FIGURE 4.

The effects of the GTPase mutations K868R and mGTP (A864R, K868N, S869R) on the in vitro membrane insertion of Toc159GM into chloroplasts. A, in vitro translated, [³⁵S]methionine-labeled Toc159 lacking the A-domain (Toc159GM) with or without the K868R or mGTP mutation were incubated with isolated Arabidopsis chloroplasts. Chloroplasts were reisolated and incubated in the absence (-) or presence (+) of 50 μg/ml of thermolysin (TL) for 30 min on ice. Samples were 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. B, quantitative analysis of data from three replicate experiments using the Quantity One® software (BioRad). The experiments were calibrated to the amount of in vitro translated radioactive protein added to the chloroplasts (is 100%). For the quantification of insertion of Toc159GM, the data were normalized based on the methionine content of Toc159GM (15) and Toc159M (8).