Figure 8.

BFA3 specifically interacts with CF₁β. A, Affinity purification of the proteins associated with BFA3. Intact chloroplasts were isolated from leaves of WT and bfa3-HA plants and incubated with (+DSP) or without (-DSP) 2.5 mm DSP on ice for 30 min in the dark. After incubation, chloroplasts were osmotically ruptured and fractionated into stromal and thylakoid fractions. Peripheral thylakoid protein released from thylakoids by 1.2% Triton X-100 and stromal fractions were mixed with anti-HA affinity matrix for immunoprecipitation, respectively. The bound proteins were washed, eluted, and detected by immunoblot analysis using the indicated antibodies. bfa3-HA represents bfa3-1 transformed with WT genomic BFA3 fused to the sequence encoding the HA epitope-tag. Thylakoid proteins corresponding to 0.2 μg chlorophyll were loaded as indicators for antibodies. B, Yeast two-hybrid analysis. BFA3 was fused to the bait vector. Subunits of the CF₁ subcomplex as well as soluble parts of CF₀I and CF₀II were fused to the prey construct. Cotransformation of pGBKT7-53 with pGADT7-T was used as a positive control, whereas cotransformation of pGBK7-Lam with pGADT7-T was used as a negative control. As another negative control (Supplemental Fig. S4) included to rule out self-activation of the prey, pGBKT7 was cotransformed with pGADT7 harboring ATP synthase subunits. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. C, In vitro pull-down assay. GST-BFA3 fusion was used as bait. MBP-tagged CF₁α, CF₁β, CF₁γ, CF₁δ, and CF₁ε subunits as well as the MBP-tagged soluble parts of CF₀I and CF₀II were used as prey. After incubation of bait and prey proteins for 1 h at 4°C, glutathione-agarose beads were added to precipitate the bait and bound prey proteins. The bound proteins were eluted and analyzed by immunoblotting with antibodies against MBP and BFA3.