  - 16
IS  - 11
PY  - 2026
DA  - 2026/06/05
SP  - e5706
C1  - Bio-protocol 2026;16:e5706
DO  - 10.21769/BioProtoc.5706
UR  - https://doi.org/10.21769/BioProtoc.5706
AB  - The activity of chloroplast ATP synthase (CFoCF1) 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 CFoCF1, a method is required to evaluate the extent of CFoCF1 reduction by Trx under controlled ΔpH conditions and to directly evaluate the redox state of CFoCF1. In this study, we present a simple in vitro procedure to assess the CFoCF1 reduction system using spinach thylakoids. The method consists of three key steps: (A) simple preparation of intact thylakoids from spinach leaves; (B) reduction of CFoCF1 on the thylakoid membrane using recombinant Trx under light irradiation; and (C) in situ determination of the redox state of CFoCF1 by labeling thiol groups with a maleimide reagent followed by protein detection using western blotting. The redox state of CFoCF1 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 CFoCF1 under fluctuating photosynthetic conditions.
KW  - Chloroplast ATP synthase
KW  - Thioredoxin
KW  - Thylakoid membrane
KW  - Redox regulation
KW  - Thiol labeling
KW  - Western blotting
KW  - Spinacia oleracea
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False