DNA damage checkpoints inhibit mitotic exit by two different mechanisms

Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.     

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.     

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     

Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.

During mitosis, a ras-related GTPase (Tem1) binds GTP and activates a signal transduction pathway to allow mitotic exit. During most of the cell cycle, Tem1 function is antagonized by a GTPase-activating protein complex, Bfa1/Bub2. How the Bfa1/Bub2 complex is regulated is not well understood. We find that Polo/Cdc5 kinase acts upstream of Bfa1/Bub2 in the mitotic exit network. Cdc5 phosphorylates Bfa1 and acts to antagonize Bfa1 function to promote mitotic exit. Bfa1 is regulated by multiple cell cycle checkpoints. The spindle assembly and spindle orientation checkpoints inhibit Bfa1 phosphorylation. DNA damage does not inhibit Bfa1 phosphorylation and instead causes a Rad53- and Dun1-dependent modification of Bfa1. Regulation of Bfa1 may therefore be a key step controlled by multiple checkpoint pathways to ensure a mitotic arrest.     

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.     

Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function

The mitotic exit network (MEN) is a signaling cascade that triggers inactivation of the mitotic cyclin-dependent kinases and exit from mitosis. The GTPase Tem1 localizes on the spindle pole bodies (SPBs) and initiates MEN signaling. Tem1 activity is inhibited until anaphase by Bfa1-Bub2. These proteins are also part of the spindle position checkpoint (SPOC), a surveillance mechanism that restrains mitotic exit until the spindle is correctly positioned. Here, we show that regulation of Tem1 localization is essential for the proper function of the MEN and the SPOC. We demonstrate that the dynamics of Tem1 loading onto SPBs determine the recruitment of other MEN components to this structure, and reevaluate the interdependence in the localization of Tem1, Bfa1, and Bub2. We also find that removal of Tem1 from the SPBs is critical for the SPOC to impede cell cycle progression. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for mitotic exit.     

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.     

In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5

The Cdc5 protein of budding yeast is a polo-like kinase that has multiple roles in mitosis including control of the mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been identified as a regulatory target of Cdc5 but there are conflicting deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1 is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited although its ability to interact with Tem1 is unaffected. Thus, in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a positive regulatory effect by increasing levels of Tem1-GTP so stimulating exit from mitosis.     

Budding Yeast DNA Damage Adaptation Mutants Exhibit Defects in Mitotic Exit

In the presence of double strand breaks, DNA damage checkpoint halts cell cycle progression. However, cells ultimately escape the checkpoint arrest and re-enter cell cycle in the presence of irreparable DNA damage. cdc5-ad was identified as a mutant that fails to adapt to the cell cycle arrest induced by DNA damage checkpoint. In budding yeast, Cdc5 protein kinase is a component of both MEN and FEAR pathways that are required for mitotic exit. It remains unclear whether the adaptation defect of cdc5-ad mutant cells is related to the function of Cdc5 in mitotic exit. Here we present evidence indicating that cdc5-ad mutant cells exhibit defects in mitotic exit. cdc5-ad mutant cells are sensitive to high dosage of Amn1, a negative regulator of MEN. It also shows synthetic growth defects with mutants in MEN pathway. Moreover, mutants in FEAR pathway exhibit defects in DNA damage adaptation. Thus, we conclude that the compromised mitotic exit pathway contributes to DNA damage adaptation defects in cdc5-ad mutant cells.     

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.     

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.     

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.     

Regulation of the Bfa1p-Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p-Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p-Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.     

Kin4 kinase delays mitotic exit in response to spindle alignment defects

For many polarized cells, it is critical that the mitotic spindle becomes positioned relative to the polarity axis. This is especially important in yeast, where the site of cytokinesis is predetermined. The spindle position checkpoint (SPOC) therefore delays mitotic exit of cells with a mispositioned spindle. One component of the SPOC is the Bub2-Bfa1 complex, an inhibitor of the mitotic exit network (MEN). Here, we show that the Kin4 kinase is a component of the SPOC and as such is essential to delay cell cycle progression of cells with a misaligned spindle. When spindles are correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite spindle pole bodies (SPBs). Bub2-Bfa1 then becomes inhibited by Cdc5 polo kinase with anaphase onset, a prerequisite for mitotic exit. In response to spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs. Kin4 now maintains Bub2-Bfa1 activity by counteracting Cdc5, thereby inhibiting mitotic exit.     

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.     

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 cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.     

Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling

In S. cerevisiae cells undergoing anaphase, a ras-related GTPase, Tem1, is located on the spindle pole body that enters the daughter cell and activates a signal transduction pathway, MEN, to allow mitotic exit. MEN activation must be reversed after mitotic exit to reset the cell cycle in G1. We find that daughter cells activate an Antagonist of MEN pathway (AMEN) in part through induction of the Amn1 protein that binds directly to Tem1 and prevents its association with its target kinase Cdc15. Failure of Amn1 function results in defects of both the spindle assembly and nuclear orientation checkpoints and delays turning off Cdc14 in G1. Thus, Amn1 is part of a daughter-specific switch that helps cells exit from mitotic exit and reset the cell cycle.     

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.     

A new function of Skp1 in the mitotic exit of budding yeast Saccharomyces cerevisiae

We previously reported that Skp1, a component of the Skp1-Cullin-F-box protein (SCF) complex essential for the timely degradation of cell cycle proteins by ubiquitination, physically interacts with Bfa1, which is a key negative regulator of the mitotic exit network (MEN) in response to diverse checkpoint-activating stresses in budding yeast. In this study, we initially investigated whether the interaction of Skp1 and Bfa1 is involved in the regulation of the Bfa1 protein level during the cell cycle, especially by mediating its degradation. However, the profile of the Bfa1 protein did not change during the cell cycle in skp1-11, which is a SKP1 mutant allele in which the function of Skp1 as a part of SCF is completely impaired, thus indicating that Skp1 does not affect the degradation of Bfa1. On the other hand, we found that the skp1-12 mutant allele, previously reported to block G2-M transition, showed defects in mitotic exit and cytokinesis. The skp1-12 mutant allele also revealed a specific genetic interaction with Deltabfa1. Bfa1 interacted with Skp1 via its 184 C-terminal residues (Bfa1-D8) that are responsible for its function in mitotic exit. In addition, the interaction between Bfa1 and the Skp1-12 mutant protein was stronger than that of Bfa1 and the wild type Skp1. We suggest a novel function of Skp1 in mitotic exit and cytokinesis, independent of its function as a part of the SCF complex. The interaction of Skp1 and Bfa1 may contribute to the function of Skp1 in the mitotic exit.