(A) Benomyl sensitivity of the yeast cells expressing 13-Myc-tagged Bub3 (2818), Mad2 (8056), Mad3 (2845-2-2), Cdc20 (2819) or Cdc27 (9545) protein; 10-fold serial dilutions of cells were spotted on YPD and YPD plus benomyl (15 μg/ml). The plates were incubated at 23°C for 4 days before being photographed. (B) Quantification of Mad2 in nocodazole-treated yeast cells. Anti-Myc immunoblot of 2-fold serial dilution of yeast whole-cell extract made from cells expressing Mad2-13Myc and recombinant Mad2-13Myc is shown. The numbers represent the amount of whole-cell extract (in μg) and the purified protein (in ng) loaded/lane. (C) The estimated number of molecules of proteins present per yeast cell arrested in M phase. (D) Gel filtration profiles of the checkpoint proteins (Bub3, Mad2, and Mad3), Cdc20, and APC (Cdc27) present in nocodazole-treated yeast cells. Whole-cell extracts were made from nocodazole-treated cells expressing Myc-tagged Bub3, Mad3, Cdc20, and Cdc27 proteins. The extracts were separated on Superose 6 column. Alternate fractions were probed with anti-Myc antibodies or anti-Mad2 antibodies. The elution profiles of the molecular weight standards in the same column are indicated by arrows. (E) Spindle checkpoint induces association of Mad2p with other proteins to form a high-molecular-weight protein complex. Wild-type cells were arrested in G₁, S, and M phase with α-factor, hydroxyurea, and nocodazole, respectively. Clarified extracts were fractionated on a Superose 6 column. Alternate fractions were probed with anti-Mad2 antibodies. The arrows indicate the elution profiles of the gel filtration standards. (F) More Mad2 coimmunoprecipitated with Cdc20 compared to Bub3 and Mad3. Cycling cultures of cells expressing Mad3-13 Myc (2845-2-2), Bub3-13Myc (2818), or Cdc20-13Myc (2819) were arrested in M phase with nocodazole. Myc-tagged proteins were immunoprecipitated from the whole-cell extracts using anti-Myc antibody-coupled beads. The whole-cell extracts, immunoprecipitates, and supernatants were probed with anti-Myc and anti-Mad2 antibodies. An untagged strain (BY4741) was used as a control. Equal amounts of whole-cell extracts and supernatants were loaded; 5-fold more protein was loaded in the IP lane.

MCC is required for the maintenance of the spindle checkpoint function. Cells expressing degron-tagged Mad2 (2850), degron-tagged Mad3 (2832-8-2), and no degron tag (wild type) (2832-10-2) were grown overnight at 23°C in YM-1 containing raffinose, galactose, and CuSO₄ (100 μM). The cycling cells were arrested in M phase with carbendazim (25 μg/ml) and nocodazole (15 μg/ml). The cells were washed, resuspended in prewarmed (37°C) complete medium containing galactose, raffinose, carbendazim, and nocodazole. Aliquots of cells were collected at the indicated time points, and protein samples were prepared. Time zero represents the time at which the cells were shifted to 37°C. (A) Anti-HA immunoblot showing the levels of the degron-tagged Mad3-HA and Mad2-HA fusion proteins. Anti-Myc antibody was used to detect Pds1-13 Myc. Immunoblotting with α-tubulin antibody was done as a loading control. Asterisks indicate nonspecific bands. (B) The percentage of large budded cells in the degron-tagged Mad2, degron-tagged Mad3, and wild-type cells at different time points as indicated above.

Mad2 associates with Mad3 and Cdc20 in the M phase independently of the functional kinetochore. (A) Flow diagram of the experiment is shown. Wild-type (3404) and ndc10-1 (3403-47-2) cells expressing P_GAL1-PDS1-MDB, Mad3-PrA, Cdc20-13Myc, and Pds1-HA were grown overnight at 23°C in YM-1 and raffinose. The cells were arrested in G₁ phase with α-factor. Following G₁ arrest at 23°C the cells were filtered, resuspended in prewarmed (37°C) YM-1 plus raffinose containing α-factor, and allowed to grow at 37°C. After 1.5 h galactose was added, and cells were grown for 30 min to induce the expression of P_GAL1-PDS1-MDB. The cells were released from α-factor arrest and allowed to grow in YM-1, raffinose, and galactose at 37°C for 90 min, and samples were collected for immunoprecipitations. (B) Flow cytometry analysis of the cells collected at the indicated time points. 1C and 2C represent the DNA content of the cells. Μad3-PrA was immunoprecipitated with IgG beads from the whole-cell extracts made from the wild-type and ndc10-1 cells. The immunoprecipitates were probed with anti-PrA and anti-Mad2 antibodies. Cdc20-13Myc was immunoprecipitated from the Mad3-PrA-depleted extracts using anti-Myc beads, and the immunoprecipitates were probed with anti-Myc and anti-Mad2 antibodies. (C) Wild-type and ndc10-1 cells were released from the α-factor arrest into galactose at 37°C as described for A in the absence (−Noc) or presence (+Noc) of nocodazole (15 μg/ml). Samples were collected every 20 min for 120 min; 0 min represents the time at which the cells were released from the α-factor arrest. Whole-cell extracts were made from the collected cells and probed with anti-HA antibodies to detect the HA-tagged endogenous Pds1. An α-tubulin immunoblot was done as a loading control.

Cdc20-Mad2 forms a complex independent of Mad3. (A) The benomyl sensitivity of the strains expressing Mad3-PrA (2873) and Mad3-PrA Cdc20-13Myc (2876-2-1) is shown. Serial tenfold dilutions of cells were spotted on YPD and YPD plus benomyl (15 μg/ml) plates. The plates were incubated at 23°C for 4 days and photographed. (B) The flow chart of the sequential immunoprecipitation from yeast whole-cell extract is shown. Mad3-PrA was immunodepleted from the extracts using IgG beads, and Cdc20-13Myc was immunoprecipitated from the resulting supernatants using anti-Myc antibody beads. (C) Mad3-PrA was immunodepleted from whole-cell extract made from nocodazole-treated cells expressing Mad3-PrA, Cdc20-13Myc (2876-2-1), and Mad3-PrA (2873). Extract made from nocodazole-treated cells expressing Cdc20-13Myc (2819) was used as the control. The whole-cell extracts (lanes 1 to 3), IgG immunoprecipitates (lanes 4 to 6), and supernatants of IgG IP (lanes 7 to 9) were probed with anti-PrA and anti-Mad2 antibodies; 50 μg whole-cell extracts and the supernatants were loaded per lane and the amount of protein loaded in the IP lane was 10-fold more than that loaded in the whole-cell extract lane. (D) Cdc20-13Myc was immunoprecipitated from the Mad3-PrA depleted extracts using anti-Myc antibody beads. The supernatants of the Mad3-PrA depleted extracts (lanes 1 to 3), anti-Myc immunoprecipitates (lanes 7 to 9), and the supernatants of anti-Myc immunoprecipitations (lanes 10 to 11) were probed with anti-Myc and anti-Mad2 antibodies. The IgG immunoprecipitates were probed with anti-Myc antibodies (lanes 4 to 6) to detect coimmunoprecipitation of Cdc20-13Myc with Mad3-PrA.

Mad3 associates with Mad2 independently of the spindle checkpoint. (A) Benomyl sensitivity of strain 2898-11-3 (Mad3-PrA P_GAL1-PDS1-MDB) was checked by spotting 10-fold serial dilutions of the cells on YPD and YPD plus benomyl (15 μg/ml). The photographs were taken after 4 days of incubation of the plates at 23°C. (B) A flow diagram of the experiment is shown. Wild-type cells (2898-11-3) containing P_GAL1-PDS1-MDB and Mad3-PrA were grown overnight in YM-1 and raffinose at 30°C. The cells were arrested in S phase with hydroxyurea (200 mM) for 3 h. Following arrest, the culture was divided into several aliquots. An aliquot of the cells were collected (HU). To another aliquot of cells, nocodazole (15 μg/ml) was added. The cells were allowed to grow and collected after 1.5 h (hydroxyurea plus Noc). To arrest cells in M phase by the activation of the spindle checkpoint, an aliquot of hydroxyurea-treated cells were washed three times and released into YM-1 with raffinose and nocodazole. The cells were allowed to grow for 1.5 h and collected (Noc). Another aliquot of cells were treated with hydroxyurea for 2.5 h, galactose was added, and the culture was allowed to grow for 30 min to induce the expression of P_GAL1-PDS1-MDB. The cells were washed three times, resuspended in YM-1 and raffinose and galactose, and harvested after 1.5 h (Gal). Cells were prepared for flow cytometry, and protein extracts were made from the samples collected. Flow cytometry profiles of the cells collected in different growth conditions is shown. 1C and 2C represent the DNA content of the cells. Mad3-PrA was immunoprecipitated from the whole-cell extracts made from the cells, and the immunoprecipitates were probed with anti-PrA and anti-Mad2 antibodies. (C) A similar experiment except after induction with galactose the culture was divided into two. One set was allowed to grow for 1.5 h in the presence of raffinose and galactose, and samples were collected (Gal [a]). Nocodazole was added to the other half in addition to raffinose and galactose. The cells were allowed to grow and collected after 1.5 h (Gal plus Noc). Flow cytometry and immunoprecipitations were done as described for B. The flow cytometry result is shown in B.