Residues Clustered in the Light-Sensing Knot of Phytochrome B are Necessary for Conformer-Specific Binding to Signaling Partner PIF3
PhyB and GAD-PIF3 were synthesized by in vitro translation in the presence of 35S-met. Co-immunoprecipitation assays were performed using GAD-PIF3 as bait and phyB Pr or Pfr as prey. A–E) Co-immunoprecipitations of wild-type phyB compared to phyB missense mutations. The upper band is phyB protein pulled down and the lower band is GAD-PIF3 protein as indicated. F–G) Quantification of binding in in vitro co-immunoprecipitation assays. Black bars represent GAD-PIF3 binding to wild-type Pr or Pfr phyB and orange bars represent binding to the mutants. The inset shows the phytochrome inputs. Binding was quantified relative to phyB input and GAD-PIF3. The wild-type phyB Pr interaction was set equal to one.