Figure 7.

Only Complexed but Not Free ³⁵S-preSSU Is Imported into Isolated Chloroplasts with High Efficiency.

(A) and (B) Freshly translated ³⁵S-preSSU was fractionated by size exclusion chromatography (see Figure 4). Fractions 11 to 13 (A) and 24 to 26 (B) from three independent runs were combined and concentrated, and equal aliquots (100 μL) were used in each import reaction. Chloroplasts equivalent to 7.5 μg of chlorophyll were incubated with preSSU at 25°C for the different times indicated. The import reaction was stopped by adding ice-cold EDTA solution to a final concentration of 37.5 mM. Chloroplasts were recovered from the import reaction by centrifugation. Chloroplast proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes, and radioactivity recovered in processed mature SSU was determined by autoradiography. A typical autoradiogram out of a set of five independent experiments is shown. TP, 10% of translation product used as internal standard; 1′, 1 min.

(C) Import efficiencies from complexed ([A], filled squares) or free ([B], open circles) preSSU as measured by the appearance of processed mature mSSU were quantified by liquid scintillation counting as above. The calculations were conducted from five independent experiments of five independent complex purifications. Values are given as percentages of the translation product supplied to each translocation reaction for the times indicated.