The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

Different levels of Bfa1/Bub2 GAP activity are required to prevent mitotic exit of budding yeast depending on the type of perturbations

In budding yeast, Tem1 is a key regulator of mitotic exit. Bfa1/Bub2 stimulates Tem1 GTPase activity as a GTPase-activating protein (GAP). Lte1 possesses a guanine-nucleotide exchange factor (GEF) domain likely for Tem1. However, recent observations showed that cells may control mitotic exit without either Lte1 or Bfa1/Bub2 GAP activity, obscuring how Tem1 is regulated. Here, we assayed BFA1 mutants with varying GAP activities for Tem1, showing for the first time that Bfa1/Bub2 GAP activity inhibits Tem1 in vivo. A decrease in GAP activity allowed cells to bypass mitotic exit defects. Interestingly, different levels of GAP activity were required to prevent mitotic exit depending on the type of perturbation. Although essential, more Bfa1/Bub2 GAP activity was needed for spindle damage than for DNA damage to fully activate the checkpoint. Conversely, Bfa1/Bub2 GAP activity was insufficient to delay mitotic exit in cells with misoriented spindles. Instead, decreased interaction of Bfa1 with Kin4 was observed in BFA1 mutant cells with a defective spindle position checkpoint. These findings demonstrate that there is a GAP-independent surveillance mechanism of Bfa1/Bub2, which, together with the GTP/GDP switch of Tem1, may be required for the genomic stability of cells with misaligned spindles.     

Bub2 Is a Cell Cycle Regulated Phospho-Protein Controlled by Multiple Checkpoints

During mitotic exit, a small GTPase Tem1 needs to be activated. During most of the cell cycle, Tem1 activity is antagonized by a GTPase activating complex (GAP) composed of Bub2 and Bfa1. Bfa1 protein has cell cycle regulated phosphorylation depending upon the Polo-like kinase Cdc5. This phosphorylation dissociates Bfa1 from Tem1 and thus relieves the inhibition of Tem1 by the GAP complex. Bub2 and Bfa1 are also required to prevent mitotic exit when there is DNA damage, spindle damage or spindle misorientation at G2/M phase. While Cdc5 inhibits Bfa1/Bub2, mutating the Cdc5 phosphorylation sites on Bfa1 does not have a strong activating effect on Bub2/Bfa1, suggesting there must be additional regulation in this pathway. Here we report that Bub2 protein also has cell cycle regulated phosphorylation. This phosphorylation is partially dependent upon the Polo-like kinase Cdc5 and is consistent with negative regulation of the Bub2/Bfa1 GAP complex. Spindle damage or spindle misorientation prevents Bub2 phosphorylation. The spindle damage effect is dependent upon the spindle assembly checkpoint components Mad2 and Mps1. Thus like Bfa1, Bub2 protein is also controlled both during mitotic exit and in response to cell cycle checkpoints. Bub2 phosphorylation is likely to be controlled by a novel kinase.

Key Words:

Bub2, Bfa1, Cdc5, Phosphorylation, Mitotic exit, Cell cycle checkpoints     

Bub2 is a cell cycle regulated phospho-protein controlled by multiple checkpoints

During mitotic exit, a small GTPase Tem1 needs to be activated. During most of the cell cycle, Tem1 activity is antagonized by a GTPase activating complex (GAP) composed of Bub2 and Bfa1. Bfa1 protein has cell cycle regulated phosphorylation depending upon the Polo-like kinase Cdc5. This phosphorylation dissociates Bfa1 from Tem1 and thus relieves the inhibition of Tem1 by the GAP complex. Bub2 and Bfa1 are also required to prevent mitotic exit when there is DNA damage, spindle damage or spindle misorientation at G(2)/M phase. While Cdc5 inhibits Bfa1/Bub2, mutating the Cdc5 phosphorylation sites on Bfa1 does not have a strong activating effect on Bub2/Bfa1, suggesting there must be additional regulation in this pathway. Here we report that Bub2 protein also has cell cycle regulated phosphorylation. This phosphorylation is partially dependent upon the Polo-like kinase Cdc5 and is consistent with negative regulation of the Bub2/Bfa1 GAP complex. Spindle damage or spindle misorientation prevents Bub2 phosphorylation. The spindle damage effect is dependent upon the spindle assembly checkpoint components Mad2 and Mps1. Thus like Bfa1, Bub2 protein is also controlled both during mitotic exit and in response to cell cycle checkpoints. Bub2 phosphorylation is likely to be controlled by a novel kinase.     

Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.     

Mitotic exit: closing the gap

Completion of mitosis is triggered by the activation of the Ras-like GTP-binding protein Tem1p. In the November 30, 2001 issue of Cell, Hu et al. suggest that Tem1p activation is achieved by inhibition of its two-component GAP Bub2p/Bfa1p via phosphorylation of Bfa1p by the Polo kinase Cdc5p. Interestingly, activation of spindle checkpoints inhibits Bfa1p phosphorylation, suggesting that these signaling pathways prevent mitotic exit by maintaining the GAP activity of Bub2p/Bfa1p.     

Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.     

Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1

The elimination of mitotic kinase activity at the end of mitosis is essential for progression to the next stage of the eukaryotic cell cycle. In budding yeast, this process is controlled by a regulatory cascade called the mitotic exit network. Extensive genetic data indicate that mitotic exit network activity is determined by a GTP-binding protein, Tem1, and its putative regulators, Bub2, Bfa1, and Lte1. Here we describe the purification and in vitro activities of Tem1, Bub2, and Bfa1. We describe the nucleotide binding properties of Tem1 and characterize its intrinsic GTPase activity. The combination of Bfa1 and Bub2 acts as a two-component GTPase-activating protein for Tem1. In the absence of Bub2, Bfa1 inhibits the GTPase and GTP exchange activities of Tem1. This inhibition is elicited by either the N- or C-terminal regions of Bfa1, which also retain some ability to co-activate GTPase activity in the presence of Bub2. Although the C-terminal region of Bfa1 binds to Bub2, no interaction of the N-terminal half of Bfa1 with Bub2 was detected despite their combined GAP activity. Therefore, we propose that Bfa1 acts both as an adaptor to connect Bub2 and Tem1 and as an allosteric effector that facilitates this interaction.     

Checkpoint control of mitotic exit—do budding yeast mind the GAP?

Cell cycle checkpoints can delay mitotic exit in budding yeast. The master controller is the small GTPase Tem1, with inputs from a proposed guanine nucleotide exchange factor (GEF), Lte1, and a GTPase-activating protein (GAP), Bub2/Bfa1. In this issue, Fraschini et al. (p. 335) show that GAP activity of Bub2/Bfa1 appears to be dispensable for inactivation of Tem1 in cells. Their results call into question the GTP/GDP switch model for Tem1 activity, as have other results in the past. The paper also focuses attention on the two spindle pole bodies as potential sites for regulation of Tem1.     

IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae

During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Delta as well as in bub2Delta, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Delta, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Delta in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Delta and bfa1Delta. The mitotic arrest defects of ibd2Delta were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Deltamad2Delta, ibd2Deltabub2Delta, and ibd2Deltadyn1Delta showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.     

Bub1 kinase targets Sgo1 to ensure efficient chromosome biorientation in budding yeast mitosis

During cell division all chromosomes must be segregated accurately to each daughter cell. Errors in this process give rise to aneuploidy, which leads to birth defects and is implicated in cancer progression. The spindle checkpoint is a surveillance mechanism that ensures high fidelity of chromosome segregation by inhibiting anaphase until all kinetochores have established bipolar attachments to spindle microtubules. Bub1 kinase is a core component of the spindle checkpoint, and cells lacking Bub1 fail to arrest in response to microtubule drugs and precociously segregate their DNA. The mitotic role(s) of Bub1 kinase activity remain elusive, and it is controversial whether this C-terminal domain of Bub1p is required for spindle checkpoint arrest. Here we make a detailed analysis of budding yeast cells lacking the kinase domain (bub1ΔK). We show that despite being able to arrest in response to microtubule depolymerisation and kinetochore-microtubule attachment defects, bub1ΔK cells are sensitive to microtubule drugs. This is because bub1ΔK cells display significant chromosome mis-segregation upon release from nocodazole arrest. bub1ΔK cells mislocalise Sgo1p, and we demonstrate that both the Bub1 kinase domain and Sgo1p are required for accurate chromosome biorientation after nocodazole treatment. We propose that Bub1 kinase and Sgo1p act together to ensure efficient biorientation of sister chromatids during mitosis.     

Bub2 regulation of cytokinesis and septation in budding yeast

Background  

The mitotic exit network (MEN) is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP) to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction.     

Mutual regulation of cyclin-dependent kinase and the mitotic exit network

The mitotic exit network (MEN) is a spindle pole body (SPB)–associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1–Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2–Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1–Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.     

The study of Bfa1p(E438K) suggests that Bfa1 control the mitotic exit network in different mechanisms depending on different checkpoint-activating signals

During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit via mitotic checkpoints. In budding yeast, mitotic exit is controlled by a regulatory cascade called the mitotic exit network (MEN). The MEN is regulated by a small GTPase, Tem1p, which in turn is controlled by a two-component GAP, Bfa1p-Bub2p. Recent results suggested that phosphorylation of Bfa1p by the polo-related kinase Cdc5p is also required for triggering mitotic exit, since it decreases the GAP activity of Bfa1p-Bub2p. However, the dispensability of GEF Lte1p for mitotic exit has raised questions about regulation of the MEN by the GTPase activity of Tem1p. We isolated a Bfa1p mutant, Bfa1p(E438K), whose overexpression only partially induced anaphase arrest. The molecular and biochemical functions of Bfa1p(E438K) are similar to those of wild type Bfa1p, except for decreased GAP activity. Interestingly, in BFA1(E438K) cells, the MEN could be regulated with nearly wild type kinetics at physiological temperature, as well as in response to various checkpoint-activating signals, but the cells were more sensitive to spindle damage than wild type. These results suggest that the GAP activity of Bfa1p-Bub2p is responsible for the mitotic arrest caused by spindle damage and Bfa1p overproduction. In addition, the viability of cdc5-2 delta bfa1 cells was not reduced by BFA1(E438K), suggesting that Cdc5p also regulates Bfa1p to activate mitotic exit by other mechanism(s), besides phosphorylation.     

Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.     

Cdc5-dependent asymmetric localization of bfa1 fine-tunes timely mitotic exit

In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: (452)S, (453)S, (454)S, and (559)S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1(4A)) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1(4D)) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1(4D) was asymmetrically localized. BFA1(4A) cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA1(4D) cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.     

The Bub2p spindle checkpoint links nuclear migration with mitotic exit

Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.     

Septins have a dual role in controlling mitotic exit in budding yeast

In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor.