FIGURE 4.

Trypsin, alkaline, and detergent sensitivities of chimeric BicA and SbtA proteins in chloroplasts. (A) Trypsin sensitivity of chimeric BicA and SbtA proteins in intact chloroplasts. Chloroplasts, equivalent to 25 μg chlorophyll, were treated with trypsin on ice for 30 min. The trypsin was inactivated and intact chloroplasts were re-isolated, resolved by SDS-PAGE, and immunoblotted with the antibody against protein A. The protease sensitivity of the outer envelope membrane protein, Toc75, was included as a positive control to confirm the validity of experiments. (B) Localization of chimeric BicA and SbtA proteins in the soluble and membrane fractions of chloroplasts. Chloroplasts were lysed in the buffer containing 0.2 M Na₂CO₃, pH 12 (Na₂CO₃), or 1% Triton X-100 (TX-100), and separated into insoluble (P) and soluble (S) fractions. The extracts were resolved by SDS-PAGE and immunoblotted with antibodies against protein A (BicA and SbtA).