FIGURE 6.

Import deficiency in TAP-Toc159GM K868R chloroplasts. A, in vitro translated, [³⁵S]methionine-labeled preprotein of the small subunit of Rubisco (pSSu) was incubated with isolated chloroplasts in the absence or presence of 0.1 mm ATP and GTP. The low concentrations of ATP and GTP allow for formation of the early import intermediate but not for complete translocation. Chloroplasts were reisolated and analyzed by SDS-PAGE and Coomassie Blue staining followed by PhosphorImager visualization and quantification. A section of the Coomassie Blue-stained gel is shown as a loading control (Coomassie). The graph shows quantitative data of three experiments. The pSSu preprotein bound to TAP-Toc159GM WT chloroplasts in the presence of ATP and GTP was set to 100%. B, chloroplasts were isolated from plants of the indicated genotypes, and used in protein import assays with the radioactive pSSu. Import was allowed to proceed for 2, 5, 10, and 15 min. Samples were analyzed by SDS-PAGE and Coomassie Blue staining followed by PhosphorImager visualization and quantification. The bands corresponding to mature SSu were quantified from triplicate experiments. The amount of imported SSu after 15 min of import into TAP-Toc159GM WT chloroplasts, was set to 100%. A section of the Coomassie Blue-stained gel is shown as a loading control (Coomassie). C, protein import into isolated chloroplasts was allowed to proceed for 5 and 15 min in the absence (-) or presence (+) of 10 mm of the non-hydrolyzable GTP analog GMP-PNP. The graph shows the quantification of mature SSu from three independent experiments. The amount of SSu after 15 min of import into TAP-Toc159GM WT chloroplasts in the absence of GMP-PNP was set to 100%. ^*, pSSu modified in the course of the import reactions.