Fig 1.
Toc75 and t75-fusion proteins used for the study.
(A) Toc75 precursor (t75-m75) carries an N-terminal extension (t75), which comprises n75 and c75, for correct targeting to the chloroplast envelope. Removals of n75 and c75 by SPP and Plsp1 yield the intermediate (c75-m75) and mature form (m75), respectively. Primary sequences of part of c75 and its variants are shown above the diagram. (B) A diagram of four chimeric proteins used in the study.
Fig 2.
In vitro import of t75-mSS variants.
(A) Radiolabeled t75-mSS variants indicated above were incubated with isolated chloroplasts under the import condition. After 30-min of import, intact chloroplasts were reisolated and separated into two aliquots. The first aliquot was kept as total chloroplasts (T). The second aliquot was hypotonically lysed and fractionated by centrifugation to a supernatant (S1) and the pellet. The pellet was then resuspended with 0.1M Na2CO3 and fractionated by centrifugation to the second supernatant (S2) and the final pellet fraction (P). Samples equivalent to 3 μg chlorophyll were separated by SDS-PAGE, and radiolabeled proteins and total proteins in each sample were visualized by phosphorimaging (PI) and Coomassie Brilliant Blue staining (CBB), respectively. The experiments were done concurrently with those shown in Fig 3A and S1 Fig. tl contained the translation product corresponding to the one used for the import assay with 3 μg chlorophyll-equivalent chloroplasts. The precursors containing the entire t75 variants, the intermediates that carrying the c75 variants, and the mature forms lacking the entire t75 variant, respectively, are indicated at right; mSS is indicated with the letter m. For the CBB panel, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll a/b-binding protein are indicated as LS and LHCP, respectively. (B) After the import reaction as described in the legend to panel (A), intact chloroplasts were reisolated and separated into six aliquots. Three of them were resuspended in import buffer containing 1 mM CaCl2 with or without 1 μg thermolysin (tlysin) per μg chlorophyll equivalent chloroplasts and 1% Triton X-100 (TX) as indicated, incubated for 30 min on ice in the dark. Other three aliquots were resuspended in import buffer with or without 0.5 μg trypsin (tryp) per μg chlorophyll equivalent chloroplasts and 1% Triton X-100 (TX) as indicated, incubated for 60 min at room temperature in the dark. The activities of thermolysin and trypsin were quenched by 10 mM EDTA and 10 μg trypsin inhibitor per μg trypsin, respectively. Samples equivalent to 3 μg chlorophyll were separated by SDS-PAGE and radiolabeled proteins visualized by phosphorimaging. The experiments were done concurrently with those shown in Fig 3B and S2 Fig. For the labels at right, see the legend to panel (A).
Fig 3.
In vitro import of t75-EGFP variants.
(A) Import of radiolabeled t75-EGFP variants into isolated chloroplasts, post-import fractionation, and analysis of the results were done as described in Fig 2A. The experiments were done concurrently with those shown in Fig 2A and S1 Fig. The precursor proteins containing the entire t75 variants, the intermediates containing the c75 variants but not n75, and the mature form that lacks the entire t75 variants are indicated as t75*-EGFP, c75*-EGFP, and m, respectively. For the CBB panel, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll a/b-binding protein are indicated as LS and LHCP, respectively. (B) Import of radiolabeled t75-EGFP variants into isolated chloroplasts, post-import protease treatment, and analysis of the results were done as described in Fig 2B. The experiments were done concurrently with those shown in Fig 2B and S2 Fig. The 27-kD protease-protected EGFP is indicated as 27. For other labels, see the legend to panel (A). (C) Import of radiolabeled t75-EGFP variants into isolated chloroplasts were performed for 5 or 30 min followed by post-import treatment with thermolysin as described in Fig 2B. The precursor proteins containing the entire t75 variants, the intermediates containing the c75 variants but not n75, and the mature form that lacks the entire t75 variants are indicated as t75-EGFP/t75GGA-EGFP, c75-EGFP/c75GGA-EGFP, and m, respectively. The 27-kD protease-protected EGFP is indicated as 27.
Fig 4.
Confocal microscopy analysis of t75-EGFP variants after transient expression in N. benthamiana.
Leaves of N. benthamiana were agroinfiltrated with constructs encoding Plsp1 (non-fluorescent control), t75-EGFP, or t75GGA-EGFP. Fluorescence from EGFP and chlorophyll was observed in infiltrated leaves by confocal microscopy. In the overlay, signals from the GFP and chlorophyll channels are depicted as green and magenta, respectively. Scale bars represent 50 μm (1× zoom) or 10 μm (5× zoom). See S4–S6 Figs for additional sets of images.
Fig 5.
Biochemical analysis of t75-EGFP variants after transient expression in N. benthamiana.
(A) Total protein extracts equivalent to ca. 12.5 mg fresh weight and total chloroplasts containing 10 μg chlorophyll prepared from leaves expressing t75-EGFP or t75GGA-EGFP were separated by SDS-PAGE and analyzed by immunoblotting with the anti-GFP antibody. The bands corresponding to proteins of 38 kD, 30 kD, and 28 kD are indicated. Different regions from the same membrane are separated by a black line. (B) Total chloroplasts after import (import) as in Fig 3A, total chloroplasts from agroinfiltrated leaves (transient) as in panel (A), and a mixture of them were separated by SDS-PAGE and detected by immunoblotting with the anti-GFP antisera (αGFP) and phosphorimaging (PI). The combined image is false-colored green for αGFP and magenta for PI. Different regions from the same membrane are separated by black lines. Import samples were loaded at 2 μg chlorophyll equivalents and transient expression samples were loaded at 10 μg chlorophyll equivalents. (C) Ten μg chlorophyll equivalent total chloroplasts (T) isolated from N. benthamiana leaves expressing t75-EGFP or t75GGA-EGFP as well as S1, S2, and P fractions as prepared in Fig 2A were separated by SDS-PAGE and proteins were detected by immunoblotting with the antisera against GFP. (D) Intact chloroplasts isolated from N. benthamiana leaves expressing t75-EGFP or t75GGA-EGFP were incubated with thermolysin (tlysin) or trypsin (tryp) with or without the presence of Triton X-100 (TX) as described in the legend to Fig 2B. Loading and detection were performed as in panel (C).
Fig 6.
Processing of t75-EGFP variants by Plsp1 in vitro.
Radiolabeled proteins indicated at top were incubated without or with recombinant Plsp1 protein for 2 h at room temperature, separated by SDS-PAGE, and detected by phosphorimaging.
Fig 7.
Far UV CD spectra t7589-112 variants at 20°C.
Each spectrum is the average of eight measurements.