Extended Data Fig. 5: Polypeptide composition of the affinity-purified PBF8-HA preparation.
From: Uncovering the photosystem I assembly pathway in land plants
a, Separation of chlorophyll–protein complexes of thylakoid membranes from the wild type (WT) and pbf8 PBF8-HA#1 by SDG ultracentrifugation. The purified thylakoid membranes were subjected to SDG ultracentrifugation; the resulting fractions were analyzed by immunoblotting using specific antibodies against PsaA, HA, and PBF8. Four chlorophyll–protein complexes were separated by SDG centrifugation in the WT and pbf8 PBF8-HA; left to right: LHCII monomer (LHCII-M), LHCII trimer (LHCII-T), PSII, and PSI–LHCI. b, Separation of chlorophyll–protein complexes of thylakoids from the WT and pbf8 PBF8-HA by BN-PAGE, and immunoblot analysis of chlorophyll–protein complexes separated by 2D BN-PAGE/SDS-PAGE using specific antibodies against PsaA and PBF8. Thylakoids isolated from the WT and pbf8 PBF8-HA#1 were solubilized using 1% (w/v) β-DM and resolved by BN-PAGE. NDH–PSI, NDH–PSI supercomplex; PSII SC, PSII supercomplexes; PSI-LHCI/PSII-D, PSI-LHCI and dimeric PSII; PSII-M, monomeric PSII; CP43 free PSII, monomeric PSII without CP43; LHCII-T, trimeric LHCII. Thylakoids from the WT and pbf8 PBF8-HA contained 10 μg chlorophyll. c, Polypeptide composition of the affinity-purified PBF8-HA preparation separated by SDS-PAGE and visualized by staining with Coomassie brilliant blue. PSI–LHCI, purified mature PSI–LHCI from the WT used as a reference. PBF8-HA (IP), affinity-purified PBF8-HA preparation from pbf8 PBF8-HA#1 plants. Control sample, protein sample used as a negative control that was obtained from the WT following the same procedure used to purify the PBF8-HA preparation from pbf8 PBF8-HA#1 plants. The arrow indicates the band corresponding to PBF8-HA. d, Polypeptide composition of the affinity-purified PBF8-HA preparation, as detected by immunoblotting. PSI–LHCI, purified mature PSI–LHCI from the WT used as a reference. PBF8-HA (IP), affinity-purified PBF8-HA preparation from pbf8 PBF8-HA#1 plants. Control sample, protein sample used as a negative control that was obtained from the WT following the same procedure used to purify the PBF8-HA preparation from pbf8 PBF8-HA#1 plants. e, Immunoblot analysis of the fractions of the affinity-purified PBF8-HA preparation after SDG ultracentrifugation (related to Fig. 4b). The purified PBF8-HA preparation was concentrated and subjected to SDG ultracentrifugation; the gradient fractions were analyzed by immunoblotting using specific antibodies against PSI subunits (PsaG, PsaJ, PsaK, PsaN, PsaO, and Lhca1–4). In a–e, three independent biological replicates were performed, and a representative replicate is shown.
