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All steps were performed on ice or at 4 °C if not otherwise stated. 50 µg mitochondria were lysed in 25 µl lysis buffer (20 mM Tris pH7.4, 0.1 mM EDTA, 10% glycerol, 1.5 w/v digitonin, 1× Complete EDTA-free protease inhibitor cocktail) for 20 min. After lysis, insoluble material was removed by centrifugation (30 min, 17,000 × g). Next, 20 µl of supernatants were transferred into a new tube, supplemented with blue native loading dye (5% Coomassie blue G, 500 mM e-amino n-caproic acid in 100 mM Bis Tris pH 7.0) and loaded onto native PAGE 3–12% (Thermo Fisher). Gels were run at 150 V using NativePAGE Running buffer kit (Thermo Fisher). The activity of mitochondrial respiratory complexes was measured in-gel by addition of buffers containing OXPHOS substrates specific for Complex I (2 mM Tris pH 7.4, 0.1 mg/ml NADH, 2.5 mg/ml iodonitrotetrazolium chloride) or Complex IV (0.5 mg/ml diaminobenzidine (DAB), 50 mM phosphate buffer pH 7.4, 1 mg/ml cytochrome C, 0.2 M sucrose, 20 µg/ml catalase) at RT. Alternatively, gels were washed 15 min in 1× wet western blot buffer (40 mM glycine, 50 mM Tris and 20 % methanol) supplemented with 1% SDS and proteins were transferred onto PVDF membrane by western blotting. Before antibody incubation, membranes were destained by three 1 min washes with methanol.

Thylakoids are washed with the solubilization buffer (25 mM Bis–Tris/HCl [pH 7.0], 20% [w/v] glycerol, 10 mM sodium fluoride, and 0.2 mM PMSF) and resuspended in the same buffer at 1 mg Chl ml⁻¹. An equal volume of 2% α-DM was added to the thylakoid solution for 15 min on ice in the dark. Traces of insoluble material were removed by centrifugation at 18,000g for 20 min at 4 °C. The Chl concentration was measured, and proteins were loaded at equal Chl content in the native gel (NativePAGE 3–12%, Bis–Tris, 1.0 mm, Mini Protein Gel, 10-well from Thermo Fisher Scientific; catalog number: BN1001BOX). Prior to loading, the samples were supplemented with sodium deoxycholate (final concentration of 0.3%). The cathode buffer is 50 mM tricine, 15 mM Bis–Tris, 0.05% sodium deoxycholate and 0.02% α-DM, pH 7.0, and anode buffer is 50 mM Bis–Tris, pH 7.0. Electrophoresis was performed at 4 °C with a gradual increase in voltage: 75 V for 30 min, 100 V for 30 min, 125 V for 30 min, 150 V for 1 h, and 175 V until the sample reached the end of the gel. The method is adapted from the study of Ref. (68 (link)).