Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

Abstract

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B-independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B-mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.

FIGURE 1:

The defect of sgo1Δ and bub1Δ strains is suppressed by BIR1 or SLI15 overexpression. (A) Overexpression of SGO1, BIR1, and SLI15 under control of their own promoter and carried on 2-μm plasmids suppresses the temperature sensitivity of bub1Δ tetraploids. Two independent clones were tested for each genotype, and cells were plated by fivefold serial dilution on plates lacking leucine (to select for plasmid retention) and containing 5-FOA (counterselection against plasmid containing the wild-type BUB1). The 2-μm LEU2 vector pRS425 (“empty”) and a LEU2-marked centromeric plasmid containing BUB1 were used as negative and positive controls, respectively. (B) Overexpression of BIR1 and SLI15 suppresses the lethality of sgo1Δ tetraploids at 37°C. The setup is identical to the previous experiment. (C) BIR1 and SLI15 overexpression suppresses the sensitivity of haploid sgo1Δ cells to the microtubule-depolymerizing drug benomyl. (D) The genetic interaction is not reciprocal, as overexpression of SGO1 does not improve the growth of sli15-3 mutants at the minimal restrictive temperature (35°C).

FIGURE 2:

Overexpression of SGO1 or SLI15 reduces the chromosome segregation defect in bub1Δ cells without restoring spindle checkpoint function. (A) Strains were grown in liquid culture and analyzed for their ability to arrest in the prolonged presence of the microtubule poison nocodazole. Whereas wild-type cells arrest as large-budded cells, mutants lacking a functional SAC reenter the cycle, which is indicated morphologically by the formation of a second bud. The accumulation of multibudded cells in the bub1Δ strain was unaffected by overexpression of SGO1 or SLI15, indicating that the SAC remains defective. (B) The elevated chromosome loss rate in a haploid bub1Δ strain bearing a YAC, measured by genetic fluctuation test, decreases significantly upon SGO1 or SLI15 overexpression. The experiments were performed at 30°C. *P < 0.05, **P < 0.01, Student’s unpaired t test, two-tailed.

FIGURE 3:

Frequent chromosome missegregation in sgo1Δ mutants is due to defects in biorientation and can be ameliorated by overexpression of BIR1 or SLI15. (A) sgo1Δ and bub1Δ tetraploid strains exhibit elevated rates of nondisjunction at chromosome IV; this defect can be suppressed by BIR1 overexpression. Chromosome IV was visualized using a TetO/TetR-GFP array, and SPBs were marked by expression of Spc29-RFP. At least 150 anaphase cells were scored for each genotype. The experiments were performed at 33°C in order to enhance the phenotype. Bar = 1 μm. (B) Haploid cells lacking SGO1 show frequent monopolar attachment of an unreplicated dicentric minichromosome, albeit less so than mutants with reduced Ipl1 kinase activity. The missegregation defect in the sgo1Δ strain can be partially suppressed by overexpression of SLI15. The experiments were performed at 33°C. The two inset images show examples of attachments: Left, the GFP-labeled attachment is positioned between the two SPBs (marked with RFP), indicating a bipolar attachment. Right, the minichromosome localizes in proximity to one of the SPBs, indicating monopolar attachment. (C) Diploid cells were released from treatment with nocodazole in order to create conditions in which a large number of syntelic attachments would be formed, even in wild-type cells. Live-cell imaging was used to monitor the initial attachment (left) and eventual segregation (right) of a GFP-marked copy of chromosome IV. sgo1Δ cells never converted monopolar attachments into bipolar attachments during the time of observation (75 min) and were consequently much more likely than wild-type cells to undergo nondisjunction in anaphase.

FIGURE 4:

The localization and activity of CPC proteins are not affected by the absence of Bub1 or Sgo1. (A) The localization of Sli15-GFP, Bir1-GFP, and Ipl1-GFP to preanaphase spindles is not diminished in the diploid sgo1Δ cells. SPBs are marked with Spc29-RFP. Bar = 1 μm. (B) Sli15-directed ChIP revealed a sevenfold enrichment of centromeric DNA relative to telomeric DNA in wild-type cells. This enrichment is not affected by absence of Bub1, similar to results obtained with Ndc10-directed ChIP. (C) Phosphorylation of Sli15-TAP is not affected by the presence or absence of BUB1 and SGO1. Cells were grown at 35°C, synchronized in α-factor (G1 phase) or hydroxyurea (S phase), harvested, and analyzed by PAGE and Western blotting. Slower migrating forms of Sli15-TAP were eliminated by mutation of the Ipl1 kinase domain but not by deletion of SGO1 or BUB1. (D) The phosphorylation of Dam1, the crucial substrate of Ip1l in the release of microtubule attachments, is unaltered by the deletion of BUB1 (top, 30°C) but is abolished in the ipl1-321 mutants at 37°C (bottom). Two independent clones of both the bub1Δ and ipl1-321 mutants were tested, and the separation between phosphoforms of Dam1-myc9 was enhanced by adding 10 μM Phos-Tag AAL-107 (Kinoshita et al., 2006) to the polyacrylamide gel mixture. The slower migrating forms of Dam1-myc9 are sensitive to alkaline phosphatase treatment (top).

FIGURE 5:

Some monopolar attachments can be corrected independently of the CPC. (A) Chromosome IV was marked with a TetO/TetR-GFP array. The percentage of cells with bioriented chromosome IV (as judged by the localization of the GFP dot in relation to the marked SPBs) among all large-budded cells was assessed after release from nocodazole. All strains carry a genomic ipl1-321 allele as well as a multicopy plasmid with either the functional IPL1 gene (black diamonds), SGO1 (light circles), or marker only (gray squares). (B) Progression through anaphase after release from nocodazole is accelerated in cells lacking functional IPL1, as evidenced by the decrease of the percentage of large-budded cells in the population (full lines). These strains also accumulate bibudded cells at higher levels than wild-type (dotted lines). Overexpression of SGO1 did not alter the cell-cycle progression of ipl1-321 mutants, even though it affected biorientation (A). The experiments were performed at 35°C.

FIGURE 6:

Sgo1 can partially bypass the role of Mps1 in chromosome segregation and appears to act in the same pathway. (A) Overexpression of SGO1 allows cells to survive transient pharmacological inhibition of Mps1 kinase during mitosis. The cells were synchronized in S phase (at the restrictive temperature for the cdc34-2 allele), released into mitosis with or without the mps1-as inhibitor and with or without nocodazole, and then spotted on nonselective plates. (B) Localization of Sgo1-GFP to preanaphase spindles requires active Mps1. The images were taken 10 min after adding dimethyl sulfoxide (control samples) or the ATP analogue 1NM-PP1 to inhibit Mps1. (C) Overexpression of Sgo1-GFP on a 2-μm plasmid increases retention of Sgo1 on the mitotic spindle, even in the absence of targeting by Mps1. (D). Quantification of the fluorescence signal on the spindle for Sgo1-GFP, overexpressed Sgo1-GFP, Sli15-GFP, and Bub1-GFP in cells with active or inactive Mps1 kinase.