Figure 4. The novel KLTF peptide motif... | Rockefeller University Press

Figure 4.

$The novel KLTF peptide motif is responsible for high-affinity Bim1 binding by Cik1-Kar3.(A) Purification of Cik1Δ69-Kar3 and Cik1Δ74-Kar3. Proteins were eluted from M2-agarose beads with 3xFlag peptide. Gel lanes show Sf9 lysate, unbound fraction (Unb.), wash 1, and wash 2 followed by the two elution fractions E1 and E2. polh, polyhedrin promoter; Mw, molecular weight marker. (B) Analysis of Bim1 binding to Cik1Δ69-Kar3 and Cik1Δ74-Kar3 in a SEC experiment. Sample fractions from Superose 6 column were resolved on SDS-PAGE and visualized by Coomassie staining. For comparison, the identical Bim1 gel is shown side by side in the middle panel. Input concentration of proteins was 5 µM for the respective Halo–Kar3–Cik1 complex and 10 µM for Bim1. (C) Analysis of Bim1 binding in a quantitative pull-down experiment. Representative gel of supernatant fractions from pull-down experiment using Bim1 immobilized on beads incubated with Cik1Δ69-Kar3 and Cik1Δ74-Kar3 in solution. Binding is well approximated with the one-site total binding model (smooth line), R2 = 0.99. Bim1 does not bind to Cik1Δ74-Kar3 under the given conditions. Experiments were performed at +4°C. Dissociation constant represents mean value of three biological repeats ± SD. N.B., no binding. (D) Schematic representation of the binding interface between Cik1-Kar3 and Bim1; the discovered elements of the binding interface are highlighted.$

The novel KLTF peptide motif is responsible for high-affinity Bim1 binding by Cik1-Kar3. (A) Purification of Cik1^Δ69-Kar3 and Cik1^Δ74-Kar3. Proteins were eluted from M2-agarose beads with 3xFlag peptide. Gel lanes show Sf9 lysate, unbound fraction (Unb.), wash 1, and wash 2 followed by the two elution fractions E1 and E2. polh, polyhedrin promoter; Mw, molecular weight marker. (B) Analysis of Bim1 binding to Cik1^Δ69-Kar3 and Cik1^Δ74-Kar3 in a SEC experiment. Sample fractions from Superose 6 column were resolved on SDS-PAGE and visualized by Coomassie staining. For comparison, the identical Bim1 gel is shown side by side in the middle panel. Input concentration of proteins was 5 µM for the respective Halo–Kar3–Cik1 complex and 10 µM for Bim1. (C) Analysis of Bim1 binding in a quantitative pull-down experiment. Representative gel of supernatant fractions from pull-down experiment using Bim1 immobilized on beads incubated with Cik1^Δ69-Kar3 and Cik1^Δ74-Kar3 in solution. Binding is well approximated with the one-site total binding model (smooth line), R² = 0.99. Bim1 does not bind to Cik1^Δ74-Kar3 under the given conditions. Experiments were performed at +4°C. Dissociation constant represents mean value of three biological repeats ± SD. N.B., no binding. (D) Schematic representation of the binding interface between Cik1-Kar3 and Bim1; the discovered elements of the binding interface are highlighted.