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. 2015 Oct 7;290(48):28778–28791. doi: 10.1074/jbc.M115.684829

FIGURE 2.

FIGURE 2.

Import of Plsp1 and Plsp11–70-Citrine into isolated chloroplasts. A, in vitro chloroplast import of Plsp1 without or with the predicted transit peptide. After import of radiolabeled proteins listed on top with 3 mm ATP in the light for 30 min at room temperature, chloroplasts were reisolated and examined by SDS-PAGE directly as total (T) or after hypotonic lysis and separation by centrifugation into soluble (S) and pellet (P) fractions. Protein signals on the same gels were visualized using phosphorimaging (PI) or Coomassie Brilliant Blue staining (CBB). Each lane contained samples equivalent to 3 μg of total chlorophyll. tl lanes were loaded with 20% of the translation product equivalent to the amount used for the assay containing 3 μg of chlorophyll. The radioactive bands corresponding to prPlsp1 (pr) and mPlsp1 (m) and Coomassie Brilliant Blue-stained bands corresponding to the stroma marker large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the thylakoid marker light-harvesting chlorophyll a/b binding protein (LHCP) are indicated at right. B, in vitro chloroplast import of Citrine without or with the N-terminal extension of Plsp1. The import assay and analysis were done as described for A. Plsp11–70-Citrine and the protein derived from Plsp11–70-Citrine after import are indicated as pr and m, respectively. Note that the translation product of Plsp11–70-Citrine (lane 5) includes a band corresponding to mature Citrine, which is due to the in-frame initiation of AUG located at the 5′-end of the Citrine-coding sequence. This protein is most likely different from imported and matured Citrine (lanes 6–8) because Citrine by itself was not imported (lanes 2–4). A minor band slightly larger than mature Citrine, whose identity remains unknown, is indicated with an arrow. C, comparison of mPlsp1 derived from prPlsp1 after chloroplast import and Plsp1Δ2–67 in the mobility on SDS-PAGE. The radiolabeled proteins indicated on top were separated by SDS-PAGE and visualized using phosphorimaging. prPlsp1 import (T) is equivalent to the sample loaded on lane 6 in A.