Figure 1.

PKS5 Interacts with J3.

(A) Schematic diagram of the PKS5, PKS5N, PKS5C, J3, J3C-219, J3C1, and J3C2 proteins used in the yeast two-hybrid analysis. KDAL, kinase activation loop; FISL, SCaBP interaction domain; PPI, phosphatase interaction domain; J-domain, DnaJ domain; G/F, domain rich in Gly and Phe residues; C-rich domain, a distal zinc finger (CxxCxGxG)4 domain.

(B) to (D) Yeast two-hybrid analysis of interactions between the full-length PKS5 protein (PKS5) or the N- (PKS5N) or C- (PKS5C) terminal portions of the protein with J3, or J3C-219, J3C1, or J3C2. Interactions between the full-length PKS5 protein or the protein with the FISL domain deleted (PKS5ΔF) and SCaBP1 were used as positive and negative controls, respectively. Yeast lines harboring the indicated plasmids were grown on synthetic complete (SC) medium without Leu and Trp (SC-LW, left panel) and on SC medium without Leu, Trp, and His (SC-LWH) and with 20 mM 3-amino-1,2,4-triazole (3-AT; right panel). Yeast cells were incubated until OD₆₆₀ = 1 and then diluted 10- or 100-fold and used for assays.

(E) Coimmunoprecipitation of PKS5 and J3 proteins in vivo. The 35SP:6×Myc-J3 plasmid was cotransformed into wild-type (Col-0) protoplasts with 35SP:3×FLAG-PKS5 (lanes 1 and 3) or 35SP:3×FLAG-TTG1 (lanes 2 and 4). Total protein extracts were analyzed with immunoblots using anti-Myc and anti-FLAG antibodies to detect the presence of PKS5, J3, or TTG1 (input). Immunoprecipitation was performed using anti-Myc agarose conjugate, and the products were analyzed with immunoblots using anti-FLAG antibody to detect coimmunoprecipitated FLAG-PKS5 (lane 3) or FLAG-TTG1 (lane 4).