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First published online July 30, 2004
doi: 10.1242/10.1242/jcs.01265

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Inner envelope protein 32 is imported into chloroplasts by a novel pathway

Department of Biology I, Botany, University of Munich, Menzinger Str. 67, Munich 80638, Germany

View larger version (26K): [in a new window]   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).

View larger version (27K): [in a new window]   Fig. 2. The N-terminus of inner envelope protein 32 (IEP32) is essential for targeting to chloroplasts. (A) The N-proximal 50 amino acids of IEP32 (GenBank accession no. AY488758) are shown in addition to the four deletion mutants. (B) Progressive N-terminal deletions of IEP32 were synthesized in vitro as radiolabelled preproteins, missing the first 10 (lanes 4-6), 20 (lanes 7-9), 30 (lanes 10-12) and 40 amino acids (lanes 13-15), respectively. Import and analysis conditions were as outlined in Fig. 1A.

View larger version (26K): [in a new window]   Fig. 3. Import of inner envelope protein 32 (IEP32) into chloroplasts is independent of protease-sensitive receptors. (A) Intact purified chloroplasts were either treated or not treated with the protease thermolysin for 20 minutes at 4°C. Chloroplasts were repurified on Percoll gradients prior to further use. Efficiency of the proteolytic treatment was controlled by immunoblotting using antisera against the outer envelope localized translocon subunits Toc159, Toc64, Toc34, Toc75III, and Toc75V and the inner envelope translocon subunit Tic110. (B) Chloroplasts from the identical batch as tested in (A) were used for a standard import reaction. All other conditions are as indicated on top of the gel and as described in the legend to Fig. 1A.

View larger version (24K): [in a new window]   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.

View larger version (57K): [in a new window]   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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