Figure 10.

Translocation of preSSU across the Inner Envelope Membrane of Chloroplasts from Antisense Plants.

Chloroplasts were prepared from 2- to 4-week-old control or Y3 seedlings grown on agar plates containing 1% Suc. Isolated chloroplasts were depleted of endogenous ATP by incubating at 26°C in the dark for 10 min. The chloroplasts were incubated with 0.1 mM ATP and 200 nM ³⁵S-preSSU at 26°C in the dark for 10 min. The chloroplasts were reisolated through 40% Percoll silica gel, resuspended in buffer containing 2 mM ATP, and incubated for 15 min at 26°C in the light. The chloroplasts were collected by centrifugation and analyzed directly by SDS-PAGE and phosphorimaging. The positions of precursor (preSSU) and mature (SSU) proteins are indicated at right. The amount of ³⁵S-preSSU bound to chloroplasts during the first incubation in the presence of 0.1 mM ATP is shown by the black bars. The amount of mature ³⁵S-SSU associated with chloroplasts after the subsequent incubation in the presence of 2 mM ATP is shown by the gray bars. The gels at top show representative fluorographs of the four used for the quantitative analysis. The import of preSSU into chloroplasts of Y3 antisense plants was inhibited selectively at the stage of translocation across the inner envelope membrane. IM, inner membrane; OM, outer membrane. Error bars indicate ±sd.