Fig 2: Immunoblot analysis (a, b) and Coomassie blue-stained SDS-PAGE gel (c) of chloroplast proteins from the wild type (WT) and the lpa2 mutants. Results from immunoblotting with antibodies against PSII–LHCII are shown in (a), where specific antibodies recognizing the PSII core subunits D1, D2, CP43, CP47, PsbO and PsbP and the LHCII antenna complexes LHCB4 (CP29), LHCB5 (CP26) and LHCBM were used. For LHCBM complexes in particular, the antibody LHCBM5 was adopted, which was reported to recognize all of the different LHCBM subunits. Results from immunoblotting with antibodies against PSI core (a-PsaA), LHCI antenna (a-LHCA3, a-LHCA4), Cyt f (a-PetA), chloroplast ATPase ß-subunit (a-AtpB), large subunit of RUBISCO (a-RbcL) and LHCSR3 are shown in (b). Each lane was loaded on a per chlorophyll (Chl) basis (1 µg), and the WT samples were loaded at three different concentrations (0.25, 0.5 and 1 µg). The levels of proteins in the lpa2#1 and lpa2#2 mutants compared with the WT are presented next to the protein bands expressed as percentages of the WT level (n = 3; the values shown are means ± SDs). The statistical significance of differences between WT and lpa2 is indicated as *P < 0.05 and **P < 0.01, determined by Student’s t-test. In (d), the protein content of D1, D2, CP43 CP47, PsaA, PetA, AtpB and RUBISCO was normalized to the D2 content, with the different ratios set as 1 in the case of the WT (black line). (e) Autoradiography of immunoprecipitated PSII complexes. Chlamydomonas reinhardtii WT and lpa2 mutant cells were grown in TAP medium under low light (70 µmol m-2sec-1) in the presence of [35S] methionine and [35S] cysteine with the different times (pulses) indicated above the figure in minutes. PSII core complexes were extracted from thylakoid membranes by membrane solubilization and immunoprecipitation using the D2 antibody. Chase experiments were performed after 60 min of pulse removing the labelled amino acids from the growth medium. The bands corresponding to D1, D2, CP43 and CP47 were identified according to their migration pattern, as reported in (f), where immunoblotting results were obtained on thylakoid membranes isolated from the WT and loaded on the same SDS-PAGE gel system as in (e).

Fig 3: Design of the atpB-3×FLAG constructThe strain harbors the pVZ322 vector containing the construct in which the PpetE promoter drives transcription of the atpB coding sequence, which is fused to a segment encoding the 3×FLAG tag followed by a stop codon, the 3' UTR of the atpT mRNA and the transcription terminator oop (Toop). Coding sequences are colored in pink, the stop codon is indicated.