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Fig. 4. Inner envelope protein 32 (IEP32) import into chloroplasts is not affected in the presence of different Toc translocon inhibitors. (A) Import of IEP32 and Rubisco small subunit pSSU was carried out in the presence of the chloroplast precursor pOE33 at a final concentration of 200 ng/µl as outlined in the Materials and Methods. Lanes 2,3 and lanes 6,7 show import reactions in the presence of a single pre-protein IEP32 and pSSU, respectively. Lanes 4 and 5 show a competition experiment in the presence of both pre-proteins. (B) Import of IEP32 was assayed in the presence of increasing pOE33 competitive concentrations as indicated. (C) Import of IEP32 and pSSU was conducted in the absence (lanes 2,3) or presence (lanes 4,5) of 5 mM spermine. In lanes 6 and 7 chloroplasts were pretreated with 1 mm CuCl₂ for 20 minutes at 4°C and used for import under standard conditions. All other conditions were as outlined and as described in Fig. 1A. (D) Chloroplasts were treated with 1 mM DEPC prior to carrying out the import reaction. All other conditions were as before. TIM: translocation intermediate.

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