Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co‐activator Cdc20 is responsible for targeting proteins for ubiquitin‐mediated degradation during mitosis. The activity of APC/C–Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C‐terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C–Cdc20 substrate and show that Kif18A degradation depends on a C‐terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo‐APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.     

Homeostatic control of mitotic arrest

The spindle assembly checkpoint (SAC) restricts mitotic exit to cells that have completed chromosome-microtubule attachment. Cdc20 is a bifunctional protein. In complex with SAC proteins Mad2, BubR1, and Bub3, Cdc20 forms the mitotic checkpoint complex (MCC), which binds the anaphase-promoting complex (APC/C) and inhibits its mitotic exit-promoting activity. When devoid of SAC proteins, Cdc20 serves as an APC/C coactivator and promotes mitotic exit. During mitotic arrest, Cdc20 is continuously degraded via ubiquitin-dependent proteolysis and resynthesized. It is believed that this cycle keeps the levels of Cdc20 below a threshold above which Cdc20 would promote mitotic exit. We report that p31(comet), a checkpoint antagonist, is necessary for mitotic destabilization of Cdc20. p31(comet) depletion stabilizes the MCC, super-inhibits the APC/C, and delays mitotic exit, indicating that Cdc20 proteolysis in prometaphase opposes the checkpoint. Our studies reveal a homeostatic network in which checkpoint-sustaining and -repressing forces oppose each other during mitotic arrest and suggest ways for enhancing the sensitivity of cancer cells to antitubulin chemotherapeutics.     

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.     

The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.     

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.     

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC also negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C^Cdc20 in response to the SAC.     

How APC/C-Cdc20 changes its substrate specificity in mitosis

Progress through mitosis requires that the right protein be degraded at the right time. One ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) targets most of the crucial mitotic regulators by changing its substrate specificity throughout mitosis. The spindle assembly checkpoint (SAC) acts on the APC/C co-activator, Cdc20 (cell division cycle 20), to block the degradation of metaphase substrates (for example, cyclin B1 and securin), but not others (for example, cyclin A). How this is achieved is unclear. Here we show that Cdc20 binds to different sites on the APC/C depending on the SAC. Cdc20 requires APC3 and APC8 to bind and activate the APC/C when the SAC is satisfied, but requires only APC8 to bind the APC/C when the SAC is active. Moreover, APC10 is crucial for the destruction of cyclin B1 and securin, but not cyclin A. We conclude that the SAC causes Cdc20 to bind to different sites on the APC/C and this alters APC/C substrate specificity.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

How cyclin A destruction escapes the spindle assembly checkpoint

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.     

Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G₂‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010. © 2010 Wiley‐Liss, Inc.     

In silico study of kinetochore control, amplification, and inhibition effects in MCC assembly

Eukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. In different organisms, a mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1/Mad3, and Cdc20 inhibits the anaphase promoting complex (APC/C) to initiate promotion into anaphase. The mechanism of MCC formation and its regulation by the kinetochore are unclear. Here, we constructed dynamical models of MCC formation involving different kinetochore control mechanisms including amplification as well as inhibition effects, and analysed their quantitative properties. In particular, in this system, fast and stable metaphase to anaphase transition can only be triggered when the kinetochore controls the Bub3:BubR1-related reactions; signal amplification and inhibition play a subordinate role. Furthermore, when introducing experimentally determined parameter values into the models analysed here, we found that effective MCC formation is not combined with complete Cdc20 sequestering. Instead, the MCC might bind and completely block the APC/C. The SACM might function by an MCC:APC/C complex rearrangement.     

Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl‐1 during an extended mitosis requires the anaphase‐promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCF^Fbw7. Using live‐cell imaging, we show that the loss of Mcl‐1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine‐arginine (IR) C‐terminal tail regulates the manner in which Mcl‐1 engages with the APC/C, converting Mcl‐1 from a Cdc20‐dependent and checkpoint‐controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl‐1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl‐1 degradation, providing an improved strategy to kill cancer cells.     

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.     

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11⁺ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that Cdc20 and C-Mad2 levels correlate at kinetochores and that depletion of Cdc20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type Cdc20 but not Cdc20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that Cdc20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-Cdc20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.     

APC/C-Mediated Degradation in Early Mitosis: How to Avoid Spindle Assembly Checkpoint Inhibition

The APC/C is an E3 ubiquitin ligase that, by targeting substrates for proteasomal degradation, plays a major role in cell cycle control. In complex with one of two WD40 activator proteins, Cdc20 or Cdh1, the APC/C is active from early mitosis through to late G1 and during this time targets many critical regulators of the cell cycle for degradation. However, this destruction is carefully ordered to ensure that cell cycle events are executed in a timely fashion. Recent studies have begun to shed light on how the APC/C selects different substrates at different times in the cell cycle. One particular problem is how the APC/C recognizes its first set of substrates, Nek2A and cyclin A, in early mitosis when, at this time, the spindle assembly checkpoint (SAC) inhibits most APC/C-dependent degradation. The answer may lie in how substrates are recruited to the APC/C. While checkpoint-dependent substrates appear to require Cdc20 for recruitment, experiments on the early mitotic substrate Nek2A demonstrate that it can bind the APC/C in the absence of Cdc20. The direct interaction of substrates with core subunits of the APC/C could allow their degradation to proceed unhindered even when the SAC is active.     

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet)

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase-anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.     

APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.