Figure 5.

Tic40 causes the release of bound transit peptides from Tic110. (A) 48 pmol of atTic110S-His₆ was incubated with 10-fold excess of ³H-labeled prFD transit peptides at 4°C for 2 h, TALON resin was added, and incubation continued for another 30 min. The resin was washed, and ∼3% of the added ³H-prFD transit peptides were bound to Tic110-His₆ resin. Washed resin was then incubated with the same molar amount (48 pmol) or 10- or 20-fold excess of GST or GST-atTic40S at 4°C for 2 h. The resin was pelleted, and ³H counts in the supernatant were measured. Approximately 14% of bound ³H-prFD was released at 20-fold GST-atTic40S. (B) Membrane-bound processed mature proteins are delayed in appearance, and their association with Tic110 is reduced in the tic40 mutant chloroplasts. Isolated wild-type and tic40 mutant chloroplasts were used to perform an import time course experiment with [³⁵S]prRBCS. Total membranes were isolated from chloroplasts of each time point and either directly analyzed by SDS-PAGE (top) or solubilized and immunoprecipitated by the anti-Tic110 antibody. (C) Quantification of the 30-min samples (B, lanes 5 and 10) of the gels shown in B. The amount of prRBCS in wild type at 30 min (prRBCS in lane 5) of each gel was taken as 1. The counts for RBCS have been corrected for the number of methionine residues compared with prRBCS.