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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Curr Biol. Author manuscript; available in PMC Aug 29, 2012.
Published in final edited form as:
PMCID: PMC3430386
NIHMSID: NIHMS400249
Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism
Ian T. Foe,1,2 Scott A. Foster,1,2,3 Stephanie K. Cheung,2 Steven Z. DeLuca,2 David O. Morgan,2,3 and David P. Toczyski2,4
2Dept. of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, 94158, USA.
3Dept of Physiology, University of California at San Francisco, San Francisco, 94158, USA.
4Corresponding author, david.toczyski/at/ucsf.edu (415) 502-1301 / (415) 502-3179
1These authors contributed equally to this work
Background
Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood.
Results
Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination.
Conclusion
We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle.
Chromosome segregation is one of the most tightly regulated events in the dividing cell. Incorrect entry into anaphase can have catastrophic cellular consequences ranging from genomic instability to cell death. Anaphase is initiated by the Anaphase-Promoting Complex/Cyclosome (APC) [1, 2], an E3 ubiquitin ligase composed of at least 13 core subunits [3, 4]. APC function is regulated by association with one of two activator subunits, Cdc20 or Cdh1 (also known as Hct1) [57]. These proteins are thought to function both in the binding of substrates to the APC [8] and APC activation [9]. Cdc20 associates with the APC in early mitosis, and triggers anaphase onset by promoting the destruction of a subset of mitotic cyclins and Securin (also known as Pds1) [10, 11], resulting in the activation of Esp1, and the separation of sister chromatids through cleavage of cohesion [12]. In late mitosis and G1, Cdh1 associates with the APC, promoting mitotic exit and maintaining low Cdk activity.
Both activators contain well-conserved motifs involved in APC and substrate binding (Figure 1A). APC binding is mediated by both a C-box motif within the activator’s N-terminus [8] and a C-terminal Isoleucine-Arginine (IR) motif [13, 14] (Figure 1A). Substrate binding is mediated by a WD40 domain that is likely to interact directly with degradation signals found within substrates [15], the most common being the Destruction box (D-box) [16] and KEN-box [17]. Processive substrate ubiquitination has also been shown to require the core APC subunit Doc1 [14, 18], which is thought to function as a co-receptor for the D-box in conjunction with the WD40 of Cdc20/Cdh1 [19, 20].
Figure 1
Figure 1
Cdc20 is turned over by the APC by Cdh1-dependent and Cdh1-independent mechanisms. (A) Diagrams of Cdc20 and Cdh1. Red, purple, blue and green boxes represent the D-boxes, the C-box, the WD40, and the C-terminal IR, respectively. (B) Three possible mechanisms (more ...)
The two mitotic APC activators are thought to function analogously, but they are regulated in distinct ways. While Cdh1 protein and transcript levels are constitutive, Cdc20 transcription and protein levels both oscillate throughout the cell cycle [21]. Cdc20 is absent in G1, but begins to accumulate in late S phase, its peak coinciding with the initiation of anaphase. Cdh1 is thought to bind an N-terminal D-box within Cdc20, leading to the destruction of Cdc20 in late mitosis and G1 [2224]. However, while Cdh1-mediated turnover of Cdc20 is likely important, several studies have suggested that Cdc20 is also turned over by Cdh1-independent mechanisms [21, 25, 26]. Regulation of Cdc20 levels is very important, as high-level over-expression of Cdc20 is lethal [27] and as little as three-fold over-expression of Cdc20 is sufficient to override the spindle assembly checkpoint [28].
Previously, we found that deletion of Cdc20’s IR domain caused a strong accumulation of Cdc20 in vivo [25], which is inconsistent with Cdc20 simply being a passive Cdh1 substrate. Here, we show that Cdc20 turnover is fully APC-dependent, but does not depend on a second activator molecule. While Cdc20 can be targeted by the APC associated with either Cdh1 or, more poorly, by a second Cdc20 molecule (i.e. in trans turnover), we find that most turnover in vivo, and ubiquitination in vitro, is promoted by direct association with the APC (in cis turnover) (Figure 1B). Consistent with this model, we show that processive ubiquitination of Cdc20 does not require Doc1. Importantly, we find that Cdc20 levels oscillate independently of CDC20 transcription and Cdh1 activity, implying that the in cis autoregulation of Cdc20 turnover changes during the cell cycle. Additionally this regulation can be influenced by the presence of APCCdc20 substrates. These findings uncover another mechanism by which the activity of the APC is tightly controlled during the cell cycle.
Cdc20 turnover depends on the APC
Cdc20 is thought to be destroyed by both APC-dependent mechanisms [21, 29, 30] and APC-independent mechanisms [30]. However, previous experiments suggesting APC-independent Cdc20 turnover were performed with temperature-sensitive APC mutants, which do not necessarily eliminate all APC function. While the APC is normally essential, we have previously shown that deletion of genes encoding two Cdc20 substrates, Pds1 and Clb5, combined with 10-fold over-expression of the Cdk inhibitor Sic1 (SIC110x), allows cells to survive in the absence of the APC [31]. To determine whether Cdc20 turnover is dependent upon a functional APC, we examined Cdc20 turnover in an apc11Δ pds1Δ clb5Δ SIC110x strain. Deletion of APC11, which encodes the essential RING finger subunit of the APC [32], abolishes APC activity in the cell. We found that, as with the known APC substrate Clb2 [33], turnover of Cdc20 was eliminated in the apc11Δ strain (Figure 1C). This strongly suggests that, under normal conditions, the majority of Cdc20 turnover depends on APC activity.
We postulated that there could be three modes of APC-dependent Cdc20 turnover (Figure 1B). First, as previously suggested, Cdh1 bound to the APC as an activator could recognize Cdc20 as a substrate through Cdc20’s D-box (Cdh1trans)[2224]. However, we found previously that while Cdc20 levels were slightly increased in cdh1Δ cells, they were more dramatically increased in Apc cells [25], suggesting that the APC targets Cdc20 by Cdh1-independent mechanisms as well. Consistent with this, we observed APC-dependent ubiquitination of Cdc20 both in the presence and absence of Cdh1 in vitro (Figure 1D).
There are two distinct mechanisms by which Cdh1-independent ubiquitination could occur. The first is similar to the Cdh1trans mechanism. Here, one molecule of Cdc20 associates with the APC as an activator and this APC-Cdc20 complex binds a second Cdc20 molecule as a substrate through a WD40/D-box interaction (Cdc20trans, Figure 1B). Alternatively, a single Cdc20 molecule bound to the APC as an activator could be ubiquitinated directly by the APC (Cdc20cis, Figure 1B).
Contribution of the Cdh1-dependent and independent mechanisms to Cdc20 turnover
We found previously that mutation of Cdc20’s IR motif increased steady-state Cdc20 levels [25], consistent with a Cdh1-independent mechanism for Cdc20 turnover. This increase in steady-state level is higher than that observed for wildtype Cdc20 in a cdh1Δ strain, suggesting that the Cdh1-independent mechanism is responsible for the majority of Cdc20 turnover (Figure 2A, lanes 5&9) [25]. The IRΔ and cdh1Δ double mutant was more stable than either single mutant, consistent with multiple mechanisms controlling Cdc20 stability (Figure 2A lanes 5–16). Since mutation of the IR decreases Cdc20 binding to the APC (data not shown), both Cdc20trans and Cdc20cis could, in principle, be affected. Consistent with this idea, we found that mutation of the IR had no effect on Cdh1-dependent ubiquitination in vitro (Supplementary Fig. 1A) but greatly inhibited autoubiquitination (Figure 2B, lanes 9–12).
Figure 2
Figure 2
Cdc20 ubiquitination and turnover in CDC20, CDH1 mutants. (A) Asynchronous PDS1 CLB5 SIC1 cells were treated with cycloheximide and samples were analyzed as in Figure 1C. Cdc20-IR denotes the Cdc20-ΔIR allele. (B) ZZ-tagged 35S-Cdc20 wildtype, (more ...)
To further assess the contribution of the Cdh1trans mechanism in isolation, we sought to create a Cdc20 mutant that was defective in binding to the APC as an activator, but could be bound as a substrate through its D-boxes. The observation that mutation of Cdc20’s IR motif has no obvious growth phenotypes is consistent with it only having a partial effect on Cdc20 binding to the APC. Mutation of C-box, however, is lethal and decreases Cdc20 binding to the APC [8, 25], suggesting that C-box mutations greatly reduce interaction with the APC. Therefore, we expected a C-box mutation to eliminate Cdc20cis and Cdc20trans mediated turnover. The minimal conserved sequence of the C-box in both Cdc20 and Cdh1 is DRYIP [8]. Previously, we characterized two C-box mutants, a weaker cdc20-I147A,P148A allele and a stronger cdc20-R145D allele (which did not translate well in vitro) [25]. We examined the turnover of Cdc20-R145D in a cdh1Δ strain. Surprisingly, while the known Cdc20 substrate Dbf4 was stabilized, the Cdc20-R145D protein was still turned over rapidly, although there was an increase in steady-state levels (Figure 2C lanes 4–9 and Supplementary Fig. 1B). It was possible that this mutation did not entirely eliminate C-box function, so we also analyzed a cdc20-D144R, R145D double mutant. This mutant turned over with similar kinetics to the cdc20-R145D allele (Supplementary Fig. 1C).
We also observed that the I147A, P148A C-Box mutant had a larger effect than the IR mutation on securin ubiquitination in vitro (Figure 2D, lanes 7–12). Yet the defect observed with the same C-box mutation is less severe than that observed with the IR mutant in autoubiquitination activity (Figure 2B, lanes 5–12). Thus, while the C-Box is essential for APC function in vivo, considerable Cdc20 turnover occurs when the C-box is mutated. Our results, together with previous evidence that the C-box, but not the IR, is essential for viability, indicates that the C-box is more important than the IR motif for substrate turnover and less critical for Cdc20 autoubiquitination.
Since neither the IR nor C-box mutation alone eliminated Cdh1-independent turnover, we generated a C-box, IR double mutant. Cdc20-IR, R145D should not be able to interact with the APC as an activator and therefore should eliminate both the Cdc20trans and Cdc20cis mechanisms of turnover. Consistent with this, the Cdc20-IR, R145D mutant was strongly stabilized in a cdh1Δ strain, but could be turned over in a CDH1 strain (Figure 3A, lanes 7–9 and 13–15). Similarly, we detected ubiquitination of a Cdc20-C-box-IR mutant in the presence of Cdh1 in vitro and this activity was entirely D-box-dependent (Figure 3B, lanes 1–6). These results are consistent with previously suggested model that Cdh1 can target Cdc20 [2224]. However, the dramatic increase in steady-state levels and the relatively slow rate of turnover in the Cdh1trans-only strain suggests that Cdh1-dependent turnover likely contributes to a small portion of normal Cdc20 turnover (Figure 3A, lanes 7–9).
Figure 3
Figure 3
The majority of Cdc20 turnover occurs by the Cdc20cis mechanism. (A) Asynchronous pds1Δ clb5Δ SIC110x cells were analyzed as in Figure 2C. Cdc20-CB denotes the Cdc20-R145D, IRΔ allele. Bands represented by Cdc20 and Cdc20-CB are (more ...)
We next sought to investigate if the Cdc20trans mechanism makes any contribution to Cdh1-independent turnover. We generated a cdh1Δ strain containing a wildtype copy of CDC20 and the cdc20-IR, R145D allele at a second locus. Turnover of Cdc20-IR, R145D should be defective in both the Cdh1trans and Cdc20cis mechanisms in this strain and should therefore be turned over exclusively by Cdc20trans. This Cdc20-IR, R145D mutant was slightly more stable than that observed in the Cdh1trans-only strain, suggesting that the Cdc20trans mechanism does occur, but likely contributes very little to Cdh1-independent turnover (Figure 3A, lanes 10–12). To further characterize the Cdc20trans mechanism, we tested whether a wildtype copy of Cdc20 can ubiquitinate this double mutant in vitro. We detected very little ubiquitination of this mutant in the presence of a wildtype copy of Cdc20 and the little stimulation seen over background was D-box-dependent (Figure 3C, lanes 4–6 & 10–12). Interestingly, while this D-box appears Cdh1-specific in terms of targeting Cdc20 as a substrate in vitro, we did see a slight defect with this mutant both in direct binding to the APC and in targeting Securin for ubiquitination in vitro, suggesting that Cdc20’s D-box may have an additional function (data not shown).
Given that total Cdc20 turnover appeared significantly faster than turnover via either Cdc20trans or Cdh1trans, we examined the contribution of the Cdc20cis mechanism using an allele of Cdc20 that could only be bound to the APC as an activator and not as a substrate. We generated a cdh1Δ strain in which the only copy of Cdc20 is mutated at its first D-box (cdc20-DB), and thus cannot function as a substrate in a Cdc20trans reaction. In this strain, where only Cdc20cis turnover occurs, Cdc20 turnover is quite fast, and steady-state Cdc20 levels are low, similar to those in a cdh1Δ strain where both Cdh1-independent mechanisms can occur (Fig. 3D, lanes 1–8). These data suggest that Cdc20cis is the dominant form of Cdc20 turnover, with the contribution of Cdc20trans being very small (Figure 3D, lanes 9–12).
To determine the extent to which the first D-box mutation eliminates Cdc20trans turnover in vivo, we examined its effect in our strain that uses Cdc20trans exclusively (see Figure 3A, lanes 10–12). We found that Cdc20-IR, R145D, DB was extremely stable in a CDC20 cdh1Δ strain, although a very low level of turnover did occur (Figure 3D, lanes 13–16). Mutation of a second N-terminal D-box had no additional effect (data not shown). Thus, the D-box mutation eliminated in trans turnover, consistent with previous reports [21, 29]. These data suggest that Cdc20cis is the dominant form of Cdc20 turnover, with the contribution of Cdc20trans being very small (Figure 3D, lanes 5–8 & 9–12).
The nonessential APC subunit Doc1 (APC10) is thought to interact directly with the D-box of substrates and enhance processivity by limiting the dissociation rate of the substrate [14, 18, 19]. Deletion of this subunit or mutation of 4 residues (Doc1–4A) within its putative substrate binding site leads to a decrease in the number of ubiquitins conjugated to the substrate, as visualized by a significant decrease in higher molecular weight substrate-ubiquitin bands and accumulation of mono-ubiquitinated substrate (Figure 3E, lanes 1–8)[19]. Cdc20 contains a D-box that has been shown to be important in Cdh1-dependent ubiquitination [21]. We tested whether a Doc1/D-box interaction was required for processive ubiquitination of Cdc20 in vitro in the absence of Cdh1. Unlike our results with all other substrates tested, mutation of Doc1 had no effect on the processivity of this reaction. Doc1 and Doc1-4A had nearly identical activity towards Cdc20 (Figure 3E, lanes 9–16), implying that Doc1 is not required for Cdh1-independent ubiquitination of Cdc20. These data strongly suggest that Cdc20 is not ubiquitinated by the APC as a canonical substrate, and can best be explained by the Cdc20cis mechanism of autoubiquitination.
Cdc20 levels oscillate independently of Cdh1 and Cdc20 transcriptional oscillation
Cdh1 activity is cell-cycle regulated, which contributes to Cdc20 periodicity. We sought to determine if Cdh1-independent mechanisms are also important for oscillations in Cdc20 levels. Since cdh1Δ cells do not arrest well in alpha factor, we examined Cdc20 levels through the cell cycle using cdh1Δ cdc15-2 cells. Cells were arrested at the non-permissive temperature in anaphase and released into the permissive temperature. Consistent with a recent report, we found that Cdh1 is not necessary for Cdc20 levels to fluctuate with the cell cycle [26] (Figure 4A, Supplementary Fig. 3A).
Figure 4
Figure 4
Cdc20 levels oscillate with the cell cycle in a Cdh1- and transcription-independent manner. (A) Asynchronous cdc15-2 or cdc15-2 cdh1Δ cells were arrested at 37°C and released into 23°C media. Time points were taken every 20 min. (more ...)
To examine the extent to which oscillations in CDC20 transcription contribute to the fluctuation of Cdc20 levels, we generated a strain with CDC20 under the control of a constitutive promoter (TEF1p). Cdc20 levels were still periodic in this strain. Moreover, Cdh1 was not required for this periodicity (Figure 4B, Supplementary Fig. 3B). While Cdh1-dependent turnover of Cdc20 and cell cycle-regulated transcription both contribute to Cdc20 cycling, Cdh1-independent turnover mechanisms appear to add significantly to Cdc20 oscillation.
Substrates inhibit Autoubiquitination
If Cdc20 targets itself while bound to the APC as an activator, then how does the cell maintain Cdc20 levels sufficient to trigger anaphase? We tested the possibility that the binding of substrates to Cdc20 might inhibit autoubiquitination, maintaining Cdc20 stability until its targets are depleted in anaphase. We generated an N-terminal fragment (aa 1–110) of budding yeast securin, containing the characterized destruction motif [11]. As expected for a competitive inhibitor, this fragment potently inhibited securin ubiquitination (IC50 ~ 200 nM) (Figure 5A). A 10 µM concentration of the securin fragment completely inhibited ubiquitination of securin (Figure 5A). This concentration of the fragment also inhibited the total activity and processivity of Cdc20 autoubiquitination (Figure 5B). These results support the notion that substrate blocks autoubiquitination, prolonging Cdc20 levels in the cell until substrates are depleted (Figure 6).
Figure 5
Figure 5
Cdc20cis mechanism is inhibited by high substrate concentrations (A) ZZ-tagged unlabeled Cdc20 generated by IVT was pre-incubated with APC (5 nM) and the specified concentration of the Securin/Pds1 fragment (referred to as Securin 1–110; values (more ...)
Figure 6
Figure 6
Model demonstrating how Cdc20cis may be regulated by substrate. Red, purple, blue and green boxes represent the D-boxes, the C-box, the WD40, and the C-terminal IR of Cdc20, respectively (see Figure 1A).
One of the first APC substrates to be identified was its own activator, Cdc20, hinting at the existence of autoregulation [21, 29]. Initial reports suggested that Cdc20 behaved similarly to other APC substrates, being targeted in part via a different activator (Cdh1) through Cdc20’s D-box [2224]. Interestingly, we show here that, unlike other APC substrates, Cdc20 is largely targeted for destruction by the APC through an autoubiquitination mechanism that occurs when Cdc20 is bound to the APC as an activator. Importantly, this mechanism appears to be regulated throughout the cell cycle, and may be influenced by the presence or absence of substrates.
The observation that Cdc20 turnover was only partially reduced in conditional APC mutants led some authors to speculate that the residual turnover observed might be mediated by an APC-independent pathway. Our work in a strain that permits the deletion of the APC11 gene shows that in unperturbed cells, Cdc20 is turned over solely by the APC. This discrepancy is likely due to the fact that conditional alleles may not be completely null for APC activity, whereas deletion of the gene encoding the catalytic subunit (APC11) eliminates activity completely.
APCCdh1 has long been assumed to be the APC complex that targets Cdc20 for destruction (Cdh1trans, Figure 1B) [17, 2224]. However, deletion of APC11 leads to much greater steady-state levels of Cdc20 than deletion of CDH1, suggesting the existence of other APC-mediated mechanisms [25]. This suggests two obvious models for turnover. First, Cdc20 bound to the APC as an activator could recognize another molecule of Cdc20 leading to ubiquitination of the substrate Cdc20 (Cdc20trans, Figure 1B). In this case, the substrate Cdc20 should behave similarly to other Cdc20 substrates. Alternatively, Cdc20 may bind to the APC as an activator and this binding alone may be sufficient for autoubiquitination (Cdc20cis, Figure 1B). To evaluate the relative contributions of the three possible modes of Cdc20 turnover, we generated strains in which only one mechanism of turnover was possible and performed in vitro experiments with similar perturbations. These experiments strongly suggested that Cdc20cis is the predominant form of Cdc20 turnover.
Previous work showed that Cdc20 not only recruits substrates to the APC, but also serves to activate the APC, since its presence was also required for the ubiquitination of the APC substrate Nek2A, which can bind the APC independently of an activator [9, 34]. Importantly, these results suggested that an N-terminal fragment of Cdc20 containing the C-box was sufficient to activate the APC toward Nek2A, and that the C-box was required for this activation [9]. Interestingly, we find that a Cdc20 C-box mutant, which does not support viability and is unable to drive Dbf4 turnover in vivo [25](Fig. 2C), is still targeted for turnover by the APC, although its turnover is compromised. This result suggests that the C-box is not absolutely required for APC activity, but is specifically required for stimulating APC activity towards other APC substrates, potentially by properly orientating either the substrate and or the catalytic arm of the APC so substrate ubiquitination can occur. Interestingly, deletion of the C-terminal IR domain, which does not result in a growth defect, has a significant effect on Cdc20 turnover, slightly greater than the defect seen for the lethal C-box mutant. The IR domain has been shown to interact with Cdc27, the terminal subunit of the TPR arm [13, 25, 35]. The non-essential nature of the IR-Cdc27 interaction could suggest that it is an intermediate in the reaction mechanism when Cdc20 is particularly susceptible to autoubiquitination. Consistent with this observation, this interaction is not required for the processive ubiquitination of other APC substrates [35]. However, the lack of affinity provided by the Cdc27-IR interaction is compensated by an interaction between the activator, substrate, and Doc1 on the APC core. However, autoubiquitination does not use Doc1, possibly, making the affinity provided by the Cdc27-IR interaction more important.
The discovery that Cdc20 is targeted for turnover by Cdh1, which is itself cell-cycle regulated, suggested a mechanism by which Cdc20’s cyclical expression could be achieved. Work from the Cross lab [26] and from experiments presented here suggests that Cdh1 may contribute to but is not necessary for Cdc20’s cell cycle oscillation. However, previous work [21] suggested that oscillation in Cdc20 levels is also achieved by transcriptional regulation. CDC20 is a member of the CLB2 cluster of genes [36], whose transcription is under the control of Fkh2/Ndd1 [37, 38]. The observation that Cdc20 levels still oscillate in cells that express CDC20 under a constitutive promoter (TEF1p) in the absence of Cdh1 implies an additional cell cycle regulated mechanism. This is strong evidence that regulation of the Cdc20cis mechanism we observe is sufficient to drive the oscillatory behavior of Cdc20 through out the cell cycle.
Previous work has shown that phosphorylation of the TPR subunits (Cdc27, Cdc16, and Cdc23) by the Cyclin-Dependent Kinase (CDK) increases the affinity of Cdc20 for the APC [39]. It is possible that these phosphorylations are regulating the Cdc20cis mechanism. However, these phosphorylations promote Cdc20 binding to the APC, and occur when CDK activity is high. If these phosphorylations are promoting the Cdc20cis turnover during the cell cycle, we would expect to see the lowest Cdc20 levels when CDK activity is highest. However, we observe that the lowest Cdc20 levels occur during the G1 phase of the cell cycle, when CDK activity is lowest. Alternatively, phosphorylation of the TPR proteins may cause Cdc20 to bind in a slightly different position on the APC, which may inhibit the Cdc20cis mechanism.
These data suggest the following model. The APC is hyperphosphorylated in early mitosis, which increases its affinity for Cdc20. As APCCdc20 runs out of substrates, Cdc20 begins to autoubiquitinate, constituting the majority of the late mitotic turnover. This model for the regulation of Cdc20 stability by the presence of substrates (Figure 6) is similar to that put forth for the ubiquitin conjugase Ube2C [40]. As cells exit mitosis, APC becomes dephosphorylated and Cdh1 becomes active, thus removing residual Cdc20. Additionally, our model for substrate inhibition of Cdc20 turnover may explain why it is advantageous for the cell to have Cdc20 binding to the APC strongly enhanced by the presence of substrates [35, 41]. In this way, Cdc20 would be unlikely to be prematurely degraded when substrates are present.
Interestingly, Cdc20 turnover has been shown to increase in the presence of spindle poisons. This turnover is dependent on an intact Spindle Assembly Checkpoint (SAC) [28]. The exact mechanism for this turnover is unknown, but it will be interesting to determine the mechanism for Cdc20 turnover during SAC activation.
Yeast Methods
Yeast were grown in Ym-1 media [42] and 2% dextrose. All cells were grown at 23°C unless otherwise noted. Cdc20 integrating plasmid was created by cloning Cdc20 and its promoter into pRS306 using standard techniques. Mutations to pRS306-Cdc20 were accomplished using quick change mutagenesis. Cdc20 plasmids were integrated at the URA3 locus into derivations of 3 strains: pds1Δ clb5Δ SIC110x cdc20Δ cdh1Δ, pds1Δ clb5Δ SIC110x cdc20Δ CDH1, or pds1Δ clb5Δ SIC110x CDC20 cdh1Δ. All strains created in this manner were checked for single integration by southern blot. Replacement of the CDC20 promoter with TEF1p was accomplished using standard PCR-based techniques, as was deletion of CDH1 and mutation of Cdc20’s IR motif in Figure 2A.
Half-life assays
Cells were grown to saturation, diluted and allowed to grow for at least 2 doublings to an OD between 0.6 and 1.0. 6 ODs of cells were collected for the zero time point. Cell pellets were washed with 1 ml cold H2O and frozen on dry ice. Cycloheximide was added to cultures for a final concentration of 50 μg/ml media. 6 ODs of cells were collected for each time point as indicated. Cell pellets processed as described below.
cdc15-2 arrest and release
Cells were grown to saturation, then diluted to an OD of 0.3 and allowed to grow to an OD between 0.6 and 1.0. 6 ODs of cells were collected as described above for an asynchronous sample. Cells were diluted to an OD of 0.5 and placed at 37°C for 3 h. Cells were examined under a microscope to confirm anaphase arrest. 6 ODs of cells were collected for the zero time point, as described above. Cells were then released into media at 23°C at an OD of 0.6, and 6 ODs of cells were collected at time points indicated. Cells were collected for flow cytometry at every time point and processed [43].
Western blots
Cell pellets were processed as follows. Cell pellets were thawed in boiling sample buffer (50mM Tris pH 7.5, 5% SDS, 5 mM EDTA, 10% Glycerol, 0.5% BME, 0.1 µg/ml pepstatin A, 0.1 µg/ml leupeptin, 0.1 µg/ml bestatin, 0.1 mM Benzamidine, 5 mM NaF, 0.5 mM Na3VO4, 40 mM β-glycerophosphate, 1 mM PMSF). Cells were boiled for 5 min, followed by bead-beating three times, 30 s each, and then boiled again for 5 min for SDS-PAGE and transfer to nitrocellulose. Western blots were performed with low salt PBST (15 mM NaCl, 1.3 mM NaH2PO4, 5.4 mM Na2HPO4, 0.05% Tween pH 6.8). All primary antibody incubations were performed overnight in 5% milk and low salt PBST unless otherwise noted. Antibodies were used as follows: Cdc20 (yC-20) from Santa Cruz at 1:1000, Cdc28 from Santa Cruz (yC-20) at 1:1000, Dbf4 (yN-15) from Santa Cruz at 1:500, Clb2 (y180) from Santa Cruz 1:1000 (Figure 4B), Cdc6 9H8/5 from Abcam at 1:2000.
APC Assays
APC was purified from a TAP-CDC16, cdh1Δ strain. E1, E2 (Ubc4), APC, and Cdh1 were expressed and purified as previously described [19, 44]. ZZ-tagged Cdc20 wildtype and mutants were transcribed and translated in vitro with TnT Quick Coupled Transcription/Translation Systems (Promega) either in the presence of 35S-methionine or unlabeled methionine. Briefly, APC assays were performed by first charging the E2 in the presence of E1 (Uba1, 300 nM), E2 (Ubc4, 50 µM), Methyl-Ubiquitin (Boston Biochem, 150 µM), and ATP (1 mM) for 20 min. E1/E2(Ubc4)/Methyl-Ubiquitin mix was added to APC (1–5 nM), ZZ-Cdc20 purified from reticulocyte lysate using IgG beads and cleaved using TEV protease, and Securin purified similarly from reticulocyte lysate. In Figure 2D, APC, Cdc20 and Securin were preincubated to increase the amount of activity observed for the mutants. For Figure 5A & B, His-tagged Securin (aa1-110) was expressed in bacteria and purified using Ni-NTA resin. After TEV protease cleavage to remove the His6-tag, the protein was further purified using cation exchange and size exclusion chromatography. APC, Cdc20, and Securin (aa1-110) were preincubated before adding IVT Securin and E1/E2(Ubc4)/Methyl-Ubiquitin mix or E1/E2(Ubc4)/Methyl-Ubiquitin mix alone. All reactions were stopped by the addition of sample buffer, separated by SDS-PAGE, and visualized and quantified with a Molecular Dynamics PhosphorImager and ImageQuant (Amersham Biosciences/GE Healthcare).
HIGHLIGHTS
  • Cdc20 can be turned over by three distinct mechanisms.
  • The most important of these mechanisms is in cis autoubiquitination of Cdc20.
  • Autoubiquitination of Cdc20 is cell cycle regulated.
  • Cdc20 autoubiquitination is inhibited in vitro by high substrate concentration.
Supplementary Material
Supplementary Material
Acknowledgements
We would like to thank C. Carroll, N. Lyons, M. Lopez, G. Yaakov, L. Holt, M. Rodrigo-Brenni, M. Matyskiela, E. Edenberg, M. Downey, T. Berens J. Lopez-Mosqueda, D. Simsek and J. Benanti for their input and advice during this project. Additionally, we would like to thank N. Lyons, E. Edenberg, and M. Downey for reading and commenting on the manuscript. This work was supported by funding from the National Institute of General Medical Sciences to D. P. T. (GM070539) and D.O.M. (GM053270).
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