FIG. 5.
The pBrp proteins are kept in an inactive state at the cytoplasmic surface of plastid envelope. (A) The A. thaliana pBrp amino-terminal domain lacks chloroplast-targeting properties. Subcellular localization of the transiently expressed Tp-RecA/GFP and Nt-pBrp/EYFP fusion proteins was monitored by confocal laser scanning microscopy. Bars, 10 μm. (B) Western blot analysis of protein fractions (∼25 μg) from spinach chloroplasts (lanes C), stroma (lanes S), and membranes (lanes M) with anti-pBrp, anti-SIG3, and anti-OE24 antibodies is shown on the right. OE24 (an outer plastid envelope protein) and SIG (SIG3, a sigma-like transcription factor) were used as membrane and stroma control proteins, respectively. Coomassie blue staining of the corresponding fractions is shown on the left. (C) Western blot analysis of protein fractions (∼25 μg) from thylakoids (lanes T) and envelopes (lanes E) with anti-pBrp and anti-IE37 antibodies. IE37 (an inner plastid envelope protein) was used as an envelope control protein. Coomassie blue staining of the corresponding fractions is shown on the left. (D) Western blot analysis of protein fractions of thermolysin-treated, Percoll-purified, intact spinach plastids with anti-pBrp, anti-OE24, and anti-L4 antibodies. Plastid L4 protein (a stromal ribosomal protein) was used as an intraplastidic control protein. The different lanes correspond to nontreated plastids (−) and plastids treated with increasing concentrations of thermolysin (Ther.) (from 10 to 400 μg/ml). (E) Bright-field images of intact spinach chloroplasts incubated with either preimmune serum (left) or anti-pBrp antibodies (1/100 dilution) (right).