Fig. 1. Bub3 interacts physically with Cdc20, Mad1 and Mad2. (A) A cycling culture (cyc) of strain ySP1444, expressing both myc-tagged Cdc20 (Cdc20myc18) and HA-tagged Bub3 (Bub3HA3), was arrested either in G₁ with α-factor (αf) or in G₂ with nocodazole (noc). As negative controls, nocodazole-arrested cells expressing either Cdc20myc18 (ySP1413) or Bub3HA3 (ySP1346) were used. The presence (+) or absence (–) of the corresponding tagged proteins in the strains is indicated in the top part of the panel. Bub3 was immunoprecipitated from the extracts with anti-HA antibodies, whereas Cdc20 was immunoprecipitated with anti-myc antibodies. Immunoprecipitates (IP) with the antibodies indicated on the left side of the figure, along with the same amounts of total and immunodepleted extracts, were then run on SDS–PAGE and immunoblotted with both anti-HA (top) and anti-myc antibodies (bottom). (B) Cycling cultures (cyc) of wild-type (wt, ySP1444), mad1Δ (ySP1506) and mad2Δ (ySP1507) cells, all expressing both Bub3HA3 and Cdc20myc18, as well as nocodazole-arrested ySP1444 cells (wt noc), were harvested and protein extracts used for immunoprecipitations with anti-HA (left panel) or anti-myc (right panel) antibodies and analysed as in (A). Polyclonal antibodies were used to detect Mad1 and Mad2. Negative controls (mock) are represented by extracts from logarithmically growing strains expressing only either Cdc20myc18 (ySP1414) or Bub3HA3 (ySP1346) (left and right panel, respectively).

Fig. 3. Bub3–Cdc20 association does not require intact kinetochores. (A) Cell cultures of either a wild-type strain (wt) or the indicated checkpoint mutants, all expressing both Bub3HA3 and Cdc20myc18 were arrested in the same stage of the cell cycle by HU. Whereas wt (ySP1444), mad1Δ (ySP1506), mad2Δ (ySP1507), mad3 (ySP1512), bub1-1 (ySP1508) and bub2Δ (ySP1513) cell cultures were arrested by HU treatment (150 mM) for 3 h at 25°C, ndc10-1 (ySP1510) and mps1-1 (ySP1553) cells were first synchronized in G₁ by α-factor at 25°C and then released in the presence of HU (100 mM) at 37°C for 4 h, to obtain a homogeneous arrest. Bub3 was immunoprecipitated with anti-HA antibodies and both total extracts and immunoprecipitates were analysed by western blot. As a negative control (mock), a strain expressing only Cdc20myc18 (ySP1414) was used. (B) Cycling cultures (cyc) of strains ySP1444 (wt), ySP2171 (ndc10-1), ySP2162 (ndc80-1), ySP2164 (spc25-1), ySP2166 (spc24-1), ySP1507 (mad2Δ) and ySP1413 (mock) were arrested in G₁ by α-factor at 25°C and released from α-factor at 37°C for 4 h in the presence of HU. Cell extracts (total) and Bub3HA3 immunoprecipitates (Bub3HA3 IP) were analysed by western blot with anti-myc (Cdc20myc18) and anti-Mad1, -Mad2 and -Mad3 antibodies. (C) Wild-type (ySP1444) and ndc10-1 (ySP1510) cycling cells (cyc) were arrested in G₁ by α-factor at 25°C (t = 0 min) and then released from α-factor at 37°C. At the indicated times cell samples were collected to produce protein extracts (upper panel), FACS analysis of DNA contents and kinetics of bipolar spindle formation and nuclear division (bottom panel). Western blot analysis with anti-myc (Cdc20myc18), anti-HA (Bub3HA3), anti-Mad1 and anti-Mad2 antibodies was performed on protein extracts (total) and Bub3HA3 immunoprecipitates (Bub3HA3 IP). The negative control (mock) is represented by strain ySP1414, expressing Cdc20myc18 and untagged Bub3.

Fig. 5. Bub3 WD40 repeats are required for interaction with Mad2, Mad3 and Cdc20. (A) Clustal W alignment of the Bub3 protein sequence from different organisms (Sc: Saccharomyces cerevisiae; Xl: Xenopus laevis; Hs: Homo sapiens; Mm: Mus musculus; Dm: Drosophila melanogaster). Lines above the ScBub3 sequence mark the three WD40 domains. Stars indicate the two Trp residues changed into Gly in the Bub3-WDd mutant protein. (B) Protein extracts from wild-type (wt, ySP2076), bub3-WDd1 (ySP2079), bub3-WDd2 (ySP2082) and bub3-WDd1,2 (ySP2085) mutant cells were used to analyse by western blot either the total levels of Mad2, Mad3, Bub3HA3 and Cdc20myc18 (total) or the amounts of the same proteins that could be co-immunoprecipitated with Bub3HA3 (Bub3HA3 IP). The negative control (mock) is represented by strain ySP1414, expressing Cdc20myc18 and untagged Bub3. Cell extracts from mad2Δ (ySP1507) and mad3Δ (ySP1577) cultures were loaded in parallel to identify aspecific bands recognized by anti-Mad2 and -Mad3 antibodies. (C) The same protein extracts as in (B) were used to also analyse the amounts of Mad3, Bub3HA3 and Mad2 in total extracts and in Cdc20myc18 immunoprecipitates (Cdc20myc18 IP). (D) Serial dilutions of exponentially growing cultures of wild-type (wt, W303), bub3Δ (ySP589), bub3-WDd1 (ySP2198), bub3-WDd2 (ySP2202) and bub3-WDd1,2 (ySP2211) strains were spotted on YEPD plates either lacking or containing benomyl and photographed after incubation at 30°C for 2 days.

Fig. 2. Bub3–Cdc20 interaction is cell cycle regulated and is stimulated upon checkpoint activation. (A) A cycling culture (cyc) of strain ySP1444 expressing both Cdc20myc18 and Bub3HA3, was either blocked with nocodazole (noc) or arrested in G₁ with α-factor (t = 0 min) and then released. At the indicated time points after α-factor release cell samples were collected to produce protein extracts (upper panel), FACS analysis of DNA contents (bottom left) and kinetics of budding and nuclear division (bottom right). Protein extracts were analysed by immunoblotting either directly to detect at each time point the total amounts of Cdc20myc18 and Bub3HA3 (total) or after immunoprecipitation of Bub3 with anti-HA antibodies (Bub3HA3 IP). The negative control (mock) is represented by strain ySP1414, expressing Cdc20myc18 and untagged Bub3. (B) Protein extracts from the following strains were used for immunoprecipitations of Bub3HA3 as described for Figure 1: wild type (wt, ySP1444), either cycling (cyc) or arrested for 3 h in nocodazole (noc), a strain carrying the galactose-inducible GAL-MPS1 construct (ySP2025) arrested by 3 h of incubation in galactose, and a negative control (mock, ySP1414). Both total extracts and immunoprecipitates (Bub3HA3 IP) were analysed by western blot. As controls, total extracts from mad1Δ (ySP1506) and mad2Δ (ySP1507) cells were similarly analysed.

Fig. 4. Co-fractionation of Mad2, Mad3, Bub3 and Cdc20 in gel filtration chromatography. (A) A protein extract from a cycling culture of a strain (ySP2156) expressing Bub1myc18, Cdc20myc18 and Bub3HA3 was fractionated by gel filtration (see Materials and methods) and fractions 1–40 were analysed by western blot with anti-HA (Bub3HA3), anti-myc (Bub1myc18 and Cdc20myc18), anti-Mad1, -Mad2 and -Mad3 antibodies. Total protein extracts from mad1Δ (ySP1506), mad2Δ (ySP1507) and mad3Δ (ySP1577) cell cultures were loaded on the same gel to identify aspecific immunoreactive bands. (B and C) Protein extracts from logarithmically growing mad2Δ (ySP2317) or mad1Δ (ySP1506) cells were fractionated and analysed as in (A).

Fig. 6. Mutations altering the WD40 repeats of Bub3 impair proper checkpoint response. Wild-type, bub3Δ (ySP589), bub2Δ (ySP1071), bub3-WDd1 (ySP2198), bub3-WDd2 (ySP2202), bub3-WDd1,2 (ySP2211), bub3-WDd1 bub2Δ (ySP2197) bub3-WDd2 bub2Δ (ySP2201) and mad2Δ bub2Δ (ySP1152) cells were arrested in G₁ by α-factor and then released at 25°C in the presence of nocodazole. At the indicated times cell samples were collected for FACS analysis of the DNA contents.