Fig. 4.

Radicicol inhibits preprotein import into chloroplasts. (A and B) Isolated pea chloroplasts were incubated with in vitro translated [³⁵S] preatSSU (A) or [³⁵S] preatTic40 (B) and 1 or 5 mM ATP at 26 °C for 20 min (lanes 2–6) in the presence of different concentrations of radicicol as indicated. Lane 1 contains 10% of the [³⁵S] preprotein added to each reaction (IVT). (C) Chloroplasts were incubated with urea-denatured preatSSU–3xFLAG in the presence or absence of radicicol and the indicated concentrations of nucleoside triphosphates (NTP). The chloroplasts were isolated, resolved by SDS/PAGE, and the precursor (pre) and mature (mat) forms of atSSU–3xFLAG were detected by immunoblotting with anti-FLAG. (D) Chloroplasts were incubated with urea-denature preatSS–3xFLAG in the presence of 0.1 mM ATP and GTP to promote formation of the early import intermediate (lane 2). In lanes 3–6, equivalent samples of chloroplasts to lane 2 were incubated with the indicated concentration of ATP and 500 μM radicicol to promote protein import. (C and D) Lane 1 contains 10% of the preprotein (pr) added to each reaction. (A–D) Each data bar represents the mean ± SEM (n = 3).