Phosphatidylglycerol

The thylakoid lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG) and phosphatidylcholine (PC), were purchased from Larodan Fine Chemicals (Malmö, Sweden) and dissolved in chloroform. Lipids were mixed in the following relative molar ratios: 41.2% MGDG, 26.7% DGDG, 15.6% SQDG, 11.5% PG, and 5% PC (Natali et al., 2016) (link). 140 µl lipids were mixed with 5.84 ml of chloroform. 500 µl of the solution was pipetted into a glass vial and the chloroform was slowly removed under N2 vapor in order to form a thin film of lipids on the inner wall of the vial. Lipids were rehydrated (using 500 µl buffer (50 mM TRIS-HCl pH 8.0, 2 mM KCl or 20 mM KCl; 50 mM MES pH 6.5, 2 mM KCl or 20 mM KCl) and vortexed (three times, each 5 min). Extrusion was performed 3 times through a 0.2 μm filter using the Extrusion was performed 3 times through a 0.2 μm nuclepore membranes (Whatman, GE Healthcare) using a homemade extrusion setup leading to liposomes of about 100 nm as measured by dynamic light scattering (Nano-S, Malvern, Sysmex, Norderstedt, Germany). 20 µl MBP-OsPTOX (1 mg protein ml -1 ) was added to 200 µl liposomes, vortexed, deposited on a sucrose gradient (30%/15%) and centrifuged at 30 000g, 1h, 4°C. 200 µl were collected from the top of the gradient and used for further analyses.