Figure 5.

Analysis of the stromal protein from bfa3 and wild-type plants. A, Immunodetection of ATP synthase subunits isolated from the thylakoid and stroma of WT plants. Intact chloroplasts of WT plants were fractionated into thylakoid membrane and stromal fractions. Stoichiometric amounts of thylakoid and stromal fractions were fractionated by SDS-urea-PAGE and detected with the specific antibodies indicated. A dilution series of thylakoid and stromal protein is shown. B, Immunodetection of ATP synthase subunits isolated from the stromal fractions of WT and bfa3 plants. Samples were loaded on an equal protein content basis. Signals of bfa3 mutants were quantified as percentages of the wild-type signal and are presented on the right. C, CN-PAGE analysis of the CF₁ subcomplex of ATP synthase. Stromal protein complexes isolated from WT and bfa3 plants were separated by CN-PAGE. After denaturation on a CN-gel, the proteins were transferred onto nitrocellulose membranes and immunodetected with antibodies against subunits of ATP synthase (CF₁α-CF₁ε), as well as the large subunit of Rubisco (RbcL). CF₁ Sub., CF₁ subcomplex of chloroplast ATP synthase. The band at the top of the CN-gel (indicated by an asterisk) may represent aggregates of the protein.