Figure 5

PBR1 regulates Ycf1 translation by binding to its mRNA. (a) PBR1-YFP (yellow fluorescent protein) signals are targeted to chloroplasts as examined in guard cells of epidermal peels. (b) The representative mass spectra for identification of Ycf1 protein in samples extracted from wild-type (WT) and pbr1-1 mutant leaves pulse-labeled with ‘heavy’ (H) and ‘medium heavy’ (M) stable isotope amino acids, respectively. The ratio of peak intensities of M versus H peptides reflects difference between the pbr1-1 mutant and WT in translation of the corresponding proteins as the newly synthesized proteins incorporate either the M or H amino acids. (c, d) The expression and protein levels of Ycf1 in WT, pbr1-1 and PBR1 overexpression line OE-5 analyzed by quantitative reverse transcriptase–PCR (c) and western blots (d), respectively. An Ycf1 polyclonal antibody was used, and equal protein loading was confirmed with antiserum against α-Tubulin (TUB). (e) Expression and purification of recombinant PBR1 protein in E. coli. (f, g) RNA-binding activity of PBR1 was detected by electrophoretic mobility shift assay (EMSA). Proteins were incubated with biotin-labeled probes derived from the 5′-UTR (untranslated region) or 3′-UTR of Ycf1 mRNA and separated by 6% native polyacrylamide gel electrophoresis. The specificity of binding bands was confirmed by cold probe competition.