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Fig. 5. Chemical crosslinking of inner envelope protein 32 (IEP32) and Rubisco small subunit pSSU pre-proteins to chloroplast translocon subunits. (A) IEP32 or pSSU [³⁵S]-labelled translation products (lanes 2,5) were incubated with 0.5 mM DSP either in the absence (lanes 1,6) or presence (lanes 3,4) of purified pea chloroplasts for 10 minutes at 3 mM ATP and separated by SDS-PAGE. Arrows indicate crosslinked protein products of IEP32 (lane 3) and Rubisco small subunit pSSU (lane 4). (B-E) Chloroplasts were incubated with [³⁵S]-labelled translation products under different conditions. Crosslinking was carried out for 30 minutes at 4°C in the presence of 0.5 mM DSP. Chloroplasts were then solubilized by 1% SDS and immunoprecipitation carried out after dilution to 0.1% SDS with antisera to various receptor/membrane proteins as indicated. (B) Chloroplasts were incubated for 5 minutes at 4°C in the presence of <20 µM ATP and <100 µM GMP-PNP. (C) Chloroplasts were incubated with preproteins for 5 minutes at 4°C in the presence of <20 µM ATP. (D) Chloroplasts were incubated with pre-proteins for 5 minutes at 25°C and 3 mM ATP. (E) Chloroplasts were incubated with pre-proteins for 15 minutes at 25°C and 3 mM ATP. C, total chloroplasts; E, eluate from protein A agarose by SDS-sample buffer in the presence of mercaptoethanol to split the crosslink product; W, wash of protein A Sepharose with buffer.

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