Figure 4.

Effects of Toc159 proteolysis on preprotein binding and import. Intact chloroplasts or chloroplasts treated with 10 μg/mL thermolysin were incubated with in vitro-synthesized [³⁵S]preSSU in a standard import reaction containing 25 μm ATP or 1 mm ATP as indicated. The chloroplasts were re-isolated and analyzed by SDS-PAGE. A, Immunoblots of envelope membrane fractions from thermolysin-treated (+) or control (−) chloroplasts. Membrane proteins (25 μg of chlorophyll) were resolved by SDS-PAGE and immunoblotted with affinity-purified antibodies to Toc86, Toc75, and Toc34. The positions of the known Toc components and the 52-kD membrane-protected fragment of Toc159 (52 kD) are indicated to the left. Tr, 10% of the [³⁵S]preSSU translation product added to each reaction. B, Phosphor imager analysis of [³⁵S]preSSU import. C, Quantitative analysis of the data presented in B. The numbers of the lanes in B that were used for quantitation are indicated at the bottom of the graph in C.