Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.     

The Mad2 conformational dimer: structure and implications for the spindle assembly checkpoint

The 25 kDa Mad2 protein is a key player in the spindle assembly checkpoint, a safeguard against chromosome segregation errors in mitosis. Mad2 combines three unusual properties. First, Mad2 adopts two conformations with distinct topologies, open (O) and closed (C) Mad2. Second, C-Mad2 forms topological links with its two best-characterized protein ligands, Mad1 and Cdc20. Third, O-Mad2 and C-Mad2 engage in a "conformational" dimer that is essential for spindle checkpoint function in different organisms. The crystal structure of the O-Mad2-C-Mad2 conformational dimer, reported here, reveals an asymmetric interface that explains the selective dimerization of the O-Mad2 and C-Mad2 conformers. The structure also identifies several buried hydrophobic residues whose rearrangement correlates with the Mad2 topological change. The structure of the O-Mad2-C-Mad2 conformational dimer is consistent with a catalytic model in which a C-Mad2 template facilitates the binding of O-Mad2 to Cdc20, the target of Mad2 in the spindle checkpoint.     

Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2

Mad2 is an essential component of the spindle assembly checkpoint (SAC), a molecular device designed to coordinate anaphase onset with the completion of chromosome attachment to the spindle. Capture of chromosome by microtubules occur on protein scaffolds known as kinetochores. The SAC proteins are recruited to kinetochores in prometaphase where they generate a signal that halts anaphase until all sister chromatid pairs are bipolarly oriented. Mad2 is a subunit of the mitotic checkpoint complex, which is regarded as the effector of the spindle checkpoint. Its function is the sequestration of Cdc20, a protein required for progression into anaphase. The function of Mad2 in the checkpoint correlates with a dramatic conformational rearrangement of the Mad2 protein. Mad2 adopts a closed conformation (C-Mad2) when bound to Cdc20, and an open conformation (O-Mad2) when unbound to this ligand. Checkpoint activation promotes the conversion of O-Mad2 to Cdc20-bound C-Mad2. We show that this conversion requires a C-Mad2 template and we identify this in Mad1-bound Mad2. In our proposition, Mad1-bound C-Mad2 recruits O-Mad2 to kinetochores, stimulating Cdc20 capture, implying that O-Mad2 and C-Mad2 form dimers. We discuss Mad2 oligomerization and link our discoveries to previous observations related to Mad2 oligomerization.     

Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer

In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C^Cdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.     

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.     

多态性蛋白Mad2与其配体Cdc20121-138的相互作用研究

多态性蛋白Mad2是有丝分裂纺锤体检测点(SAC)的关键蛋白,也是多态性蛋白质家族中研究最广泛的成员之一.Mad2有两种不同的天然构象:O-Mad2和C-Mad2.Mad2构象间的转变及其与配体Cdc20间的相互作用对SAC发挥其生物学功能至关重要.本文利用荧光各向异性技术对O-Mad2和C-Mad2与配体TAMRA-Cdc20~(121-138)间相互作用的热力学及动力学过程进行了系统表征.结果表明:在无盐和低盐溶液(100 mmol/L NaCl)中,Mad2两种构象与Cdc20~(121-138)的平衡解离常数(K_D)均在10~(-6) mol/L,但C-Mad2与Cdc20~(121-138)结合的K_D值约为O-Mad2的1/5;在高盐(300 mmol/L NaCl)溶液中,Mad2两种构象与TAMRA-Cdc20~(121-138)结合的K_D值无明显差别.动力学实验结果显示,在同一种缓冲液中Mad2两种构象与Cdc20~(121-138)相互作用的解离速率常数k_d没有显著差别,而C-Mad2与Cdc20~(121-138)的结合速率常数k_a却比O-Mad2高一个数量级,这表明C-Mad2与Cdc20~(121-138)不仅结合力更强,且结合速率要快很多.Mad2与Cdc20~(121-138)突变体间的相互作用以及离子强度对二者相互作用的影响结果提示,Mad2和Cdc20间的相互作用不是通过静电相互作用,而可能是通过疏水相互作用来实现的.本研究为揭示多态性蛋白Mad2的构象转变机理及其在有丝分裂过程中的作用机制提供了重要的实验基础.     

Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.     

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.     

p31comet blocks Mad2 activation through structural mimicry

The status of spindle checkpoint signaling depends on the balance of two opposing dynamic processes that regulate the highly unusual two-state behavior of Mad2. In mitosis, a Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 dimerization and converts Mad2 to a conformer amenable to Cdc20 binding, thereby facilitating checkpoint activation. p31(comet) inactivates the checkpoint through binding to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting the dissociation of the Mad2-Cdc20 complex. Here, we report the crystal structure of the Mad2-p31(comet) complex. The C-terminal region of Mad2 that undergoes rearrangement in different Mad2 conformers is a major structural determinant for p31(comet) binding, explaining the specificity of p31(comet) toward Mad1- or Cdc20-bound Mad2. p31(comet) adopts a fold strikingly similar to that of Mad2 and binds at the dimerization interface of Mad2. Thus, p31(comet) exploits the two-state behavior of Mad2 to block its activation by acting as an "anti-Mad2."     

Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1

The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.     

Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation-specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.     

Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2-Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1-Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2-Cdc20 complex, and propose that the Mad1-Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1-Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1-Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1-Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2-ligand complexes.     

The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.

Mad2 participates in spindle checkpoint inhibition of APC(Cdc20). We show that RNAi-mediated suppression of Mad1 function in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Mad1 and Cdc20 contain Mad2 binding motifs that share a common consensus. We have identified a class of Mad2 binding peptides with a similar consensus. Binding of one of these ligands, MBP1, triggers an extensive rearrangement of the tertiary structure of Mad2. Mad2 also undergoes a similar striking structural change upon binding to a Mad1 or Cdc20 binding motif peptide. Our data suggest that, upon checkpoint activation, Mad1 recruits Mad2 to unattached kinetochores and may promote binding of Mad2 to Cdc20.     

Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.     

Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31(comet) (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31(comet) protein antagonizes the ability of Mad2 to inhibit APC/C(Cdc20) in vitro and in Xenopus egg extracts. Interestingly, p31(comet) binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31(comet) to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31(comet) forms a transient complex with APC/C(Cdc20)-bound Mad2. Purified p31(comet) enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31(comet) counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.     

Shugoshin is a Mad1/Cdc20-like interactor of Mad2

Mammalian centromeric cohesin is protected from phosphorylation-dependent displacement in mitotic prophase by shugoshin-1 (Sgo1), while shugoshin-2 (Sgo2) protects cohesin from separase-dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so-called template model, Mad1-Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20-like Mad2-interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine-tuning of Sgo2's sub-centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.     

Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae

Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.     

The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint

Background: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. Results: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. Conclusions: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.     

Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model

The inheritance of a normal assortment of chromosomes during each cell division relies on a cell-cycle surveillance system called the mitotic spindle checkpoint. The existence of sister chromatids that do not achieve proper bipolar attachment to the mitotic spindle in a cell activates this checkpoint, which inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) and delays the onset of anaphase. The mitotic arrest deficiency 2 (Mad2) spindle checkpoint protein inhibits APC/C through binding to its mitotic-specific activator, Cdc20. Binding of Mad2 to Cdc20 involves a large conformational change of Mad2 and requires the Mad1-Mad2 interaction in vivo. Two related but distinct models of Mad1-assisted activation of Mad2, the "two-state Mad2" and the "Mad2 template" models, have been proposed. I review the recent structural, biochemical, and cell biological data on Mad2, discuss the differences between the two models, and propose experiments that test their key principles.     

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.