(A) Wild-type (KH186), mad1Δ (MB076), mad2Δ (KH141), mad3Δ (KH173), bub1Δ (KH127), bub3Δ (MB003), and bub1ΔK (JF098) strains were plated out in 10-fold serial dilutions on YPDA media or on YPDA media containing 8 μg/ml benomyl.
(B) The indicated strains were grown in YPDA media containing 30 μg/ml nocodazole, and the viability of the cells was measured as percentage of cells able to form colonies on YPDA media lacking microtubule drugs.
(C) Wild-type (JF004), bub1ΔK (JF125), bub1Δ (JF140), and mad3Δ (EK013) strains containing a GFP-marked chromosome were synchronised in G1 using α-factor, then released into YPDA with 30 μg/ml nocodazole at 23 °C. We tested ability of the cells to maintain a spindle checkpoint arrest by scoring cells that could keep their GFP-marked sister chromatids cohesed for 3 h in nocodazole (i.e., one GFP dot). The percentage of cells with two GFP dots was counted at the release from G1 (grey bars) and after 3 h in nocodazole (black bars) (n = 100 cells for each repeat experiment). Error bars indicate standard deviation.
(D) Wild-type (JF004) and bub1ΔK (JF125) strains containing Pds1-18myc were arrested in G1 using α-factor and synchronously released into YPDA media or YPDA media containing 30 μg/ml nocodazole at 23 °C. Samples were taken at indicated times. Levels of Pds1 were monitored by immunoblotting using A14 α-myc antibody and α-PGK1 as a loading control.

(A) Wild-type (AMY1110) and bub1ΔK (JF038) strains containing Sgo1-9Myc and Ndc10-6HA (to mark the kinetochores) were arrested in metaphase with 15 μg/ml nocodazole and 30 μg/ml benomyl at 23 °C for 3 h. Chromosome spreads were performed and stained with α-myc antibody (CM-100), α-HA antibody (HA11), and DAPI to recognize the DNA. Spreads with clear Ndc10-6HA staining were categorized as showing colocalization with Sgo1-9myc only if they contained multiple (>2) overlapping foci. Fifty spreads per strain were analysed.
(B) Sgo1-9myc colocalises with the SPB in the bub1ΔK mutant. Spreads were prepared as in (A) and stained with α-myc antibody, anti-Spc110 antibody to detect SPBs, and DAPI. Scale bar represents 2 μm.
(C) Schematic of primers sets for Sgo1-6HA ChIP analysis showing the CEN3, centromeric region; R3, pericentromeric region; and c281, the negative arm region.
(D) PCR on ChIPs of “no tag” (KH186), Sgo1-6HA (AMY209), and bub1ΔK, Sgo1-6HA (JF211) strains arrested with 15 μg/ml nocodazole and 30 μg/ml benomyl at 23 °C for 3 h, showing reduced Sgo1p levels associated with CEN3 and R3 regions in the bub1ΔK mutant compared to wild type.
(E) The graph shows quantification of the ChIP data from the “no tag” (KH186), wild-type (AMY209), and bub1ΔK (JF211) strains. The “binding ratio” was calculated as a ratio of the ChIP PCR signal to the PCR signal from a 1:500 diluted input fraction. The error bars indicate standard deviation (SD) of the mean from five different PCR reactions from two separate experiments.

(A) We analysed the indicated strains for unattached kinetochores following nocodazole treatment. Wild-type (SBY4340), bub1ΔK (JF123), sgo1Δ (JF202), and ndc80–1 (SBY4342) cells were arrested in media containing 30 μg/ml benomyl and 15 μg/ml nocodazole for 3 h at 23 °C. The microtubule drugs were then washed out three times and released into rich media at 30 °C for 20 min. The control ndc80–1 cells were grown at 36 °C for 1 h before fixation. Cells were then fixed in 3.7% formaldehyde for 5 min. We scored cells with kinetochores (Mtw1-3GFP staining) off the spindle axis as “unattached kinetochores.” All strains, apart from the ndc80–1, had mostly attached kinetochores aligned on the spindle. A total of 8% of wild-type cells contained unattached kinetochores at this timepoint (n = 212); bub1ΔK, 8% (n = 252); and sgo1Δ, 4% (n = 157) compared to ndc80–1, which had 79% (n = 96) unattached kinetochores. Right hand panel shows examples of unattached kinetochores for each strain. Scale bar represents 2 μm.
(B) Schematic of the principle of the experiment showing GFP marked centromere “breathing” upon chromosome biorientation (i.e., two green dots).
(C) Images of separated SPBs (Spc42-Tomato) containing either one GFP foci in between two SPBs (empty white triangle, scored as nonbioriented) or one GFP foci by one SPB (red empty triangle, scored as nonbioriented) or finally two GFP foci between two SPBs (filled triangle, scored as bioriented chromosome). Scale bar represents 2 μm.
(D) Wild-type (JF152), bub1ΔK (JF154), sgo1Δ (JF156), and bub1ΔK,sgo1Δ (JF202) strains carrying cenIV-GFP, Met-Cdc20, and Spc42-Tomato were synchronised in G1 for 3 h and then depleted for Cdc20 by incubating them in α-factor and 8 mM methionine for 2 h before releasing them into media containing 30 μg/ml benomyl, 15 μg/ml nocodazole, and 8 mM methionine at 23 °C for 3 h. The nocodazole was then washed out and samples were taken at indicated times. Only cells that had separated SPBs were counted for each sample (n = 100) and scored for biorientated chromatids (two GFP foci between two SPBs) versus nonbioriented chromatids (one GFP foci between two SPBs and one GFP foci by one SPB). Error bars indicate standard deviation. Standard deviations are based on five separate experiments with all strains apart from sgo1Δ,bub1ΔK double mutant strain, which was used in two separate experiments.
(E) This graph represents the cells in which the single GFP dot resided next to the SPB (red triangle) as opposed to in between the SPBs (white empty triangle), calculated as a percentage of the total cells with only one GFP dot. The data plotted are the average from two separate experiments.

(A) Wild-type (JF004), mtw1–1 (SBY1646), mtw1–1, bub1ΔK (JF100), and mtw1–1, ipl1-321 (SBY1724) strains carrying the temperature-sensitive allele mtw1–1 and Pds1-18Myc were synchronised in G1 for 2.5 h then incubated at the restrictive temperature 36 °C for 30 min before release. The levels of Pds1 were monitored by immunoblotting with A14 anti-myc antibody and anti-PGK1 as a loading control.
(B) Wild-type (VBI545), bub1ΔK (JF023), mad2Δ (VBI560), and sgo1Δ (JF224) strains, containing Pds1-myc, were synchronised in G1 with α-factor in media containing 2% galactose and 2% raffinose to maintain Mcd1 expression. Mcd1 was then turned off by incubating them in media containing glucose and α-factor for 3 h. The cells were then released into rich media containing glucose, and samples for immunoblotting were taken at indicated times. Pds1 and PGK1 levels were monitored as previously described.
(C) Wild-type (YBS151), bub1ΔK (JF110), and mad2Δ (YBS406) strains, carrying a LEU2-marked linear minichromosome (LMC) were plated in 10-fold serial dilutions on −LEU plates and on −LEU plates with 10 μg/ml doxycycline. In this strain doxycycline represses a dominant CDC20 allele that is insensitive to the spindle checkpoint proteins [13]. Therefore this CDC20 allele makes cells insensitive to the presence of LMCs. These LMCs are thought to be unable to withstand spindle forces and separate producing kinetochore-microtubule attachments that are no longer under tension. In the presence of doxycycline, wild-type cells grow very poorly because of their checkpoint response to the LMCs, but the bub1ΔK mutant grows far better, indicating that their ability to respond to attachments lacking tension is impaired.

(A) Wild-type (JF004) and bub1ΔK (JF125) cells were released from G1 into media containing 30 μg/ml nocodazole and incubated at 23 °C for 3 h. Cells were subsequently released into anaphase by washing out the nocodazole. Samples were fixed in 3.7% formaldehyde for 1 h, 30 min after release, and stained with α-GFP (GFP-marked chromosome) and α-tubulin (red spindle) antibodies. DNA was stained with DAPI (blue). Percentage of nondisjunction of the GFP-marked chromatid at the first anaphase following nocodazole arrest was 2% in wild-type cells and 33% in bub1ΔK cells (n ≥ 50 anaphase cells). Scale bar represents 3 μm.
(B) Wild-type (JF004) and bub1ΔK (JF125) strains were synchronised in G1 as previously described and cells with one GFP dot (empty triangle) versus two dots (filled triangle) were counted (n = 400). Scale bar represents 2 μm.
(C) Cells from (B) were then released and incubated in media containing 30 μg/ml nocodazole at 23 °C for 3 h and released into media containing α-factor to score cells in the following G1. The number of cells with one GFP dot versus two dots were scored (n = 400).
(D) Wild-type (KH186), bub1Δ (KH127), bub1ΔK (JF098), sgo1Δ (JF188), and bub1Δ, sgo1Δ (JF185) strains were plated out in 10-fold serial dilutions on rich media and on rich media containing 8 μg/ml benomyl.
(E) Strains carrying the SUP11 artificial chromosome were grown overnight in CSM-URA media, then diluted back to OD₆₀₀ 0.2 and grown in YPDA media at 30 °C for 3 h. Cells were then plated out on YPD at a density of ∼500 cells per plate. Only colonies that were at least half red were scored for losing the test chromosome at the first division.

(A) Simple linear pathway: Bub1 kinase phosphorylates Factor X in such a way that Sgo1 is efficiently targeted to centromeres. This in turn ensures efficient recruitment of the Passenger proteins, which act to monitor and correct any inappropriate kinetochore-microtubule attachments.
(B) Parallel pathways: Bub1 kinase, Sgo1p, and the Passenger proteins act in a concerted fashion to ensure efficient biorientation of sister kinetochores. This model better explains why localization of the different proteins are not entirely dependent upon one another. It also suggests that Bub1 kinase, and/or Sgo1p, might act to break inappropriate kinetochore attachments independently of Aurora kinase.