As06 172

Proteins were extracted from approximately 20 million cells, collected via centrifugation (12,000×g, 10 min) and resuspended in protein extraction buffer (50 mM Tris–HCl; pH 8, 2% SDS, 10 mM EDTA). After resuspension, the cells were frozen in liquid nitrogen, followed by thawing in a 45 °C water bath. This was repeated three times in rapid succession, after which the debris was removed via centrifugation (15,000×g, 5 min).
The relative amounts of PSII and PSI were estimated from Western blots with antibodies for two of their core proteins, CP43 and PsaA, respectively. Proteins were first separated with SDS-PAGE, using 1 µg (for CP43) or 2 µg (for PsaA) of protein per well. These amounts of protein were found to be optimal for detection through dilution series. Primary antibodies for CP43 (Agrisera, product no. AS06 110) and PsaA (Agrisera, product no. AS06 172) were used in concentrations of 1:6000 and 1:5000, respectively. The secondary antibody, goat-anti rabbit IgG (H + L), alkaline phosphatase conjugate (Life technologies, REF G21079) was used in final concentration of 1:50 000 and the binding was detected via luminescence caused by alkaline phosphatase. Relative amounts of the proteins were calculated from signal intensities, quantified with the image processing software Fiji (Fiji Is Just ImageJ, v. 1.52).

The intracellular contents of ATP and ATP + ADP were measured according to a method reported previously^{27 (link)}. The level of F_oF₁ accumulation was assessed using immunoblot analysis with the membrane fraction and antibodies against the β-subunit of F_oF₁ as described previously^{27 (link)}. At the late-log phase, 1 L of cells was harvested via centrifugation, and these cells were flash-frozen in liquid nitrogen and stored at − 80 °C until use. The cells were resuspended in a buffer containing 20 mM of Hepes–KOH (pH 8.0), 10 mM of NaCl, 0.1 mM of MgCl₂, and 0.1 mM of ATP and broken up via vortexing with zircon beads, after which the homogenate was centrifuged for 10 min at 3000 × g and 4 °C to remove cell debris. The supernatant was then centrifuged for 30 min at 125,000 × g and 4 °C to precipitate the thylakoid membranes. The chlorophyll concentration of the membrane fraction obtained was then quantified using the methanol extraction method described above and subsequently used for immunoblot analysis. For PSII and PSI analyses, whole-cell extracts and the antibodies against PsbA and PsaA were used (AS06 124A and AS06 172; supplied by Agrisera, Sweden).

Algal cells were broken twice using a Cell Disruptor (Constant System Cell Disruptor) with a pressure of 20 kPSI (1350 bar). Thylakoid membranes were purified by sucrose cushion centrifugation as described previously⁵⁷ . The thylakoids in the appropriate buffer were immediately frozen in liquid nitrogen and stored at −80 °C until further use. Care was taken to avoid repeated freezing and thawing of the thylakoid membrane samples, and the sample preparation for native gel electrophoresis was performed rapidly under dim light at 4 °C. Aliquots of 5 μg Chl of thylakoid membrane samples were solubilized by 2% β-dodecylmaltoside and subsequently separated by blue-native PAGE. Polyclonal antibodies for PsaA (AS06172), D1 (AS10704), and Cyt b₆ (AS184169) (Agrisera, Sweden) were used to identify components in the various electrophoresis bands by immunoblot analysis. The polypeptide composition in PAGE bands of interest was determined upon band excision and in-gel tryptic digestion, followed by identification by mass spectrometry.