CDC20 and CDH1: A Family of Substrate-Specific Activators of APC-Dependent Proteolysis

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Science  17 Oct 1997:
Vol. 278, Issue 5337, pp. 460-463
DOI: 10.1126/science.278.5337.460


Proteolysis mediated by the anaphase-promoting complex (APC) triggers chromosome segregation and exit from mitosis, yet its regulation is poorly understood. The conserved Cdc20 and Cdh1 proteins were identified as limiting, substrate-specific activators of APC-dependent proteolysis. CDC20 was required for the degradation of the APC substrate Pds1 but not for that of other APC substrates, such as Clb2 and Ase1. Conversely, cdh1Δmutants were impaired in the degradation of Ase1 and Clb2 but not in that of Pds1. Overexpression of either CDC20 orCDH1 was sufficient to induce APC-dependent proteolysis of the appropriate target in stages of the cell cycle in which substrates are normally stable.

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. In budding yeast, degradation of the Pds1 protein is required for the transition from metaphase to anaphase in the cell cycle (1). Exit from mitosis is initiated by proteolysis of mitotic cyclins (2). Destruction of the mitotic spindle–associated protein Ase1 is important for the disassembly of the mitotic spindle during exit from mitosis (3). Degradation of all these proteins is mediated by a 20Sparticle called the cyclosome or APC, which functions as a ubiquitin-protein ligase (4, 5). The stability of substrates of the APC-dependent proteolysis machinery varies greatly during the cell cycle. Substrates are stable during S phase, G2 phase, and early mitosis (M) phase but are highly unstable during exit from M phase and G1 phase (1, 3, 6–8). Work in embryonic extracts has shown that APC activity is cell cycle regulated, suggesting that the activity of the APC determines whether APC substrates are degraded or not (4,5). However, degradation of different APC substrates is not coordinated, because temporal differences are observed (9-11). The proteolysis of APC substrates, therefore, cannot simply be regulated by the activation or inactivation of APC. Regulation must also occur at the level of specific substrates. We identified two highly conserved Saccharomyces cerevisiaeproteins that function in this manner. Cdc20 and Cdh1 are substrate-specific activators of APC-dependent proteolysis.

To identify regulators of APC-dependent proteolysis, we screened previously identified temperature-sensitive cell division cycle (cdc) mutants for defects in APC-dependent proteolysis. Among these, mutants defective in the CDC20 gene, which encodes a protein containing seven WD40 repeats (12), were of particular interest. Saccharomyces cerevisiae cdc20-1mutants and Drosophila mutants defective in thefizzy gene, a CDC20 homolog, arrest in metaphase before the activation of the APC-dependent proteolytic program (11-14). To analyze the effects of the cdc20-1mutation on APC-dependent proteolysis, we took advantage of the fact that in yeast all known APC substrates (the mitotic cyclins Clb2 and Clb3, Pds1, and Ase1) are unstable during G1 phase because of continuous APC-dependent proteolysis (1, 3,7, 15). Wild-type and cdc20-1 cells were arrested at the permissive temperature in G1 phase by exposure to the mating pheromone α-factor, and the half-lives of Clb2, Pds1, and Ase1 were determined after transient expression of their respective genes from the galactose (Gal)-inducible GAL1-10 promoter and inactivation of CDC20 by temperature shift (16). The cdc20-1 mutation had little if any effect on the half-life of Clb2 (Fig. 1, A and B), which is consistent with the finding that thecdc20-1 mutation does not affect ubiquitination of Clb2 in G1 phase extracts (15). Similarly, thecdc20-1 mutation had little if any effect on the half-life of Ase1 during G1 phase (Fig. 1, C and D). In contrast, the half-life of Pds1 was prolonged in the cdc20-1 mutant; Pds1 protein declined rapidly in wild-type cells but not incdc20-1 mutants (Fig. 1, E and F). These results indicate that cdc20-1 mutants are defective in the degradation of Pds1 but not in that of Clb2 and Ase1 during the G1 phase.

Figure 1

Defective degradation of Pds1, but not Clb2 and Ase1, during G1 phase in cdc20-1mutants. Wild-type (WT) and cdc20-1mutants carrying either CLB2 (K4017 and A977),PDS1 (A914 and A1008), or ASE1 (A1045 and A1046), under the control of the GAL1-10 promoter (16), were arrested in G1 phase with α-factor pheromone at the permissive temperature. After 3.5 hours, Gal was added for 30 min to induce production of either Clb2, Pds1, or Ase1, followed by a shift to the restrictive temperature (35°C) for 45 min. The half-life of Clb2 (A and B) was determined by pulse-chase analysis (7). The half-lives of Ase1 (C and D) and Pds1 (E andF) were determined by promoter turn-off. After transient induction of Pds1 and Ase1 from the GAL1-10promoter for 30 min, 2% glucose and cycloheximide (1 mg/ml) were added (t = 0 min) to repress transcription and translation, respectively. Pds1 and Ase1 proteins were visualized by protein immunoblot analysis. Cdc28 protein was used as an internal loading control. (B, D, and F) DNA content analysis.

Because CDC20 was required for the degradation of Pds1, we hypothesized that overexpression of CDC20 might hasten the degradation of Pds1. To test this hypothesis, we treated exponentially growing cells carrying CDC20 under the control of theGAL1-10 promoter (17) with Gal to induce increased expression of CDC20. Although overexpression ofCDC20 did not affect the amount of PDS1 RNA (18), the bulk of Pds1 protein disappeared within 60 min of Gal addition (Fig. 2A). In contrast, overexpression of CDC20 had little if any effect on the total amounts of Clb2 or Ase1 pro tein (Fig. 2A). The decline in the amount of Pds1 brought about by overexpression of CDC20 was prohibited in cells carrying a temperature-sensitive allele ofCDC23 (cdc23-1, which encodes a component of the APC), suggesting that the decline was dependent on APC-dependent proteolysis. Similar results were obtained with cells arrested in S phase by hydroxyurea, in which Clb2 and Pds1 are stable (1,7). Overexpression of CDC20 led to a decrease in the amount of Pds1 in a CDC23-dependent manner, although somewhat less efficiently than in exponentially growing cells (Fig.2B). In contrast, overexpression of CDC20 did not affect the total amounts of Clb2 or Ase1 protein (Fig. 2B). In cells treated with the microtubule-depolymerizing drug nocodazole, Clb2, Pds1, and Ase1 are stable (1, 3, 7). Overexpression of CDC20 in this arrest induced a decline in the amount of Pds1, Clb2, and, to a lesser degree, Ase1 (Fig. 2C). Whereas Clb2 normally has a half-life of more than 120 min in nocodazole-arrested cells (7), the bulk of Pds1 and Clb2 protein decayed within 30 min, suggesting that rapid protein degradation was responsible for the decreased amounts of Pds1 and Clb2. Indeed, degradation was delayed (in the case of Pds1) or prevented (for Clb2 and Ase1) in cells carrying a cdc23-1mutation (Fig. 2C). These results indicate that overexpression ofCDC20 is sufficient to induce APC-dependent degradation of Pds1 in stages of the cell cycle when Pds1 is normally stable but that it does not affect Clb2 and Ase1 protein. However, this specificity is partially lost in cells arrested with nocodazole.

Figure 2

Degradation of Pds1 induced by overexpression ofCDC20. Wild-type cells (A1015) and cells carrying aGAL-CDC20 fusion (17), either wild type for CDC23 (A1016) or carrying a cdc23-1 allele (A1022), were grown to exponential phase (A) or incubated in the presence of hydroxyurea (10 mg/ml) (B) or nocodazole (15 μg/ml) (C). After 3 hours, Gal was added (t = 0 min), and samples were withdrawn at the indicated times to determine total amounts of Pds1, Clb2, and Ase1 protein by protein immunoblot analysis. Kar2 protein was used as an internal loading control. The experiments were carried out at 23°C because expression of CDC20 from the GAL1-10 promoter was weak at 37°C. However, cdc23-1 mutants are partly defective in Clb2 degradation even at 23°C (29). Because the experiment was carried out at 23°C, cdc23-1 mutants presumably retained some activity that could account for the incomplete inhibition of Pds1 degradation in the nocodazole arrest.

Our findings raised the question whether factors exist that regulate the degradation of Clb2 and Ase1. A FASTA search (19) against all translated open reading frames (ORFs) of the S. cerevisiae genome identified the ORF YGL003C as similar to that ofCDC20 (20). We termed this ORF CDH1for CDC20 homolog 1. Computer searches of protein sequence databases with BLASTP (21) revealed that CDC20and CDH1 are highly conserved among all eukaryotes and thatSchizosaccharomyces pombe and Caenorhabditis elegans also contain at least two members of theCDC20/CDH1 family.

Deletion of CDH1 (22) had little effect on viability and cell cycle progression. Cells grew more slowly, and there was a slight increase in cells with long anaphase spindles in exponentially growing cultures (20% compared with 14% in wild-type cells). We also analyzed the consequences of deleting CDH1for the stability of Pds1, Clb2, and Ase1. Wild-type G1phase cells obtained by centrifugal elutriation (23) lacked Clb2, Pds1, and Ase1 protein because of the lack of transcription and continuous proteolysis (Fig. 3A) (1, 3, 7). In contrast, elutriated cdh1Δ cells lacked Pds1 but contained abnormally large amounts of Clb2 and Ase1 (Fig. 3A). In cdh1Δ cells, similar amounts of Clb2 and Ase1 were present in G1, S (hydroxyurea arrest), and M phases (nocodazole arrest), suggesting that their abundance does not fluctuate substantially during the cell cycle. This may be a consequence of defects in protein degradation rather than deregulation of transcription. Even overexpression of CLB2 orASE1 from the GAL1-10 promoter does not lead to accumulation of the protein in G1 phase cells, because of rapid proteolysis (3, 7). Thus, one interpretation of this result is that cells lacking CDH1 had degraded Pds1 during exit from M phase but that they had failed to degrade Clb2 and Ase1.

Figure 3

Presence of Clb2 and Ase1 during G1phase in cdh1Δ mutants. Wild-type cells (A1015) orcdh1::HIS3 (cdh1Δ) mutants (A1120) were elutriated to obtain small G1 phase cells or arrested with nocodazole (Noc.) or hydroxyurea (HU) (23). (A) Clb2, Pds1, and Ase1. Cdc28 was used as an internal loading control. Cyc., cycling. (B) DNA content analysis.

Although the amount of Clb2 did not fluctuate much during the cell cycle in cdh1Δ cells, cdh1Δ mutants were viable, suggesting that degradation of mitotic cyclins might not be absolutely required for exit from M phase. This observation is consistent with previous findings. Moderate amounts of nondestructible Clb2 do not affect exit from M phase (7). Under conditions at which proteolysis of mitotic cyclins is impaired, mitotic kinase inhibition by the kinase inhibitor Sic1 (24) might be sufficient to trigger exit from M phase. Thus, inactivation of both Sic1 and proteolysis of mitotic cyclins (by deletion ofCDH1) should be lethal. Indeed, we found such cells to be inviable. cdh1Δ, sic1Δ double-mutant spores germinated but arrested as large budded cells after one to three divisions.

If CDH1 is important for Clb2 and Ase1 degradation, overexpression of CDH1 might induce ectopic degradation of Clb2 and Ase1. Exponentially growing cells carrying aGAL-CDH1 fusion (22) were treated with Gal to induce the expression of CDH1. Within 60 min of Gal addition, the bulk of Clb2 and Ase1 disappeared (Fig.4A). This decline was absent in acdc23-1 mutant, suggesting that it was a result of APC-dependent proteolysis. In contrast, overexpression ofCDH1 had only minor effects on the total amount of Pds1 (Fig. 4A). Similar results were obtained with cells arrested in S phase with hydroxyurea (Fig. 4B). In nocodazole-arrested cells, overexpression of CDH1 induced degradation of Clb2, Ase1, and, to some extent, Pds1 (Fig. 4C). It is unlikely that mechanisms other than APC-dependent proteolysis are responsible for the loss of Clb2 and Ase1. The half-life of Clb2 is normally more than 2 hours in hydroxyurea- or nocodazole-arrested cells (7). Furthermore, loss of Clb2 and Ase1 brought about by overexpression ofCDH1 was dependent on a functional APC. We conclude that overexpression of CDH1 is sufficient to induce the degradation of Clb2 and Ase1 in exponentially growing and hydroxyurea-arrested cells but not to induce the degradation of Pds1. The loss of specificity in the nocodazole arrest suggests thatCDC20 and CDH1 are capable, at least when overexpressed, of partially substituting for each other in this arrest point. The reasons for the loss in specificity are at present unclear.

Figure 4

Degradation of Clb2 and Ase1 in cells overexpressingCDH1. Wild-type cells (A1015) and cells carrying aGAL-CDH1 fusion (22), either wild type forCDC23 (A1110) or carrying a cdc23-1 allele (A1112), were grown to exponential phase (A) or incubated in the presence of hydroxyurea (B) or nocodazole (C). After 3 hours, Gal was added (t = 0 min), and samples were withdrawn at the indicated times to determine the total amount of Pds1, Clb2, and Ase1. Kar2 was used as an internal loading control.

Overexpression of CDH1 was lethal and caused a specific cell cycle arrest. This arrest was similar to that caused by lack of the mitotic cyclins CLB1, CLB2, CLB3, and CLB4(25, 26). Cells replicated their DNA but arrested with an elongated bud, lacking a bipolar mitotic spindle (Fig.5). This phenotype is consistent with our findings that overexpression of CDH1 induces degradation of mitotic cyclins.

Figure 5

Terminal phenotype of cells overexpressing CDH1. The mitotic spindle and nuclear morphology of cells overexpressing CDH1 (A1110) were analyzed 2 hours after Gal addition by indirect immunofluorescence with antibodies to tubulin (A) or by 4′,6′-diamidino-2-phenylindole (DAPI) staining (B). (C) DNA content 4 hours after Gal addition.

We have identified a family of highly conserved WD40-repeat proteins as limiting activators of APC-dependent proteolysis. Cdc20 and Cdh1 were not general activators of APC-dependent proteolysis, but instead they appeared to be substrate specific. The WD40-repeat protein Cdc4 also serves as a specificity factor in the degradation of substrates of the Skp1/Cdc53-dependent proteolysis machinery (27). Specificity factors may facilitate the differential regulation of various substrates ubiquitinated by the same ubiquitination machinery. Cdc20 and Cdh1 may ensure that different substrates of the APC are degraded at the right time during M phase. Cdc20 may target APC substrates whose degradation is required for the metaphase-anaphase transition (such as Pds1) for degradation, whereas Cdh1 may trigger destruction of substrates whose degradation is important for exit from M phase (such as Clb2 and Ase1). Cdc20 homologs in other species may also be substrate-specific activators of APC-dependent proteolysis.


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