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Fig. 1. Import of inner envelope protein 32 (IEP32) into chloroplasts requires ATP. (A) Purified pea chloroplasts equivalent to 15 µg chlorophyll were incubated simultaneously with in vitro translated [³⁵S]-labelled pre-proteins (TL) Rubisco small subunit pSSU and IEP32 in the presence of 3 mM ATP (lanes 2,3) or <20 µM ATP (lanes 4,5), which was carried over from addition of the translation mixture. A post-ribosomal supernatant was used (see Materials and Methods). After incubation for 15 minutes at 25°C chloroplasts were repurified by centrifugation through a Percoll cushion at 4°C. Chloroplasts were then either treated or not treated with the protease thermolysin (Therm.-Post) as indicated. Intact chloroplasts were recovered by centrifugation and products analysed by SDS-PAGE and phospho-imager. Lanes 1 and 6 show 1/10 of the translation product (TL) added to each import reaction. pSSU was used as an endogenous control and was imported and processed as determined by the presence of the lower molecular weight form (mSSU). (B) IEP32 translation product (TL lane 1) was incubated with 6 M urea and separated into a soluble (S) and pellet (P) fraction by centrifugation for 10 minutes at 250,000 g. A post-ribosomal supernatant was obtained from the IEP32 translation product by centrifugation for 10 minutes at 250,000 g. Aggregated IEP32 recovered from the pellet (P) or soluble IEP32 from the supernatant (S) (lanes 2,3) was either not treated (lanes 4,5) or treated (lanes 6,7) with the protease thermolysin (Therm). (C) [³⁵S]-labelled IEP32 translation product was imported into chloroplasts as outlined in (A). Chloroplasts were then extracted by 6 M urea for 15 minutes at 25°C. Urea-insoluble proteins in the pellet (P) were separated from soluble proteins in the supernatant (S) by centrifugation and products analysed as outlined above. (D) Translation products were either treated (lanes 4,5) or not treated (lanes 2,3) with the ATP hydrolysing enzyme apyrase prior to the import reaction. All other conditions were as outlined in (A). (E) Chloroplasts were fractionated after import into outer (O.E) and inner envelope (I.E) membranes and the stroma (St).

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