Article Figures & Data
Figures
- Fig. 1.
In vitro protein import assay of pFdI and pFdIII in the light versus in the dark. Intact chloroplasts were incubated with in vitro-synthesized pFdI (A) or pFdIII (B) in a standard import reaction either in the light or in the dark as described in “Materials and Methods.” Reactions, which were incubated in the dark, had 5 mm Mg-ATP added to them prior to incubation. The chloroplasts were reisolated and protease accessibility after import reaction was assayed by the incubation of reisolated chloroplasts with 60 μg/mL trypsin on ice for 20 min. Proteins were separated by centrifugation and analyzed by SDS-PAGE followed by fluorography.
- Fig. 2.
pFdIII accumulated in the light was present in a soluble compartment of chloroplasts. A, pFdIII was incubated with chloroplasts as described in Figure 1. After the incubation, chloroplasts were recovered by centrifugation and treated with or without trypsin. The chloroplasts were reisolated and resuspended in a hypotonic buffer. Soluble (S) and membrane (P) fractions were separated by centrifugation and analyzed by SDS-PAGE followed by fluorography. Note that all trypsin-resistant pFdIII proteins were recovered in the soluble fraction of chloroplasts. B, After import of pFdIII either in the light or in the dark, trypsin treatment (0, 10, or 20 μg/mL of trypsin, on ice for 5 min) was performed as indicated either in the presence or absence of 0.2% (w/v) Triton X-100.
- Fig. 3.
pFdIII accumulated in the intermembrane space of chloroplasts in the light. After import of pFdIII in the light, trypsin treatment was carried out either on ice or at room temperature (r.t.) for 10 min as indicated. Proteins were separated by SDS-PAGE and then analyzed by fluorography (A) or by immunoblotting using antisera raised against ferredoxin (B), Tic110 (C), Toc34 (D), or Toc75 (E).
- Fig. 4.
Accumulated pFdIII did not undergo subsequent import into the stroma in the dark. A, Chase experiment of accumulated pFdIII. After import of pFdIII in the light (first incubation), chloroplasts were recovered and further incubated in the dark (second incubation) and analyzed as described in Figure 1. B, Chloroplasts were preincubated in the import buffer in the light for the indicated time. Then in vitro-synthesized pFdIII was added to each reaction mixture. The mixture was further incubated for 20 min in the light or in the dark and analyzed as above.
- Fig. 5.
Gel filtration chromatography of pFdIII accumulated in the light. After import of pFdIII in the light or in the dark, chloroplasts were recovered and their soluble fractions purified as described in Figure 2. Gel filtration analysis was performed with a Superose 6 column (Pharmacia Biotech, Piscataway, NJ) that had been equilibrated with 20 mm HEPES-KOH (pH 8.0) and 150 mm KCl using a SMART system (Pharmacia Biotech). Fractions obtained by gel filtration were analyzed as described in Figure 2. As a control, in vitro-translated pFdIII was also chromatographed and analyzed.
- Fig. 6.
pFdIII accumulated in the light was able to be processed by stromal processing protease. The soluble fraction of the chloroplast, which contained the pFdIII accumulated under light conditions, was incubated with a concentrated stromal fraction (10-fold excess amounts as compared with that had been contained in the chloroplasts used in the initial import reaction) that had been prepared from maize chloroplasts as indicated B. As control experiments, in vitro-synthesized pFdIII (A) and pFdI (C) were also separately incubated with the concentrated stromal fraction.
- Fig. 7.
pFdIII recovered after light-induced accumulation in the intermembrane space was able to be reimported into chloroplastic stroma in the dark. The soluble fraction of the chloroplasts, which contained this pFdIII, was incubated with fresh chloroplasts either in the light or in the dark and then analyzed as described in Figure1.
- Fig. 8.
The presequence of pFdIII was responsible for accumulation of the unprocessed form of precursor in the intermembrane space in the light. Two chimeric proteins were translated in vitro: One consists of the presequence of pFdI and mature domain of FdIII (pFdI-III) and the other consists of the presequence of pFdIII and mature domain of FdI (pFdIII-I). These chimeric proteins and authentic pFdI and pFdIII were used independently in in vitro import assays with chloroplasts in the light or in the dark. After import, chloroplasts were recovered and analyzed as described in Figure 1.
- Fig. 9.
Accumulation of precursor proteins in the intermembrane space in the light was observed in the case of pFNRII. In vitro import assays of pFNRI (A) and pFNRII (B) with maize chloroplasts were carried out as described in Figure 1.
- Fig. 10.
Light-induced mis-sorting of precursor proteins to the intermembrane space also occurred in chloroplasts prepared from pea. A, Maize pFdI and pFdIII and pea pFNRI were used independently in in vitro import assays with chloroplasts in the light or in the dark for 20 min. After import, chloroplasts were recovered, and protease-resistant proteins were analyzed as described in Figure 1. B, After import of pFdIII in the light into the pea chloroplasts, trypsin treatment was carried out either on ice or at room temperature (r.t.) for 10 min as indicated. Proteins were separated by SDS-PAGE and then analyzed by fluorography for FdIII (upper) or by immunoblotting using antisera raised against pea Tic22 (lower). Note that an asterisk corresponds to a position of an unknown thylakoid protein, which was cross-reacted with the anti-pea Tic22 serum.
