Fig. 4.

Ppr1 interacts with both Rip1-PDZ and Anti-SigM. (A) Interaction of Ppr1 with RsmA and Rip1-PDZ. Yeast strains carrying a LexA-LacZ reporter plasmid and plasmids encoding AD fusions to Ppr1 with the amino acid boundaries listed above each column and LexA-DNA BD fusion to Rip1-PDZ, RsmA-C, or RskA-C. The strains are shown on X-gal–containing media with either glucose (Upper) or galactose (Lower), which induces expression of the AD fusion proteins. (B) Mixtures of His-SUMO-RslA-C/Ppr1 (Left) or His-SUMO-RsmA-C/Ppr1 (Right) were purified by metal-affinity chromatography. Bound proteins were eluted with imidazole, and the input protein (I), wash (W), and elution (E) were separated by SDS/PAGE and proteins visualized by staining with Coomassie blue. (C) Mixtures of Ppr1/His-SUMO/RsmA-C or Ppr1/His-SUMO-Rip1-PDZ-/RsmA-C were purified by metal-affinity chromatography. Bound proteins were eluted with imidazole, and the input protein (I), wash (W), and elution (E) were separated by SDS/PAGE and proteins visualized by staining with Coomassie blue. (D) Specificity of the Ppr1-RsmA interaction. Ppr1-Myc was coexpressed in E. coli with the C-terminal fragments of RsmA-His, RskA-His, or RslA-His. Soluble lysates from the indicated combinations of expressed proteins or vector controls were subjected to metal-affinity chromatography to purify histidine-tagged proteins. Supernatants (sn) and eluted proteins (e) were analyzed by immunoblotting with anti-Myc or anti-His antibodies. The lanes labeled “-” are no-IPTG control samples, and the lanes labeled “x” are blank lanes.