12132 h rotor

C. reinhardtii was grown mixotrophically in Tris-acetate-phosphate (TAP) medium at 25 °C with vigorous shaking 90 rpm under 50 µmol photon m⁻² s⁻¹ photosynthetically active radiation^{75 (link)}. Cultures (50 mL, 5 replicates) from the exponential phase were centrifuged at 4,000 g for 15 min at 4 °C (Beckman Coulter Allegra® X-15R Centrifuge (Pasadena, CA, USA); rotor: 4750 A), then stored at −80 °C. Cells were broken by sonication in lysis buffer (15 mM Tris, 0.1 mM EDTA at pH 7.5) supplemented by protease inhibitors 40 µg mL⁻¹ (Sigma-Aldrich, P2714); the homogenate was centrifuged at 11,000 g, 4 °C for 30 min (2–16KC centrifuge using a 12132-H rotor, Sigma-Aldrich, Saint-Louis, MO, USA) to isolate the supernatant that mainly contained non-membrane proteins.

Acid-resistant proteins were extracted by treating with 10% perchloric acid (PCA) or 5% trichloroacetic acid (TCA), on ice for 15 min. The heat-resistant proteins were extracted by boiling the samples at 98 °C for 5 min, 30 min and 1 h. After cooling to room temperature, the insoluble fractions were removed by centrifugation at 11,000 g, 4 °C for 30 min (2–16KC centrifuge using a 12132-H rotor, Sigma-Aldrich, Saint-Louis, MO, USA) and thus, only the soluble extracts containing heat-resistant or acid-resistant proteins were kept and analyzed further^{35 (link)}. Protein concentration was determined, using the Bio-Rad reagent protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard.