Figure 8.

Import of preSSU into Isolated Chloroplasts of Antisense Plants.

Chloroplasts were prepared from 2- to 4-week-old control or Y3 seedlings grown on agar plates containing 1% Suc.

(A) Import of preSSU in control and antisense plants. In vitro–translated ³⁵S-preSSU was incubated with chloroplasts (50 μg of chlorophyll) from control or Y3 antisense plants in a standard protein import reaction for 30 min. The samples were divided in half and treated with 100 μg/mL thermolysin (+ T-lysin) or buffer (− T-lysin) on ice for 30 min. The chloroplasts were reisolated through 40% Percoll silica gel and analyzed directly by SDS-PAGE and phosphorimaging. One-tenth of the ³⁵S-preSSU in vitro translation product (St.) added to each reaction is shown in lane 1. The level of preSSU import was reduced in Y3 antisense chloroplasts.

(B) Time course of ³⁵S-preSSU import into isolated chloroplasts from control or Y3 antisense plants. Import was performed as in (A). Samples corresponding to 25 μg of chlorophyll were collected at the times indicated, treated with 100 μg/mL thermolysin on ice for 30 min, reisolated through 40% Percoll silica gel, and analyzed directly by SDS-PAGE and phosphorimaging. One-tenth of the ³⁵S-preSSU in vitro translation product (St.) added to each reaction is shown in lane 1. The positions of precursor (preSSU) and mature (SSU) proteins are indicated at left. The chloroplasts from Y3 antisense plants exhibited a slower rate of preSSU import than control plants.

The gel at the top of each panel shows one representative fluorograph of the four used for the quantitative analysis. Error bars indicate ±sd.