Figure 3.

Localization of the STN kinases within the thylakoid membrane. (A) Analysis of STN7 and STN8 localization by thylakoid membrane sub-fractionation. Thylakoids of PSII- or PSI-light adapted WT plants were fractionated into grana lamellae (10 K), grana margins (40 K) and stroma lamellae (150 K) by differential ultracentrifugation after digitonin solubilization. Proteins were separated by SDS-PAGE and analyzed by immunolabeling using antibodies against STN7, STN8, PsaF, PetC, PsbA, and AtpB. (B) Analysis of STN7 and STN8 distribution by 2D BN/SDS-PAGE. Thylakoid membranes of WT equivalent to 50 μg of Chl were solubilized with β-DM, subjected to 2D BN/SDS-PAGE and analyzed by Western blotting. STN7, STN8 and various subunits of the major photosynthetic complexes (AtpB, Lhcb2, PsaB, PetC, PetB, and PsbD) were detected with specific antibodies. Protein-Chl complexes are indicated by black arrowheads. The bands detected were identified as specific protein complexes in accordance with previously published profiles (Armbruster et al., 2013): PSI-NDH supercomplex (PSI-NDH), PSII supercomplexes (PSII super), PSI monomers and PSII dimers (PSI mon and PSII di), PSII monomers and dimeric Cyt b₆/f (PSII mon and Cyt b₆/f di), PSII monomers w/o CP43 (CP43-free PSII), multimeric (LHCII multi), trimeric (LHCII tri), and monomeric (LHCII mon) LHCII.