Figure 4. Mad2 prevents Cdc20 from interacting with the APC/C. (A) The hydrophobic core of the KILR motif is essential to bind Mad2. E. coli extracts expressing GST, GST fused to the N terminus of wild-type Cdc20, or the indicated mutants were incubated with recombinant human Mad2 for 30 min at 4°C and purified with glutathione–Sepharose, and the amount of Mad2 was analyzed by quantitative immunoblotting. Relative amount of Mad2 bound is shown at the bottom. Results are representative of three independent experiments. Asterisk shows a truncated form of Cdc20 that does not bind Mad2. (B) The IL motif is required to bind to the APC/C. In vitro translated (IVT) full-length 3×Flag–wild-type Cdc20 or the indicated mutants were analyzed as in Fig. 2 C. Results are representative of three independent experiments. (C) The KILR and C box motifs are required for the N terminus of Cdc20 to interact with the APC/C. GST fusion proteins of the N terminus of Cdc20 (N151), wild type, and the indicated mutants were incubated for 40 min at 4°C with mitotic extracts depleted of endogenous Cdc20. Proteins retained on the beads were analyzed by quantitative immunoblotting with the indicated antibodies. Results are representative of three independent experiments. (D and E) Mad2 competes with the APC/C for binding to the KILR motif. (D) GST or GST fusion proteins of the N terminus of wild-type Cdc20 or the ΔKILR mutant were incubated with recombinant Mad2 and mitotic HeLa cell extracts as in C. The APC/C bound to the beads was analyzed by quantitative immunoblotting with the indicated antibodies (Western blot [WB]). Recombinant proteins were also detected by Coomassie blue staining (CBB). (E) GST or GST fusion proteins were prebound to gluthatione–Sepharose and incubated with mitotic extract plus recombinant Mad2. “Pre” indicates the fusion protein was incubated with Mad2 before the mitotic extract. Mad2 was added in fourfold excess over the amount of GST-N151. Samples were analyzed by quantitative immunoblotting with the indicated antibodies. The relative amount of the APC/C and Mad2 bound to the beads is shown on the bottom where the amount of APC/C bound at 40 min or the amount of Mad2 bound at 40 min in the “pre” sample is set to 1. Binding assays in D and E are representative of three experiments. end, endogenous; IP, immunoprecipitation; WT, wild type.
Figure 4.

Mad2 prevents Cdc20 from interacting with the APC/C. (A) The hydrophobic core of the KILR motif is essential to bind Mad2. E. coli extracts expressing GST, GST fused to the N terminus of wild-type Cdc20, or the indicated mutants were incubated with recombinant human Mad2 for 30 min at 4°C and purified with glutathione–Sepharose, and the amount of Mad2 was analyzed by quantitative immunoblotting. Relative amount of Mad2 bound is shown at the bottom. Results are representative of three independent experiments. Asterisk shows a truncated form of Cdc20 that does not bind Mad2. (B) The IL motif is required to bind to the APC/C. In vitro translated (IVT) full-length 3×Flag–wild-type Cdc20 or the indicated mutants were analyzed as in Fig. 2 C. Results are representative of three independent experiments. (C) The KILR and C box motifs are required for the N terminus of Cdc20 to interact with the APC/C. GST fusion proteins of the N terminus of Cdc20 (N151), wild type, and the indicated mutants were incubated for 40 min at 4°C with mitotic extracts depleted of endogenous Cdc20. Proteins retained on the beads were analyzed by quantitative immunoblotting with the indicated antibodies. Results are representative of three independent experiments. (D and E) Mad2 competes with the APC/C for binding to the KILR motif. (D) GST or GST fusion proteins of the N terminus of wild-type Cdc20 or the ΔKILR mutant were incubated with recombinant Mad2 and mitotic HeLa cell extracts as in C. The APC/C bound to the beads was analyzed by quantitative immunoblotting with the indicated antibodies (Western blot [WB]). Recombinant proteins were also detected by Coomassie blue staining (CBB). (E) GST or GST fusion proteins were prebound to gluthatione–Sepharose and incubated with mitotic extract plus recombinant Mad2. “Pre” indicates the fusion protein was incubated with Mad2 before the mitotic extract. Mad2 was added in fourfold excess over the amount of GST-N151. Samples were analyzed by quantitative immunoblotting with the indicated antibodies. The relative amount of the APC/C and Mad2 bound to the beads is shown on the bottom where the amount of APC/C bound at 40 min or the amount of Mad2 bound at 40 min in the “pre” sample is set to 1. Binding assays in D and E are representative of three experiments. end, endogenous; IP, immunoprecipitation; WT, wild type.

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