(A) Cycling cells with Bub1-myc were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were digested with trypsin and analyzed by mass spectrometry. The coverage of the identified peptides was 23%. The phosphorylation site was confirmed as threonine 566 (T566) on the basis of the MS/MS data. The phosphorylated peptide sequence and characteristic ions representing loss of phosphoric acid (-H₃PO₄) are shown. (B) Antiphosphorylated T566 antibodies. Strains with Bub1-myc or Bub1-T566A-myc were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were incubated with and without calf intestinal phosphatase (CIP) and analyzed with anti-phospho-T566 antibody (α-pT566). The membrane was then stripped and immunoblotted with antibody to the myc epitope (α-myc). (C) Phosphorylation of T566 on Bub1 requires Cdc28. Wild-type and cdc28-1N cells with Bub1-myc were grown at 25°C and then shifted to 37°C for 90 min in the presence of nocodazole (15 µg/mL). Cells were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were analyzed with anti-phospho-T566 antibody (α-pT566). The membrane was then stripped and immunoblotted with antibody to the myc epitope (α-myc). DNA content was measured by FACS analysis. (D) Cdc28 phosphorylates T566 on Bub1 in vitro. Wild-type cells with and without myc-tagged Cdc28 were incubated with nocodazole (15 µg/mL) at 30°C for 90 min. Cells were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were incubated with 100 µM ATP, 0.2 µCi [gamma-³²P]ATP with and without histone H1, MBP fused recombinant protein Bub1_400-700-MBP (MBP-fused Bub1 fragment 400–700 amino acids), and Bub1_400-700-T566A-MBP (MBP-fused Bub1 fragment 400–700 amino acids with T566A change). Coomassie Brilliant Blue staining (CBB) is shown as a loading control. A background band is indicated by the asterisk (*).

(A) BUB1-T566A mutant cells were sensitive to nocodazole on a plate. Wild-type (WT), BUB1-T566A, and bub1Δ mutant cells were spotted in 5-fold dilutions from 4×10⁷ cells per spot on YPD plates containing nocodazole (2.5 and 10 µg/mL). (B) Wild-type (WT), bub1Δ, and BUB1-T566A cells were incubated with nocodazole (10 µg/mL) for 1.5, 2, 3, 4 and 5 h; at the indicated times, samples were taken for FACS analysis. (C) Cell and nuclear morphologies. Wild-type (WT) and BUB1-T566A cells were incubated with nocodazole (10 µg/mL) for 1.5, 2, 3, 4 and 5 h; at the indicated times, samples were fixed with 4% paraformaldehyde and stained with DAPI. Percentages of indicated morphologies are presented. (D) Representative pictures of cell and nuclear morphologies analyzed in Figure 2C. 1: an unbudded cell without a nucleus, 2: an unbudded cell with a nucleus, 3: a large budded cell with a nucleus in the mother cell, 4: a large budded cell with a nucleus at the neck in the mother cell, 5: a large budded cell with a nucleus at the neck between the mother cell and the daughter cell, and 6: a rebudded cell with a nucleus. Bar, 5 µm. (E) Clb2 levels in nocodazole-treated cells. Wild-type (WT) and BUB1-T566A cells were incubated with nocodazole (10 µg/mL) for 1.5, 2, 3, 4, 5 and 6 h; at the indicated times, samples were taken for Western blot analyses with Clb2 antibody. Cdc28 was used as a loading control.

(A) Wild-type (WT) and BUB1-T566A cells were arrested using alpha-factor and released into medium containing 2.5 µg/mL nocodazole at 30°C. After 1 h, alpha-factor was added to the medium and samples were taken for FACS analysis at the indicated times. (B) Graph representing the G1 and rebudded cells shown in Figure 3A. The percentages of G1 and rebudded cells were scored in samples taken at the indicated times (n = 100). (C) Wild-type (Video S1) and BUB1-T566A (Video S2) mutant cells were arrested by alpha-factor and released from G1 onto plates containing 2.5 µg/mL nocodazole at 30°C. Time-lapse images of representative examples of each strain were taken at the indicated times.

(A) BUB1-T566A mutant cells do not show substantial delay in recovering from nocodazole arrest. Wild-type (WT) and BUB1-T566A mutant cells were incubated with nocodazole (15 µg/mL) at 30°C for 90 min and then released from nocodazole arrest; at the indicated times, samples were taken to measure rebudded cells. (B) BUB1-T566A mutant cells show no significant sensitivity to nocodazole in a survival assay. Wild-type (WT), bub1Δ, and BUB1-T566A cells were incubated with nocodazole (15 µg/mL); at the indicated times, cells were washed out and approximately 200 cells were plated on a YPD plate. Cell viability was calculated by dividing the number of colonies formed at the 2.5 and 5 h time points by that formed in the absence of nocodazole (0 h) at 30°C. (C) Phosphorylation of T566 is important for degradation of Bub1 during anaphase but not during G1. cdc15-2 cells with HA-tagged Bub1 or Bub1-T566A expressed by the GAL1 promoter (GAL-BUB1 and GAL-BUB1-T566A) were arrested in anaphase (cdc15-2) or in G1 (alpha-factor) and then transferred to medium containing galactose for 2 h to induce Bub1 expression. Glucose was added to shut-off Bub1 expression; samples were taken at the indicated times for Western blot analyses with antibody to the HA epitope. Tubulin was used as a loading control. (D) The Bub1-T566A protein is more stable than wild-type in the presence of nocodazole. Wild-type and BUB1-T566A mutant cells (Bub1 and Bub1-T566A are tagged with myc) were incubated in the presence of nocodazole (10 µg/mL) at 30°C; at the indicated times, samples were taken for Western blot analyses with antibody to the myc epitope. Tubulin was used as a loading control.

(A) BUB1-T566A mutation does not alter kinase activity. Cells expressing Bub1-myc or Bub1-T566A-myc were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were analyzed by performing a kinase assay with 100 µM ATP, 0.2 µCi [gamma-³²P]ATP in the presence or absence of human histone H2A recombinant. Coomassie Brilliant Blue staining (CBB) is shown as a loading control. (B) BUB1-T566A mutant cells do not display a chromosome missegregation phenotype. The colony color assay was performed as previously described [27], [43]. Briefly, wild-type, bub1Δ and BUB1-T566A mutant cells containing a single SUP11-marked chromosome fragment were plated at a density of ∼200 colonies per plate on minimal (SD) medium containing a limiting amount of adenine (6 µg/mL) and grown at 30°C. A colony consists of cells containing the chromosome fragment (white) and cells that have lost it (red), resulting in a white-and-red sectored phenotype. (C) BUB1-T566A mutant cells do not lose their endogenous chromosome. Diploid strains at MAT do not mate because of codominant suppression of haploid-specific cell differentiation pathways. Loss of either the MATa or MATalpha allele results in mating competence, where mating type is determined by the remaining allele [29], [44]. The indicated diploids cells were mated with haploid MATa (17/14) and MATalpha (17/17) tester strains and the mating products were selected. Two independent clones of BUB1-T566A/BUB1-T566A mutant cells are shown. (D) BUB1-T566A mutant cells do not show elevated a-like faker frequency. Loss of the MATalpha locus leads to the default mating type. MATalpha cells that lose the MAT locus will mate as a-type cells [29], [44]. Indicated MATalpha strains were mated with the MATalpha tester strains (17/17) and mating products were selected.