Fig. 5. Interaction of FPB1 and PAM68 with chloroplast proteins.

a Co-immunoprecipitation of FPB1/PAM68-containing assembly intermediates. WT thylakoids were solubilized with 1% β-DM and incubated with CNBr-activated agarose coupled with preimmune serum (Pre) as well as purified FPB1 and PAM68 antibodies. Co-immunoprecipitated proteins were separated by SDS-PAGE and then immunoblotted with various antibodies. Thylakoids corresponding to 0.1 µg chlorophyll (IB for D1, D2, CP43, and CP47) or 2.5 µg chlorophyll (IB for FPB1, PAM68, and Alb3) were used as reference of the blots. IgG eluted from the beads or unknown proteins are indicated with asterisks. b Immunoprecipitation of FPB1/PAM68-crosslinked proteins. Thylakoid proteins were crosslinked by DSP and then solubilized with SDS. The samples were immunoprecipitated and analysed as in a. c Immunoprecipitation of crosslinked (+ DSP) and not crosslinked (-DSP) proteins with CNBr-activated agarose coupled with purified YCF4 antibody. Immunoblots were performed as in (a and b). The migration of PAM68 compared to the 15-kDa molecular weight marker is slightly different in SDS-PAGE (a-c) and SDS-urea-PAGE (Supplementary Figs. 5 and 6c). d Split-ubiquitin yeast two hybrid assays for protein-protein interactions. The Cub was fused to the N-terminus of mature FPB1 (Cub-FPB1) as well as the C-terminus of Alb3 (Alb3-Cub) and SecY1 (SecY1-Cub) as baits. Other proteins were fused to NubG at their C- or N-termini as preys. Bait and prey constructs were cotransformed into yeast NMY32 strains and the positive clones were grown on the SD-leu-trp-his-ade medium for 2–4 days at 28 °C. Alg5-NubI and Alg5-NubG were used as positive and negative controls, respectively. A series of dilutions were spotted as indicated. Data are representative of two independent biological replicates. Source data are provided as a Source Data file.