Figure 5.

Thermolysin Treatment and Alkaline Extraction of Chloroplasts Purified from Transfected Protoplasts.

(A) Thermolysin treatment. Chloroplasts were isolated from transfected protoplasts that expressed the CTs of Toc159 or Toc132 fused to the C terminus of EGFP or the transit peptide of ferredoxin fused to the N terminus of EGFP. Purified chloroplasts were incubated with or without 10 µg of thermolysin on ice for 30 min prior to reisolation of the chloroplasts on a Percoll gradient. The reisolated chloroplasts were subjected to immunoblot analysis using an anti-EGFP antibody and an antibody against Rubisco large subunit (RbcL) as loading controls.

(B) Alkaline extraction. Chloroplasts were isolated from transfected protoplasts that expressed the CTs of Toc159 or Toc132, the entire Toc34 protein fused to the C terminus of EGFP, or OEP7 fused to the N terminus of EGFP. Purified chloroplasts were incubated in carbonate buffer, pH 11.5, or HEPES buffer, pH 7.3, on ice for 10 min. The precipitated membrane pellets were subjected to immunoblot analysis using an anti-EGFP antibody and an antibody against Toc34 as loading controls.