Figure 7.

ATP preloading can partially rescue the tic40 mutant import defect. (A) Isolated chloroplasts from wild-type and tic40 mutant plants were preincubated with 3 mM ATP (closed bar) or import buffer (open bar) for 5 min at room temperature in the dark, reisolated, and used to perform import experiments with [³⁵S]prRBCS. The amount of mature RBCS imported at 30 min was quantified, and the ratio of imported RBCS in tic40 chloroplasts to that in wild-type chloroplasts was plotted. (B) Hsp93 has a higher ATPase activity in the ATP concentration after chloroplasts have been preloaded with ATP. The ATP concentration within chloroplasts was calculated to be ∼0.07 mM before and 2.1 mM after preloading with 3 mM ATP (see Materials and methods). ATPase activity of Hsp93 was assayed under these two ATP concentrations. The ATPase activity at 0.07 mM ATP was taken as 1. (C) Association of Tic40 with Hsp93 decreases in the presence of ADP. 138 nM Hsp93-His₆ was incubated with 690 nM ATP, ADP, or AMP-PNP at room temperature for 10 min. An equal amount of atTic110S-His₆, prFD transit peptide, and GST-atTic40S was added with a 1-h incubation at 4°C after each addition. Tic40-containing complexes were recovered by glutathione resin, washed in PBS containing the nucleotides, eluted by glutathione, and analyzed by immunoblotting with antibodies against Hsp93 and Tic40.