Figure 3.

The N Terminus of OEP80 Is Necessary for the Import of the IMS-Localized POTRA Domains But Not the Transmembrane Domain.

(A) In vitro-translated, radiolabeled sections of AtOEP80 (illustrated below the gels) were incubated with isolated chloroplasts (import). Chloroplasts were reisolated, then treated with or without thermolysin or solubilized with 1% (v/v) Triton X100 (TX100) and treated with thermolysin. The protease was stopped with 10-mM EDTA, and intact chloroplasts were reisolated. Proteins in the solubilized sample were precipitated in 80% (v/v) acetone. Samples were separated by SDS-PAGE next to 10% of the input translation product (TL). Radioactive bands were detected with autoradiography. 80TP, transit peptide of OEP80; u, uncharacterized region between the transit peptide and the first POTRA. The three POTRA domains are labeled as “1,” “2,” and “3.” The pound sign (#) indicates additional processed bands, other than the mature-sized one.

(B) In vitro-translated, radiolabeled OEP24 and OEP80-OEP24 chimeras were incubated with isolated chloroplasts, then treated as in (A). pr, precursor; m, mature.

In both (A) and (B), the asterisks (*) near the TL lane indicates bands resulting from translation starting at the second and/or third methionines in the coding sequence.