The regulation of Cdc20, like that of most key regulatory proteins, depends on its short lifetime in the cell. This instability arises primarily from self-inhibition by autoubiquitination, which targets the protein for destruction in the proteasome. In a normal cell cycle, autoubiquitination is the primary mechanism underlying the drop in Cdc20 levels that occurs in late mitosis (Foe et al., 2011
). Surprisingly, however, we found that cells expressing the poorly ubiquitinated Cdc20-5K mutant are viable and display no significant cell cycle defects, despite the presence in these cells of high Cdc20 levels that decline only slightly outside of mitosis. Thus, rapid Cdc20 turnover is not essential in a normal cell cycle. The same is not true in a spindle checkpoint, however. The CDC20-5K
strain failed to establish a spindle checkpoint arrest, to a similar extent as mutants lacking the checkpoint genes MAD2
A lysine-free form of human Cdc20 also bypasses the SAC (Nilsson et al., 2008
), but it has been suggested that this bypass might be due to a slight decrease in Mad2 affinity caused by mutation of two lysines in the Mad2-binding region (Varetti et al., 2011
). However, budding yeast Cdc20 does not contain these lysines and instead contains one lysine N-terminal of the conserved interaction motif (Luo et al., 2002
). Furthermore, we found that wild-type Cdc20 and Cdc20-5K interact equally well with checkpoint proteins in vitro (). We therefore conclude that the yeast Cdc20-5K mutant is not defective in its interaction with checkpoint proteins.
How, then, does the Cdc20-5K mutant allow progression through mitosis despite activation of the checkpoint? The most likely possibility, which is consistent with previous evidence that moderate CDC20
overexpression drives yeast cells through a checkpoint arrest (Pan and Chen, 2004
), is that the higher levels of Cdc20-5K protein somehow provide resistance to activated checkpoint proteins. One simple possibility is that checkpoint proteins are limiting, allowing high levels of Cdc20 to outnumber Mad2 to allow formation of active APC/C-Cdc20 complexes despite the checkpoint activation. This seems unlikely, however, given the ability of mnd2
Δ cells to arrest normally despite accumulating higher Cdc20 levels within the arrest (). A more appealing possibility stems from our observation that Mad2 does not inhibit the activity of preformed APC/C-Cdc20 complexes in vitro (data not shown), presumably because the binding affinity of Cdc20 is so high that Mad2 cannot gain access to its binding site on Cdc20. Thus, the downregulation of Cdc20 from late mitosis through S phase may be required to keep Cdc20 levels at a low level that prevents the premature formation of checkpoint-resistant APC/C-Cdc20 complexes. Thus, our results argue that the degradation of Cdc20 outside mitosis is required for efficient establishment of a checkpoint arrest.
Checkpoint proteins are required for rapid Cdc20 turnover during a checkpoint arrest (King et al., 2007
; Pan and Chen, 2004
) (Figure S2C
). To explore the underlying mechanism, we reconstituted the effects of Mad2 and the Mad3-Bub3 complex with purified components. We found that Mad2 alone inhibited Cdc20 binding and autoubiquitination. The incomplete effect of Mad2, even at apparently saturating concentrations, suggests that the target of Mad2 may be just one of the multiple contact points that mediate Cdc20 binding to the APC/C. Given that the Mad2 binding motif in Cdc20 lies in the amino-terminal region near the C-box, a likely possibility is that Mad2 somehow interferes with C-box function.
Checkpoint proteins clearly synergize in forming the Cdc20-Mad2-Mad3-Bub3 complex (MCC). Although Mad2 inhibits Cdc20 binding to the APC/C, the Mad3-Bub3 complex reverses this effect and stimulates Cdc20 binding and autoubiquitination. We find that the three budding yeast checkpoint proteins are sufficient to promote robust Cdc20 autoubiquitination in the absence of additional components, in contrast to the dependence on p31comet
in human cells and lysates (Reddy et al., 2007
; Varetti et al., 2011
). Yeast do not contain a clear homolog of p31comet
, and so vertebrate cells may have evolved additional control mechanisms. The synergistic actions of checkpoint proteins are likely to depend on interactions between Mad2 and Mad3, as suggested by recent studies supporting a direct interaction between purified human Mad2 and BubR1 (Tipton et al., 2011
). Recent structural analysis of fission yeast MCC components (Mad3, Mad2, and Cdc20) also revealed Mad2-Mad3 interactions that would explain the synergistic effect (Chao et al., 2012
Interestingly, the full set of checkpoint proteins has opposite effects on two APC/C-dependent activities: inhibition of securin ubiquitination and stimulation of Cdc20 autoubiquitination. Thus, the MCC is not a global inhibitor of the APC/C, but inhibits only its substrate-targeting function. How is this possible? KEN boxes in Mad3 were proposed to function as pseudosubstrate inhibitor motifs that interfere with substrate binding to Cdc20 (Burton and Solomon, 2007
), and recent structural data provides evidence for engagement of a Mad3 KEN box by the WD40 domain of Cdc20 (Chao et al., 2012
). Interestingly, other studies suggest that the MCC causes a shift in the position of Cdc20 on the APC/C, away from the Doc1/Apc10 subunit that contributes to substrate binding (Herzog et al., 2009
; Izawa and Pines, 2011
); thus, the MCC might reduce substrate binding in part by separating Cdc20 from Doc1. We found that the majority of autoubiquitination sites lie within the Cdc20 N-terminal region, which is predicted to be unstructured and also contains the C-box motif, which recent studies suggest could interact with the Apc2 subunit at a location that is close to the site of E2 binding – and thus in a good position to attack the E2-ubiquitin conjugate (da Fonseca et al., 2011
), as also suggested recently for human Cdc20 (Zeng and King, 2012
). Perhaps MCC binding shifts Cdc20 to a position that reduces substrate interactions while favoring autoubiquitination.
The stimulation of Cdc20-APC/C binding by Mad3-Bub3 suggests that Mad3 (and/or Bub3) interacts with the APC/C core. Structural analysis of the APC/C-MCC complex suggests that the MCC could contact multiple subunits (Herzog et al., 2009
). We found that Doc1/Apc10 is not required for the stimulation of Cdc20 binding by Mad3, suggesting that this subunit does not contribute to MCC binding – and consistent with the notion, mentioned above, that the MCC shifts Cdc20 away from Doc1. We also tested another nonessential subunit, Mnd2, based on recent evidence that a related human subunit, Apc15, is required for Cdc20-MCC turnover in the checkpoint (Mansfeld et al., 2011
). Mnd2 has been suggested to interact with Apc1, Apc5, and Cdc23 (Hall et al., 2003
), three subunits in the APC/C region where the MCC appears to bind (Chao et al., 2012
; Herzog et al., 2009
; Schreiber et al., 2011
). Furthermore, Cdc23/Apc8 is particularly important in Cdc20 binding in the checkpoint (Izawa and Pines, 2011
). Deletion of Mnd2 had a striking and specific effect: autoubiquitination and activity toward securin were largely unaffected in the absence of checkpoint proteins, but the loss of Mnd2 blocked the ability of Mad3-Bub3 to stimulate autoubiquitination in the presence of Mad2. Interestingly, we found that the loss of Mnd2 did not prevent the stimulation of Cdc20 binding to the APC by Mad3-Bub3. These results argue strongly that Mnd2 (and perhaps Apc15 in the human APC/C) is required for the MCC to shift the position of Cdc20 for checkpoint-induced autoubiquitination.
Surprisingly, despite the rapid turnover of Cdc20 that occurs in checkpoint-arrested cells, steady-state levels of Cdc20 appear constant (). Thus, a high rate of mitotic Cdc20 synthesis balances increased destruction. As recently proposed (Varetti et al., 2011
), this constant flux of Cdc20 is likely to be important for reversing the effects of the checkpoint when all sister-chromatid pairs achieve correct spindle attachment. Cells lacking MND2
provided us with an effective approach to explore this possibility. In these cells, the lack of Mnd2 did not greatly affect Cdc20 oscillations in a normal cell cycle, and thus these cells do not have the general increase in Cdc20 levels that we observed in the CDC20-5K
cells. Instead, mnd2
Δ cells displayed a more specific defect in autoubiquitination and Cdc20 turnover in the presence of checkpoint proteins. These cells establish and maintain a checkpoint arrest, indicating that MCC-dependent autoubiquitination and rapid Cdc20 degradation are not required for the arrest. These results also support the notion, discussed above, that the Cdc20-5K mutant bypasses the arrest because of its high levels throughout the cell cycle.
Δ cells arrest in the checkpoint, they are less efficient than wild-type cells in inactivation of the checkpoint when spindle poisons are removed, as observed in human cells depleted of Apc15 (Mansfeld et al., 2011
). Thus, inactivation of the checkpoint might depend, at least in part, on MCC-dependent Cdc20 autoubiquitination. How does ubiquitination promote checkpoint inactivation? In purified reactions, we have not seen evidence that polyubiquitination causes dissociation of Cdc20 from the checkpoint complex and APC/C (Figure S6
). Instead, we suspect that MCC removal is an active process mediated in part by the proteasome and other factors, as suggested by recent evidence for the involvement of ATP hydrolysis and proteolysis (Ma and Poon, 2011
; Miniowitz-Shemtov et al., 2010
; Teichner et al., 2011
; Visconti et al., 2010
; Zeng et al., 2010
). In vertebrates, checkpoint inactivation also depends on the protein p31comet
, which has been proposed to provide a functionally redundant mechanism for driving MCC disassembly (Jia et al., 2011
). We speculate that when the SAC signal is extinguished, the ATP-dependent removal of polyubiquitinated Cdc20, together with its checkpoint partners, helps allow newly synthesized Cdc20 to reactivate the APC/C. In this way, the dynamic features of the checkpoint – balanced high rates of Cdc20 synthesis and destruction – allow cells to rapidly initiate anaphase upon checkpoint inactivation.