Thylakoid membrane extraction, protein electrophoresis, and immunoblot analysis

The total protein extracts of the Synechococcus cells were obtained according to the previous methods^71,72: cells suspended in isolation buffer (50 mM HEPES-NaOH, pH 7.5, 30 mM CaCl₂, 800 mM sorbitol, and 1 mM ε-amino-n-caproic acid) were disrupted by vortexing with glass beads (212–300 μm in diameter) at 4 °C and centrifuged at 3,000 × g for 5 min to remove the glass beads and unbroken cells. From the total protein extracts thylakoid membranes and soluble proteins were fractioned as described previously:¹² membranes were pelleted by centrifugation at 17,000 × g for 30 min and resuspended in storage buffer (50 mM Tricine-NaOH, pH 7.5, 600 mM sucrose, 30 mM CaCl₂, and 1 M glycinebetaine).

Protein complexes in their native form from isolated membrane fractions of Synechococcus were studied by Blue Native (BN)-PAGE according to the previous methods⁷²: isolated membranes were washed with washing buffer (330 mM sorbitol, 50 mM Bis-Tris, pH 7.0, and 250 µg mL⁻¹ of Pefabloc, Sigma) and suspended in 20% glycerol (w/v), 25 mM Bis-Tris, pH 7.0, 10 mM MgCl₂, and 0.01 unit mL⁻¹ RNase-Free DNase RQ1 (Promega) at the final concentration of 20 µg protein µL⁻¹. The samples were incubated on ice for 10 min, and an equal volume of 3% n-dodecyl-b-D-maltoside was added. Samples were first solubilized for 10 min on ice and after that for 20 min at room temperature following centrifugation at 18,000 × g for 15 min to remove insoluble material. The supernatant was collected and mixed with 1/10 volume of 0.1 M EDTA and 1/10 volume of sample buffer (5% Serva blue G, 200 mM Bis-Tris, pH 7.0, 75% sucrose, and 1 M ε-amino-n-caproic acid). Samples were applied to NativePAGE Bis-Tris protein gels with 4−16% gradient (Thermo Scientific) and voltage was gradually increased from 50 V up to 200 V during the gel run. After electrophoresis, the proteins were transferred to a PVDF membrane (Immobilon-P; Millipore) and examined with protein-specific antibodies; α-PsaB (AS10 704 Agrisera) in dilution of 1:1000 and α-PsbD (AS06 146, Agrisera) in dilution of 1:4,000. Secondary antibody (AS09 602, Goat anti-Rabbit IgG (H&L), HRP conjugated, Agrisera) was applied in dilution of 1:10,000. 200 µg of proteins from the thylakoid membrane fraction per sample were loaded to BN-PAGE gels. For immunoblot analysis, 50 µg of proteins from the thylakoid membrane fraction per sample were loaded to BN-PAGE gels.

For denatured electrophoresis, crude thylakoid membrane proteins were denatured according to the previous methods;⁷³ Sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris-HCl, pH 6.8, 8% SDS, 400 mM dithiothreitol (DTT), 0.02% bromophenol blue) was added to thylakoid membrane samples and incubated at 75 °C for 10 min. Then, proteins were separated by 15% (w/v) SDS-PAGE, transferred to a PVDF membrane (Immobilon-P, Millipore) and analyzed with the protein-specific antibodies; α-PsaB (AS10 695, Agrisera) in dilution of 1000, α-PsbA (AS05 084, Agrisera) in dilution of 1:4000, α-PsbD (AS06 146, Agrisera) in dilution of 1:4,000, α-PetC (AS08 330, Agrisera) in dilution of 1:1500, α-AtpD (AS05 085, Agrisera) in dilution of 1:3,000, α-NdhV (a generous gift from Dr. Hualing Mi) in dilution of 1:4000 and α-GFP (A-11122, Invitrogen) in dilution of 1:5,000. Secondary antibody (AS09 602, Goat anti-Rabbit IgG (H&L), HRP conjugated, Agrisera) for all other primary antibodies besides for α-GFP was applied in dilution of 1:10,000 and secondary antibody (AS11 1772; Goat anti-Mouse IgG, HRP Conjugate, Agrisera) for α-GFP in dilution of 1:10,000.