Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast
Cell Division volume 8, Article number: 9 (2013)
The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.
Here we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation.
Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.
Meiosis is a specialized developmental program during which diploid nuclei undergo two consecutive meiotic divisions to produce haploid gametes. In the budding yeast, spore wall assembly follows the second meiotic nuclear division producing four haploid spores encased in a protective ascus . Similar to differentiation programs in higher eukaryotes, meiotic progression is regulated by the transient expression of genes that are either meiosis specific or expressed during both meiotic and mitotic divisions (reviewed in ). In addition, progression through the meiotic divisions is also driven by the degradation of key regulatory proteins directed by the highly conserved multi-complex ubiquitin ligase called the anaphase promoting complex/cyclosome (APC/C) (reviewed in [3–6]).
During meiosis, the APC/C is sequentially activated by two of the three known Trp-Asp activator (WD40) proteins, Cdc20p (reviewed in [7, 8]), and Ama1p, the latter of which is only expressed during meiosis [9, 10]. The Cdc20p activated APC/C (written APC/CCdc20) mediates the degradation of several key regulatory proteins including Pds1p (securin) and the S-phase cyclin Clb5 during both meiosis I (MI) and meiosis II (MII) [8, 11]. Ama1p directs the ubiquitylation of the B-type cyclin Clb1p , Cdc20p  plus other unknown substrates  and co-ordinates exit from MII . APC/CAma1 also activates Smk1p, the meiotic MAP kinase required for spore wall morphogenesis  and is required for the early stages of spore wall assembly [11, 13, 15]. The third APC/C activator Cdh1p, is not required for normal meiosis .
It has been well documented that APC/C activator proteins recognize substrates through two conserved degrons called the “Destruction-box” (D-box, DB) and “KEN box” that bind the WD40 domain in the activator [17, 18]. In addition, Doc1p (Apc10), a conserved component of the APC/C complex, also recognizes these degrons. These findings have lead to the model that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of an activator protein and Doc1p ( and reviewed in ). During meiosis, Ama1p recognizes the D-box as well as variant of the KEN box called GxEN [10, 12] whereas Cdc20p recognizes the D-box and the KEN box [21, 22]. However, in Xenopus egg extracts the APC/C recognizes destruction motifs directly, in both a Cdc20p and Cdh1p-independent manner . Similarly, much is known about how the activator proteins bind to the APC/C . Structural analysis of Cdh1p has shown that a domain called the C-box interacts with Apc2p . Another domain termed the IR motif promotes the association of the activator with the TPR region of several APC/C subunits (Cdc16p, Cdc23p and Cdc27p) [25–28]. Doc1p (Apc10p), a subunit of the APC/C, also associates with the TPR subunits via its IR tail [29, 30]. During meiosis, both the C-box and IR domains are required for Ama1p and Cdc20p function . However, mutational analysis revealed that the C-box in Ama1p is significantly more important for meiotic progression than the IR motif . Similarly, during mitotic cell division, the IR box of Cdc20p is not required for function but contributes to APC/C dependent turnover [3, 6].
Although much is known about how the APC/C is activated during meiotic divisions (reviewed in ), considerably less is known about how this ligase is inactivated as cells complete meiotic program. This is an important question as APC/C inactivation at the end of meiosis may be critical to allow the spore to reenter the mitotic cell cycle. Our previous studies have shown that both Ama1p and Cdc20p are down regulated as cells exit from meiosis II [10, 12]. Furthermore, Cdc20p degradation is mediated by APC/CAma1 . In this report, we present evidence that Ama1p down regulation occurs via ubiquitin-mediated degradation directed by APC/CCdc20. Taken together, these results indicate that the cell has solved the problem of APC/C inactivation in a linear differentiation pathway by evolving a mutual degradation system for the activators.
Cdc20p activates the APC/C to mediate Ama1p degradation
We have previously reported that Ama1p levels are reduced as cells complete the second meiotic division . As APC/C activators have been reported to be down-regulated by APC/C mediated proteolysis during mitotic and meiotic cell divisions (reviewed in [7, 8]), we first asked if the reduction in Ama1p levels was APC/C dependent. The meiotic levels of Ama1p-T7  were monitored in a strain harboring a temperature sensitive allele of CDC16 (cdc16-1), an essential component of the APC/C  that is required for meiosis . To inactivate Cdc16-1p, the cells were switched to the restrictive temperature (34.5°C) 4.5 h after meiotic entry as previously described [8, 10, 32]). As a control, Ama1p degradation was also examined in identically treated wild-type cells. Immunoblot analysis revealed that Ama1p-T7 levels remained elevated in the cdc16-1 strain compared to wild type (Figure 1A, quantitated in Figure 1B). Similar results were obtained when these experiments were repeated in a cdc20-1 strain (Figure 1A). Furthermore, these results are consistent with those obtained when Ama1p levels were monitored in a strain where Cdc20p was inactivated during meiosis by placing it under the control of CLB2 promoter . Taken together, these results indicate that APC/CCdc20 is required for the down regulation of Ama1p-T7 in meiosis.
A caveat to this interpretation is that Ama1p-T7 stabilization in the cdc20-1 mutant is an indirect effect of the metaphase I arrest associated with this mutation . To address this issue, two approaches were taken. First, we examined Ama1p stability in a cdc20-1 mutant shifted to the restrictive temperature following meiosis II (15 h timepoint). These results show that Ama1p remains stable in the cdc20-1 strain at restrictive temperature even following 30 h in SPM (Figure 1C). To confirm that the cdc20-1 cells had completed the meiotic divisions by this timepoint, the transcription profiles of meiosis-specific genes were monitored using Northern blot analysis. By 15 h in SPM, maximal transcriptional accumulation of SPS4 was observed (Additional file 1) which is an indicator that the meiotic divisions are completing . Similarly, SPS100 mRNA induction, which correlates with spore wall formation , occurs 18 h after meiotic entry.
For the second approach, we analyzed the meiotic degradation of Clb5p, a known substrate of APC/CCdc20 . Clb5p-HA levels were followed by immunoblot analysis in wild type and cdc20-1 cultures using the same temperature shift protocol as described in panel A. The results show that, compared to wild-type cells, Clb5p was stabilized following Cdc20p-1 inactivation (Figure 1D). In contrast, Clb1p, a known substrate of APC/CAma1 , is destroyed in cdc20-1 cells using the same conditions (Figure 1D). The slower induction kinetics observed for both cyclins is due to the fact that expression of early-middle, middle gene mRNAs is significantly reduced as well as delayed in this strain background . Taken together, these results support a model that APC/CCdc20 mediates the degradation of Ama1p as cells complete the meiosis and begin spore morphogenesis.
Cdh1p is not required to mediate the degradation of Ama1p during meiosis
To determine whether Cdh1p plays a role in Ama1p proteolysis during meiosis, Ama1p protein levels were monitored in cdh1∆ cells during meiosis. The results show that cdh1∆ cells both progress through meiosis (Additional file 2: Figure S2A, S2B and S2C) and degrade Ama1p with the same kinetics as wild type (Additional file 2: Figure S2D and see Tan et al.  for Northern analysis). Interestingly, dissection of the resulting cdh1∆ tetrads revealed that, different to previously published results , cdh1∆ spores exhibit a significant reduction in their ability to form colonies (Additional file 2: Figure S2E). These results indicate that Cdh1p does not control Ama1p stability but does play a role in promoting spore viability.
Ama1p contains functional degradation signals
Ama1p contains two motifs, the destruction box (Db) and GxEN, that are recognized by APC/CCdc20 (reviewed in ), see Figure 2A). To determine if these sequences are required for Ama1p-T7 degradation, wild-type cells expressing either Ama1pDb1∆-T7 or Ama1pGxEN-T7 mutant proteins were induced to enter meiosis and their degradation profiles monitored by immunoblot analysis. These studies revealed no difference in decay kinetics for the single mutant derivatives compared to wild type (Figure 2B) indicating that individually the Db1 or GxEN motifs are not essential for Ama1p degradation. We have recently shown that the APC/CAma1 mediates Cdc20p degradation through more than one degron . To determine if Cdc20p also recognizes multiple Ama1p degrons, wild-type cells expressing a double Db1 and GxEN AMA1 derivative were examined as just described. The results (Figure 2B, quantified in Figure 2C) show that combining the GxEN and Db1 mutations protected Ama1p-T7 from degradation similar to that observed in cdc16-1 cells (compare to Figure 1A). These results indicate that either Db1 or GxEN is sufficient to target Ama1p for degradation. No difference in the rate of meiotic progression (Figure 2D) or spore viability (Figure 2E) was noted indicating that stabilizing Ama1p did not have an adverse effect on the process.
Ama1p is a substrate of APC/CCdc20 in vitro
To further confirm that APC/CCdc20 mediates the degradation of Ama1p, in vitro ubiquitylation assays were performed (see Methods for details). As Ama1p is an activator of the APC/C , the assays were performed with an in vitro transcription coupled translation produced 35-S labeled Ama1p derivative deleted for its two APC/C binding domains (C-box and IR motif). These motifs are required for Ama1p function  and their mutation reduces its association with the APC/C (see Figure 3B and C). To ensure that the added Cdc20p is the only activator in the reaction, the APC/C core complex was purified from mitotically dividing cdh1∆ cells. Furthermore, Mnd2p (Apc15p) was not present in the extracts as it inhibits meiotic APC/C activity . As predicted from the in vivo studies, Ama1pCB∆/IR∆ is ubiquitylated by APC/CCdc20 in vitro (Figure 4A, lanes 1, 2 and 3 and see Additional file 3 for input), but also that Cdc20p is required for this event (Figure 4A – lane 12).
The in vivo stability assays just described (Figure 2) indicated that either Db1 or the GxEN motif is sufficient to induce Ama1p degradation. Consistent with this result, deletion of either of these motifs in the Ama1pCB/IR mutant still allowed ubiquitylation to occur (Figure 4A, lanes 4-6 for GxEN, 8 and 9 for Db1). However, Ama1p mutated for both Db1 and GxEN was still ubiquitylated in vitro by APC/CCdc20 (Figure 4A, lanes 10 and 11). This result was unexpected as this mutant is not targeted for degradation in vivo (Figure 2B). These results led us to test if the second destruction box degron (Db2) on Ama1p can mediate Cdc20p-dependent in vitro ubiquitylation. This was indeed the case as the mutation of Db2, in addition to Db1 and GxEN, rendered Ama1p resistant to APC/CCdc20-dependent ubiquitylation (Figure 4A, lane7). Taken together, these results reveal that Cdc20p can recognize degrons Db1, Db2 and GxEN using in vitro assays. However, Db2 is not recognized by Cdc20p as a degron in vivo during meiosis.
The APC/C core component Doc1p forms part of the bipartite degron receptor in yeast [19, 25, 30]. Therefore, we addressed whether Doc1p is required for APC/CCd20 mediated ubiquitylation of Ama1p. The ubiquitylation assays were repeated using Ama1pC-Box∆/IR∆ as the substrate and APC/C was prepared from cdh1∆ mnd2∆ doc1∆ cells. The results show a slight qualitative reduction in Ama1pC-Box∆/IR∆ ubiquitylation when the APC/C was prepared from cdh1∆ mnd2∆ doc1∆ extracts compared to those prepared from a cdh1∆ mnd2∆ strain (Figure 4B, compare lane 3 to 6). These results suggest that Doc1p is dispensable for Ama1p ubiquitylation in vitro.
Ama1p association with the APC/C through its C-box and IR motif is not required for its degradation
Significant structural analysis of the APC/C and its substrates has found two distinct locations within the cavity of the core APC/C complex that are occupied by the activator protein and the substrate. Our findings that Ama1p is both an activator and a substrate of the APC/C raised the question of its location within the APC/C cavity before it was destroyed. To address this question, we took advantage of the observation that the conserved APC/C binding domains of Ama1p (C-box and IR motif) are required for APC/CAma1 function and normal association with the APC/C . Therefore, we reasoned that if Ama1p was destroyed while in its activator binding pocket, then disruption of this interaction should protect the protein from degradation. Immunoblot blot analysis of ama1∆ cells harboring either wild-type Ama1p or Ama1pCB∆/IR∆-T7 during meiosis revealed no differences in the kinetic profile of Ama1p accumulation and degradation (Figure 3A). These results indicate that Ama1p association to the APC/C via the CB and IR motifs is not a pre-requisite for its degradation. These results also suggest that the majority of Ama1p degradation is not mediated by auto-ubiquitylation as Ama1pCB∆/IR∆-T7 is still degraded in the absence of a functional copy of Ama1p.
To further address this question, co-immunoprecipitation performed assays were performed between Cdc27p-9myc and either Ama1p, Ama1pCB∆-T7, Ama1pIR∆-T7, or Ama1pCB∆/IR∆-T7. The results showed that Ama1pCB∆-T7 and Ama1pCB∆/IR∆-T7, which complemented an ama1∆ allele with 11 and <0.5% sporulation efficiency, respectively , exhibited reduced Cdc27p-9myc binding (Figure 3B). Conversely, Ama1pIR∆-T7, which exhibited only slight reduction in activity , binds Cdc27p-9myc with similar affinity as wild-type Ama1p. These results were somewhat unexpected as deleting the IR and Cbox motifs in Cdh1p eliminates its ability to bind the APC/C . In addition, these results suggest the presence of additional APC/C binding motif(s) in Ama1p. Consistent with this possibility, we found that a GST-Ama1p fusion construct containing the divergent amino third of Ama1p (codons 1-200) , can co-immunoprecipitate with Cdc27p-9myc (Figure 3C) whereas GST alone cannot (lanes 3 and 4). Again, we only observe a slight reduction in Cdc27p-9myc association when a GST-Ama1p1-200CB∆ fusion construct (Figure 3C, lane 6). These results indicate that the amino-terminal region of Ama1p is sufficient for APC/C association and contains an uncharacterized APC/C binding motif(s).
Cdc20p and Ama1p are degraded with the same kinetics during meiosis
We have previously demonstrated that APC/CAma1 directs the degradation of meiotic Cdc20p . Our results here indicate that in a reciprocal fashion APC/CCdc20 also mediates the degradation of Ama1p as cells exit meiosis II. If Ama1p and Cdc20p are required for each other’s degradation, one prediction of this model is that their degradation kinetics should be similar. To test this hypothesis, a strain was constructed harboring integrated alleles of CDC20- 18myc and AMA1-3HA under the control of their own promoters. Our previous studies found that Ama1p-3HA is both functional and has the same degradation kinetics as Ama1p-T7 . A meiotic timecourse was conducted and Cdc20p-18myc and Ama1p-3HA expression profiles were determined by immunoblot blot analysis. These studies revealed that the accumulation and subsequent degradation of both proteins were remarkably similar (Figure 3D). These results are consistent with the model that Ama1p and Cdc20p simultaneously mediate each other’s degradation, thus terminating APC/C activity as the cells complete meiosis and form quiescent spores.
The APC/C ubiquitin ligase is required for the meiotic nuclear divisions in yeast. Previous studies have found that the two APC/C activators in meiosis, Ama1p and Cdc20p, are down regulated as cells complete meiosis II. Cdc20p is targeted for degradation by APC/CAma1 . In this study, we demonstrate that the reverse is true in that APC/CCdc20 is required for Ama1p degradation. Using a combination of stability assays and in vitro ubiquitylation experiments, we show that Cdc20p, but not Cdh1p, targets Ama1p through either one of two degrons, Db1 and GxEN. We also provide evidence to support a model in which degradation of Ama1p does not occur by auto-ubiquitylation as the non-functional Ama1pCB∆/IR∆ mutant is still degraded with wild-type kinetics in ama1∆ cells. Finally, we show that the degradation of Ama1p and Cdc20p at MII exit occurs with similar kinetics. Taken together, these results suggest a model in which the mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast.
Understanding how the APC/C is regulated during both mitotic and meiotic divisions is important as unscheduled APC/C activity can lead to mis-segregated chromosomes and aneuploid gametes. Many studies have been devoted dissecting the precise mechanisms by which the APC/C is both activated and inactivated in mitotic cells (reviewed in ). These studies revealed that the complete inactivation of the APC/C late in G1 is driven by inhibition of Cdc20p and Cdh1p. This system not only resets the APC/C clock, which is critical for maintaining ploidy as it ensures that the pre-replication complex is assembled prior to S phase (reviewed in ). Cdh1p inactivation is achieved by phosphorylation (reviewed in ). However, Cdc20p regulation is more complex. Initially, it was shown that Cdc20p is inactivated by transcriptional oscillation and turnover by APC/CCdh1 (reviewed in ). However, recently it was shown that APC/CCdh1 only partially contributes to Cdc20p degradation during anaphase . Instead, Cdc20p degradation is predominantly mediated by an auto-ubiquitylation event [6, 39]. Ama1p degradation does not seem to take the same course as the non-functional CB∆/IR∆ is still degraded in ama1∆ cells (Figure 4A).
Even less is known about how the APC/C is inactivated as cells exit meiosis II. This is an important question as APC/C inactivation is important for normal embryonic development in Drosophila . Similarly, we find that the two APC/C activators are degraded late in meiotic development. However, we find no significant effect on meiosis II fidelity or overall spore viability when either Cdc20p or Ama1p degradation is inhibited ( and Figure 2). These observations suggest that either APC/C inactivation is not required for the normal execution of meiosis and spore formation or that this ubiquitin ligase is disabled by redundant systems. In support of the latter possibility, several mechanisms are known to control APC/C function including inhibitory phosphorylation [41–44], APC/C specific inhibitors [45–52], or removal of the activator from the APC/C complex . The roles these mechanisms play as cells exit the meiotic program are not well understood. However, in Xenopus and S. pombe, inhibitors of meiotic Cdc20p have been identified [54, 55].
Model for substrate recognition by APC/C activators
Extensive studies have been devoted to understanding the molecular mechanisms of APC/C activator binding and substrate recognition (reviewed in ). Currently, two non-mutually exclusive models have been proposed. In the bi-partite model (outlined in model A, Figure 5), the substrate binds to both the activator and to Doc1p in the inner cavity of the APC/C. This dual association increases the affinity of the substrate enzyme complex [19, 24, 25, 30]. However, Doc1p it is not essential for substrate binding in yeast  and its contribution to meiosis is not well documented. In the second model, coined the allosteric model, binding of the activators to the APC/C induces a conformational change which leads to substrate recognition . Currently, the bipartite model is favored but the two models can co-exist as the bi-partite model can still accommodate activator association promoting conformational changes.
That being said, how does Ama1p fit into these models when it becomes a substrate of the APC/C? Recently, work by Foe et al.  has shed some light on this question. This group demonstrated that the majority of the late mitotic turnover of Cdc20p occurs while Cdc20p is bound as an activator and is driven by auto-ubiquitylation (see model in Figure 5C, cis-model). Consistent with this model, Cdc20pIR∆ mutants show increased steady state levels and reduced auto-ubiquitylation [3, 6]. In contrast, we present evidence that Ama1p degradation is independent of APC/C binding via the CB and/or IR motifs (see Figures 4 and 3A). As the CB and IR motifs associate with Cdc27p/Cdc23p and Apc2p, respectively , our data support a model (outlined in Figure 5B, trans-model) in which Ama1p disassociates from Cdc27/23 and Apc2 before it is recognized as a substrate by APC/CCdc20. Thus, the residual association that we observed between Cdc27p and Ama1pCB∆/IR∆ (Figure 3B and C) could be due to Ama1p associating with the APC/C in the substrate location. This suggests a model in which C-box and IR motifs anchor Ama1p in the activator position but in their absence, Ama1p switches into the substrate position binding the APC/C via as yet uncharacterized motifs. The mechanism that triggers this disassociation remains unknown but recently it has been shown that phosphorylation of Cdc20p prevents its CB-dependent activation of the APC/C in Xenopus egg extracts . Lastly a “cis-dimer” model (Figure 5D) where Ama1p remains in the activator position and is degraded when an APC/CCdc20 complex forms a dimer partner is also possible. This model is not favored as although yeast APC/C exist as dimers, recent work has shown that the monomers associate along the backbone of the “arc lamp” thus positioning the substrate binding sites in opposite directions [19, 60].
Finally, the observation that Cdc20p and Ama1p both regulate each other leads to the mechanistic question of which protein is the last one to be degraded. Analysis of both proteins under the control of their own promoters in a single meiotic timecourse experiment showed that they were down regulated at the same time. These results suggest that it may not be critical as to which activated APC/C molecule is the last one. To conclude, these data presented here allow us to propose a model of how APC/C activators are recognized as substrates of the APC/C during meiosis. It remains to be seen if this model is conserved during gametogenesis in other systems.
Yeast strains and plasmids
The strains used in this study (Table 1) are isogenic to RSY335  and are derived from an SK1 background . The only exception to this is RSY1337 that is isogenic a W303a-related strain RSY10 . The Cdc27-9myc::LEU2 strains (KCY328 and RSY1337) were made by inserting CDC27-9myc tagged allele (P. Hieter) into RSY335 and RSY10 respectively. The mnd2∆:: KANMX cdh1∆::LEU2 CDC16-TAP strain (KCY1381) was made as follows. First, the TAP cassette was inserted into the carboxyl terminus of CDC16 by recombining PCR products from pFA6a-TAP-kanMX6 (D. Barford) to create KCY456. Next, the mnd2∆:: KANMX haploid (KCY419) was created in the opposite mating type using the gene disruption . These two haploids were then mated and an mnd2∆::KAN CDC16::TAP:: KANMX haploid (RSY1248) spore clone was identified that showed 2:2 distribution of the KANMX allele following tetrad analysis. CDH1 was deleted from RSY1248 using pWS176 (W. Seufert) to create RSY1381. Finally DOC1 was deleted from this strain using standard gene disruption techniques  to create RSY1748. The temperature-sensitive cdc20-1 strain (RSY809) has been previously described  . The temperature-sensitive cdc16-1 strain RSY954 was made by back crossing H20c1a5  into the RSY335 strain background eight times. The strain harboring integrated epitope-tagged alleles of both AMA1 and CDC20 (RSY750) was made by using integrating plasmids containing functional AMA1-3HA  and CDC20-18myc (from W. Zachariae), respectively. Tables 2 and 3 list the oligonucleotides and plasmids used in this study, respectively. Details of plasmid constructions are available on request. In brief, all the AMA1-T7 tagged plasmids were derived from pKC3036 . The Ama1p expressing plasmids used for ubiquitylation assays were derived from pME67 (D. Morgan). The Cdc20p plasmid used for ubiquitylation assays was pME41 (D. Morgan). The CLB5-3HA plasmid (pKC440) was made by cloning an Xho1-Cla 1 fragment containing Clb5-3HA (from C. Wittenberg) under the control of its own promotor and terminator into Ycplac222. The Clb1-9HA plasmid was made by first cloning a Pst 1-Pst 1 fragment from a CLB1/CLB6 contig (from C. Wittenburg) into pRS315 and then inserting 9 repeats of the HA epitope just upstream of the stop codon to create pKC427. The galactose inducible GST-Ama11-200 fusion construct (pKC3113) has been previously described . In brief, AMA1 was introduced into pEG[KT], which contains GST under the control of the galactose promotor (a gift from M. Solomon). Site directed mutagenesis was used to delete the C-box in this construct to make pKC3071. All mutations were introduced using the Quikchange Site-directed Mutagenesis (SDM) Kit (Stratagene) according to the manufacturer’s protocol. All introduced mutations were verified by DNA sequencing (MWG/Operon).
Meiotic and mitotic timecourse experiments
Growth and sporulation conditions were accomplished as previously described . To permit cdc20-1 and cdc16-1 cultures to exit mitosis and enter the meiotic program, these cells were maintained at 23°C following transfer to sporulation medium for the amount of time indicated in the text before switching to the restrictive temperature (water bath). Quantitation of meiosis I and II was achieved by analyzing 4’, 6-diamidino-2-phenylindole (DAPI) stained cells as described . A Nikon E800 fluorescence microscope was used for all experiments at a final magnification of 1000X. At least 200 cells were counted per timepoint. For the experiments using the galactose inducible GST expression constructs (Figure 4C), cells were grown to 1 × 107 cells/ml in 2% raffinose, 2% galactose medium as previously described .
Northern blot analysis, protein extract preparation, co-immunoprecipitation and Immunoblot analysis
Northern blot analysis was executed as previously described . Protein extracts for co-immunoprecipitation and Western blot analyses (referred to as Immunoblot in text) were prepared as described . Immunoblot analysis and co-immunoprecipitation experiments were conducted with 100 μg and 1 mg of soluble protein, respectively. Immunoblot signals were detected using goat anti-mouse secondary antibodies conjugated to alkaline phosphatase (Sigma) and the CDP-Star chemiluminescence kit (Tropix, Bedford, MA). Quantitation of Ama1p immunoblot signals from the mem brane was performed with an Image Station 4000R (Kodak Inc.) using Molecular Imaging Software (4.0.5) and standardized to tubulin. For all comparative immunoblot analyses, the membranes were treated with the same probe at the same time and the resulting signals were developed to the same extent.
In vitro ubiquitylation assays
The in vitro ubiquitylation assays were performed as previously described [32, 70]. In brief, the APC/C complex was purified from yeast extracts utilizing tandem affinity purification (TAP) tagged Cdc16p, a core component of this ubiquitin ligase. The ligase was incubated with E. coli produced ubiquitin conjugating enzyme (made from His6-Ubc4p (from M. Solomon) and in vitro transcription/translation produced Cdc20p. The Ama1p substrates were synthesized by in vitro transcription/translation (Promega) but in the presence of 35S-methionine. As previously described , 1 μl of the substrate was used per reaction (see Additional file 3 for input). The ubiquitylation reactions were conducted for the times indicated with fixed Cdc20p amounts (2.5 μl). The reactions were stopped by addition of 2X sample buffer and separated by SDS PAGE. The gels were fixed, soaked in Amplify® (Amersham Biosciences), then dried and subjected to autoradiography.
Anaphase promoting complex
Destruction box (degron)
C-box (APC/C binding motif)
(APC/C binding motif)
Herskowitz I: Life style of the budding yeast Saccharomyces cerevisiae . Microbiol Rev 1988, 52: 536–553.
Vershon A, Pierce M: Transcriptional regulation of meiosis in yeast. Curr Opin Cell Biol 2000, 12: 334–339. 10.1016/S0955-0674(00)00104-6
Thornton BR, Ng TM, Matyskiela ME, Carroll CW, Morgan DO, Toczyski DP: An architectural map of the anaphase-promoting complex. Genes Dev 2006, 20: 449–460. 10.1101/gad.1396906
Yu H: Cdc20: a WD40 activator for a cell cycle degradation machine. Mol Cell 2007, 27: 3–16. 10.1016/j.molcel.2007.06.009
Barford D: Structural insights into anaphase-promoting complex function and mechanism. Philos Trans R Soc Lond B Biol Sci 2011, 366: 3605–3624. 10.1098/rstb.2011.0069
Foe IT, Foster SA, Cheung SK, DeLuca SZ, Morgan DO, Toczyski DP: Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism. Curr Biol 2011, 21: 1870–1877. 10.1016/j.cub.2011.09.051
Pesin JA, Orr-Weaver TL: Regulation of APC/C activators in mitosis and meiosis. Annu Rev Cell Dev Biol 2008, 24: 475–499. 10.1146/annurev.cellbio.041408.115949
Cooper KF, Strich R: Meiotic control of the APC/C: similarities & differences from mitosis. Cell Div 2011, 6: 16. 10.1186/1747-1028-6-16
Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown PO, Herskowitz I: The transcriptional program of sporulation in budding yeast. Science 1998, 282: 699–705.
Cooper KF, Egeland DE, Mallory MJ, Jarnik M, Strich R: Ama1p is a Meiosis-Specific Regulator of the Anaphase Promoting Complex/Cyclosome in yeast. Proc. Natl. Acad. Sci. USA 2000, 97: 14548–14553. 10.1073/pnas.250351297
Diamond AE, Park JS, Inoue I, Tachikawa H, Neiman AM: The anaphase promoting complex targeting subunit Ama1 links meiotic exit to cytokinesis during sporulation in Saccharomyces cerevisiae. Mol Biol Cell 2009, 20: 134–145. 10.1091/mbc.E08-06-0615
Tan GS, Magurno J, Cooper KF: Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II. Mol Biol Cell 2011, 22: 315–326. 10.1091/mbc.E10-04-0360
Rabitsch KP, Toth A, Galova M, Schleiffer A, Schaffner G, Aigner E, Rupp C, Penkner AM, Moreno-Borchart AC, Primig M, et al.: A screen for genes required for meiosis and spore formation based on whole-genome expression. Curr Biol 2001, 11: 1001–1009. 10.1016/S0960-9822(01)00274-3
McDonald CM, Cooper KF, Winter E: The Ama1-Directed Anaphase-Promoting Complex Regulates the Smk1 Mitogen-Activated Protein Kinase During Meiosis in Yeast. Genetics 2005, 171: 901–911. 10.1534/genetics.105.045567
Coluccio A, Bogengruber E, Conrad MN, Dresser ME, Briza P, Neiman AM: Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae. Eukaryot Cell 2004, 3: 1464–1475. 10.1128/EC.3.6.1464-1475.2004
Kamieniecki RJ, Liu L, Dawson DS: FEAR but not MEN genes are required for exit from meiosis I. Cell Cycle 2005, 4: 1093–1098.
Glotzer M, Murray AW, Kirschner MW: Cyclin is degraded by the ubiquitin pathway. Nature 1991, 349: 132–138. 10.1038/349132a0
Pfleger CM, Lee E, Kirschner MW: Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev 2001, 15: 2396–2407. 10.1101/gad.918201
Buschhorn BA, Petzold G, Galova M, Dube P, Kraft C, Herzog F, Stark H, Peters JM: Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1. Nat Struct Mol Biol 2011, 18: 6–13. 10.1038/nsmb.1979
Barford D: Structure, function and mechanism of the anaphase promoting complex (APC/C). Q Rev Biophys 2011, 44: 153–190. 10.1017/S0033583510000259
King EM, van der Sar SJ, Hardwick KG: Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint. PLoS One 2007, 2: e342. 10.1371/journal.pone.0000342
Bolte M, Dieckhoff P, Krause C, Braus GH, Irniger S: Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1–3 proteins in Saccharomyces cerevisiae. Microbiology 2003, 149: 1205–1216. 10.1099/mic.0.26062-0
Yamano H, Gannon J, Mahbubani H, Hunt T: Cell cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts. Mol Cell 2004, 13: 137–147. 10.1016/S1097-2765(03)00480-5
Da Fonseca PC, Kong EH, Zhang Z, Schreiber A, Williams MA, Morris EP, Barford D: Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature 2011, 470: 274–278. 10.1038/nature09625
Passmore LA, McCormack EA, Au SW, Paul A, Willison KR, Harper JW, Barford D: Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J 2003, 22: 786–796. 10.1093/emboj/cdg084
Burton JL, Tsakraklides V, Solomon MJ: Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box. Mol Cell 2005, 18: 533–542. 10.1016/j.molcel.2005.04.022
Kraft C, Vodermaier HC, Maurer-Stroh S, Eisenhaber F, Peters JM: The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates. Mol Cell 2005, 18: 543–553. 10.1016/j.molcel.2005.04.023
Izawa D, Pines J: How APC/C-Cdc20 changes its substrate specificity in mitosis. Nat Cell Biol 2011, 13: 223–233. 10.1038/ncb2165
Wendt KS, Vodermaier HC, Jacob U, Gieffers C, Gmachl M, Peters JM, Huber R, Sondermann P: Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex. Nat Struct Biol 2001, 8: 784–788. 10.1038/nsb0901-784
Carroll CW, Enquist-Newman M, Morgan DO: The APC subunit Doc1 promotes recognition of the substrate destruction box. Curr Biol 2005, 15: 11–18. 10.1016/j.cub.2004.12.066
Lamb JR, Michaud WA, Sikorski RS, Hieter PA: Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J 1994, 13: 4321–4328.
Mallory MJ, Cooper KF, Strich R: Meiosis-specific destruction of the Ume6p repressor by the Cdc20-directed APC/C. Mol Cell 2007, 27: 951–961. 10.1016/j.molcel.2007.08.019
Oelschlaegel T, Schwickart M, Matos J, Bogdanova A, Camasses A, Havlis J, Shevchenko A, Zachariae W: The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1. Cell 2005, 120: 773–788. 10.1016/j.cell.2005.01.032
Hepworth SR, Ebisuzaki LK, Segall J: A 15-base-pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae. Mol Cell Biol 1995, 15: 3934–3944.
Law DTS, Segall J: The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation. Mol Cell Biol 1988, 8: 912–922.
Harper JW, Burton JL, Solomon MJ: The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev 2002, 16: 2179–2206. 10.1101/gad.1013102
Schwab M, Neutzner M, Mocker D, Seufert W: Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J 2001, 20: 5165–5175. 10.1093/emboj/20.18.5165
Robbins JA, Cross FR: Regulated degradation of the APC coactivator Cdc20. Cell Div 2010, 5: 23. 10.1186/1747-1028-5-23
Foster SA, Morgan DO: The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation. Mol Cell 2012, 47: 921–932. 10.1016/j.molcel.2012.07.031
Pesin JA, Orr-Weaver TL: Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator. PLoS Genet 2007, 3: e202. 10.1371/journal.pgen.0030202
Rudner AD, Murray AW: Phosphorylation by cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol 2000, 149: 1377–1390. 10.1083/jcb.149.7.1377
Tang Z, Shu H, Oncel D, Chen S, Yu H: Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint. Mol Cell 2004, 16: 387–397. 10.1016/j.molcel.2004.09.031
Chung E, Chen RH: Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint. Nat Cell Biol 2003, 5: 748–753. 10.1038/ncb1022
Labit H, Fujimitsu K, Bayin NS, Takaki T, Gannon J, Yamano H: Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C. EMBO J 2012, 31: 3351–3362. 10.1038/emboj.2012.168
Reimann JD, Freed E, Hsu JY, Kramer ER, Peters JM, Jackson PK: Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 2001, 105: 645–655. 10.1016/S0092-8674(01)00361-0
Martinez JS, Jeong DE, Choi E, Billings BM, Hall MC: Acm1 is a negative regulator of the CDH1-dependent anaphase-promoting complex/cyclosome in budding yeast. Mol Cell Biol 2006, 26: 9162–9176. 10.1128/MCB.00603-06
Choi E, Dial JM, Jeong DE, Hall MC: Unique D box and KEN box sequences limit ubiquitination of Acm1 and promote pseudosubstrate inhibition of the anaphase-promoting complex. J Biol Chem 2008, 283: 23701–23710. 10.1074/jbc.M803695200
Ostapenko D, Burton JL, Wang R, Solomon MJ: Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation. Mol Cell Biol 2008, 28: 4653–4664. 10.1128/MCB.00055-08
Enquist-Newman M, Sullivan M, Morgan DO: Modulation of the mitotic regulatory network by APC-dependent destruction of the Cdh1 inhibitor Acm1. Mol Cell 2008, 30: 437–446. 10.1016/j.molcel.2008.04.004
Burton JL, Solomon MJ: Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev 2007, 21: 655–667. 10.1101/gad.1511107
Sczaniecka M, Feoktistova A, May KM, Chen JS, Blyth J, Gould KL, Hardwick KG: The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C). J Biol Chem 2008, 283: 23039–23047. 10.1074/jbc.M803594200
Lara-Gonzalez P, Scott MI, Diez M, Sen O, Taylor SS: BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner. J Cell Sci 2011, 124: 4332–4345. 10.1242/jcs.094763
Jaquenoud M, Van Drogen F, Peter M: Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1). EMBO J 2002, 21: 6515–6526. 10.1093/emboj/cdf634
Schmidt A, Duncan PI, Rauh NR, Sauer G, Fry AM, Nigg EA, Mayer TU: Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of APC/C activity. Genes Dev 2005, 19: 502–513. 10.1101/gad.320705
Kimata Y, Kitamura K, Fenner N, Yamano H: Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast. Mol Biol Cell 2011, 22: 1486–1494. 10.1091/mbc.E10-09-0774
Hwang LH, Murray AW: A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis. Mol Biol Cell 1997, 8: 1877–1887. 10.1091/mbc.8.10.1877
Passmore LA, Barford D: Coactivator functions in a stoichiometric complex with anaphase-promoting complex/cyclosome to mediate substrate recognition. EMBO Rep 2005, 6: 873–878. 10.1038/sj.embor.7400482
Matyskiela ME, Morgan DO: Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism. Mol Cell 2009, 34: 68–80. 10.1016/j.molcel.2009.02.027
Schreiber A, Stengel F, Zhang Z, Enchev RI, Kong EH, Morris EP, Robinson CV, Da Fonseca PC, Barford D: Structural basis for the subunit assembly of the anaphase-promoting complex. Nature 2011, 470: 227–232. 10.1038/nature09756
Passmore LA, Booth CR, Venien-Bryan C, Ludtke SJ, Fioretto C, Johnson LN, Chiu W, Barford D: Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation. Mol Cell 2005, 20: 855–866. 10.1016/j.molcel.2005.11.003
Cooper KF, Mallory MJ, Strich R: Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires the Phosphatidylinositol-specific phospholipase C and the 26S proteasome. Mol Cell Biol 1999, 19: 3338–3348.
Cooper KF, Mallory MJ, Guacci V, Lowe K, Strich R: Pds1p is required for meiotic recombination and prophase I progression in Saccharomyces cerevisiae. Genetics 2009, 181: 65–79.
Cooper KF, Mallory MJ, Smith JS, Strich R: Stress and developmental regulation of the yeast C-type cyclin UME3 ( SRB11 / SSN8 ). EMBO J 1997, 16: 4665–4675. 10.1093/emboj/16.15.4665
Longtine MS, McKenzie AR, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR: Additional modules for versatile and economical PCR-based gene deletion and modification. Saccharomyces cerevisiae 1998, Yeast 14: 953–961.
Shirayama M, Zachariae W, Ciosk R, Nasmyth K: The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae . EMBO J 1998, 17: 1336–1349. 10.1093/emboj/17.5.1336
Schwab M, Neutzer M, Mocker D, Seufert W: Yeast Hct1 recognises the mitotic Clb2 and other substrates of the ubiquitin ligase APC. EMBO 2001, 20: 5165–5175. 10.1093/emboj/20.18.5165
Burton JL, Solomon MJ: D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. Genes Dev 2001, 15: 2381–2395. 10.1101/gad.917901
Cooper KF, Strich R: Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p is required for efficient induction and execution of meiotic development. Eukaryot Cell 2002, 1: 66–74. 10.1128/EC.01.1.66-74.2002
Prinz S, Hwang ES, Visintin R, Amon A: The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis. Curr Biol 1998, 8: 750–760. 10.1016/S0960-9822(98)70298-2
Passmore LA, McCormack EA, Au SWN, Paul A, Willison KR, Harper JW, Barford D: Doc1 mediates the activity of the anaphase promoting complex by contributing to substrate recognition. EMBO 2003, 22: 786–796. 10.1093/emboj/cdg084
We thank D. Bradford, P. Hieter, D. Morgan, W. Seufert, M. Solomon, C. Wittenberg and W. Zachariae for plasmids. This work was supported by ACS grant # CCG106162 to K. F. C. and by Public Health Service grant #’s CA-099003 and GM086788 from the National Institutes of Health, U.S.A. to R. S.
The authors declare that they have no competing interests.
GT performed the experiments outlined in Figure 1A, B and C, 2 and 3A and B. RL performed the experiments outlined in Figure 4. MM performed the experiment in Figure 1D and 3C. KFC and RS wrote the manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1: Analysis of cdc20-1 during meiosis. A: Northern blot analysis of cdc20-1 cells progressing through meiosis at 23°C showing the expression of early (IME2), early middle (NDT80), middle (SPS4) and late genes (SPS100). ENO1 represents the loading control. (TIFF 474 KB)
Additional file 2: Cdh1p is not required to degrade Ama1p during meiosis. A: Fluorescence and Nomarski (Nom.) images (1000X magnification) of DAPI stained wild type (RSY335) and cdh1∆ (RSY777) diploids 24 h after transfer to sporulation medium. B: Rate of appearance of bi- and tetranucleated cells in wild type and cdh1∆ cells after entry into the meiotic program. Percentage of cells in the culture executing at least one meiotic division, presented as a function of time following transfer to sporulation medium. MI, Meiosis I; MII meiosis II. C: % mono, bi and tetranucleated cells in the total population after 24 h in sporulation medium. D: cdh1∆ strain (RSY777) harboring Ama1p-T7 (pKC3036) was induced to enter meiosis and timepoints taken as indicated. Immunoblot analysis of immunoprecipitated protein extracts was conducted to detect Ama1p-T7. Immunoblot analysis of Tub1p was used as a loading control. E: Viability of wild type (RSY335) and cdh1∆ (RSY777) tetrad spores. (TIFF 684 KB)
Additional file 3: 35 S labeled Ama1p input for ubiquitylation assays. 1 μl of 35S labeled in vitro transcription/translation Ama1p prepared from either pKC3095 (lane 1), pKC3122 (lane 2) pKC3148 (lane 3) or pKC3124 (lane 4) or zero DNA control was visualized by autoradiography. (TIFF 297 KB)
About this article
Cite this article
Tan, G.S., Lewandowski, R., Mallory, M.J. et al. Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast. Cell Div 8, 9 (2013). https://doi.org/10.1186/1747-1028-8-9
- Anaphase Promoting Complex