Figure 6

PBR1 knockdown impairs accumulation of the Ycf1 protein in thylakoid membranes and association of the Ycf1 transcript with polysomes. (a) Analysis of thylakoid membrane protein complexes by Blue-Native polyacrylamide gel electrophoresis. Thylakoid membranes were isolated from wild-type (WT) and pbr1-1 mutant leaves and equal amounts of samples (3 μg chlorophyll) were separated. (b) The protein levels of Ycf1 analyzed by western blots in thylakoid membranes of the WT and pbr1-1 with an polyclonal antibody against Ycf1. Red arrows indicate the reduction in the protein levels of Ycf1 in the corresponding complexes in the pbr1-1 mutant compared with WT. (c–e) Association of the Ycf1, rbcL and psaA transcripts with polysomes. Whole-cell extracts were fractionated in linear 15–55% sucrose gradients by ultracentrifugation. Ten fractions with equal volume were collected from top to bottom of the sucrose gradients, and equal proportions of each fraction were analyzed by northern blotting with specific probes of Ycf1 (c), rbcL (d) and psaA (e), respectively. (f) RNA gel blots of polysome gradient fractions were stained with ethidium bromide (EtBr) to visualize distribution of rRNAs.