Figure 4.

Assembly of the Chloroplast ATP Synthase Is Less Efficient in bfa1 Mutants.

(A) In vivo pulse labeling of the thylakoid proteins of the wild type (WT) and the bfa1 mutants. Primary leaves of 12-d-old plants were labeled with [³⁵S]Met in the presence of cycloheximide, and the thylakoid proteins were isolated and separated using SDS-urea-PAGE. The gels were stained with Coomassie blue (left panel), and labeled thylakoid proteins were detected by autoradiography (right panel).

(B) Immunoblot analysis of the thylakoid proteins used in (A) probed with antibodies against CF₁β and RbcL.

(C) Pulse and pulse-chase labeling of the thylakoid proteins in wild-type and bfa1 plants. After labeling for 20 min, the leaves were further incubated with unlabeled Met for the indicated durations (0 to 2 h). Labeled thylakoid proteins were detected by autoradiography.

(D) Analysis of the assembly of the thylakoid protein complexes. After pulse labeling for 20 min (indicated as 0 min) and a subsequent chase of 15 and 30 min, thylakoid proteins were separated by 2D BN/SDS-urea-PAGE and labeled proteins were detected using autoradiography. Arrows indicate intact CF_o-CF₁ ATP synthase, the CF₁ subcomplex containing CF₁α/β/γ/ε but lacking CF₁δ (CF₁ Sub), and the Rubisco complex (asterisks). The positions of CF₁α and CF₁β are indicated by horizontal arrows in the panels of wild-type samples.