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Genetics, Vol. 178, 589-591, January 2008, Copyright © 2008
doi:10.1534/genetics.107.083642

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Differential Regulation of Anaphase Promoting Complex/Cyclosome Substrates by the Spindle Assembly Checkpoint in Saccharomyces cerevisiae

Brice E. Keyes*, Christopher M. Yellman{dagger} and Daniel J. Burke*,1

* Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, Charlottesville, Virginia 22908 and {dagger} Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511

1 Corresponding author: Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, 1300 Jefferson Park Ave., Box 800733, Charlottesville, VA 22908.
E-mail: dburke{at}virginia.edu

Manuscript received October 22, 2007. Accepted for publication November 7, 2007.


   ABSTRACT
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 ABSTRACT
LITERATURE CITED
 
The anaphase promoting complex (APC) targets proteins for degradation to promote progression through the cell cycle. Here we show that Clb5, an APCCdc20 substrate, is degraded when the spindle checkpoint is active, while other APCCdc20 substrates are stabilized, suggesting that APCCdc20 inhibition by the spindle checkpoint is substrate specific.

ORDERED temporal degradation of proteins is an important regulatory mechanism that controls progression through the eukaryotic cell cycle (REED 2006). The anaphase promoting complex/cyclosome (APC/C) is the E3 subunit of an ubiquitin-conjugating enzyme composed of at least thirteen subunits that targets proteins for proteolysis during the cell cycle (PETERS 2006). APC/C function is critical for progression through mitosis where it degrades Pds1 (securin in higher eukaryotic cells) and other substrates to promote anaphase and the exit from mitosis (PINES 2006). APC/C cofactors Cdc20 and Cdh1 are important for conferring substrate specificity during different stages in the cell cycle (PETERS 2006). It is unclear, however, how the APC/C chooses substrates for ubiquitylation and the specific role of each subunit in this process (ACQUAVIVA and PINES 2006). The spindle assembly checkpoint (SAC) ensures the formation of a bipolar spindle and proper attachment of kinetochores (LEW and BURKE 2003). The SAC, when activated, opposes the activity of the APC/C by inactivating Cdc20 (HWANG et al. 1998). The SAC prevents the onset of anaphase by stabilizing Pds1 by inhibiting APCCdc20 activity, allowing time for establishment of proper attachment, orientation, and alignment of chromosomes (LEW and BURKE 2003).

The B-type cyclin Clb5 and Cdc28 protein kinases are responsible for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae (EPSTEIN and CROSS 1992). Clb5 peaks in late G1 and is degraded in mitosis by APCCdc20 soon after metaphase (SCHWOB and NASMYTH 1993). To study Clb5 degradation during the cell cycle, we placed CDC20 under the control of the GAL1 promoter. Cdc20 expression from the GAL1 promoter was increased 32-fold as compared to the endogenous promoter (data not shown). Clb5 and Pds1 were expressed and degraded after release from {alpha}-factor arrest (Figure 1A). Repressing Cdc20 stabilized Clb5 and Pds1, confirming that both are substrates of APCCdc20 (Figure 1C), as previously described (VISINTIN et al. 1997; SHIRAYAMA et al. 1999). The slight turnover in Clb5 and Pds1 is likely due to APCCdh1 activity late in the cell cycle. Cells were also released into media containing 15 µg/ml of nocodazole to activate the SAC (Figure 1, B and D). Pds1 was stabilized by SAC activation (compare Figure 1, A and B), since nocodazole treatment inhibits both APCCdc20 and APCCdh1. Interestingly, Clb5 turned over when the SAC was activated with similar kinetics as in untreated cells (Figure 1B). Cells lacking Cdc20 and treated with nocodazole stabilized both Clb5 and Pds1 (Figure 1D). These results show that Clb5 is a substrate of APCCdc20, but Clb5 turnover is not inhibited by activating the SAC.


Figure 1
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  		FIGURE 1.— Clb5, an APCCdc20 substrate, is not regulated by the SAC. Protein expression was followed by Western blot with cells containing PDS1-3HA and CLB5-TAP (A and B) or CLB5-9MYC tagged (C and D). Cells were grown in media containing 2% raffinose and galactose (Raff/Gal) to induce the expression of Cdc20 (ADE2-pGAL1-CDC20-13MYC). Cells grown to midlog at 30° in Raff/Gal media were arrested in G1 with {alpha}-factor. Cells were then released into media with Raff/Gal (A), Raff/Gal with nocodazole (B), glucose (C), or glucose with nocodazole (D). {alpha}-Factor was added back to the media after cells had budded to restrict the analysis to a single cell cycle. Samples were collected for Western blot analysis every 25 min and Western blots were performed as previously described in (YELLMAN and BURKE 2006). Below each Western is the accompanying flow cytometry following the cell cycle progression by DNA content for each experiment. Percentage of undivided nuclei was scored by flourescence microscopy from cells stained for flow cytometry. Flow cytometry was performed as previously described by (YELLMAN and BURKE 2004).

 
We followed DNA content by flow cytometry and nuclear morphology as independent measures of cell cycle progression. Cells released into galactose (Figure 1A, bottom) progressed through G2/M as shown by flow cytometry, divided their nuclei and began to accumulate in G1 of the next cell cycle in the late stages of the time course. Cells released into galactose with nocodazole, glucose, and glucose with nocodazole arrested with 2N DNA content with undivided nuclei, suggesting a preanaphase arrest.

One possibility was that Clb5 turnover in nocodazole-treated cells was somehow due to the artificial expression of Cdc20. We therefore examined Clb5 proteolysis in a strain with endogenously expressed Cdc20. Cells were arrested in medium containing {alpha}-factor and released into medium with and without nocodazole. Cells without nocodazole progressed through the cell cycle and degraded both Clb5 and Pds1, although Clb5 was degraded more slowly than in cells with excess Cdc20 (Figure 2A). Cells became large budded and exited mitosis (Figure 2C). Cells released into nocodazole-containing media stabilized Pds1, but Clb5 turned over (Figure 2B). Large-budded cells accumulated, verifying SAC activation (Figure 2D). Quantification of the Western blots is shown (Figure 2E). These results support the conclusion that APCCdc20-dependent Clb5 and Pds1 proteolysis are differentially regulated by the SAC.


Figure 2
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  		FIGURE 2.— Differential regulation of APCCdc20 substrates. MATa bar1::loxP his3 leu2-3,112 trp1 ura3 CLB5-9MYC-TRP1 PDS1-3HA-URA3 cells were grown to midlog and arrested in G1 with {alpha}-factor. Cells were released into media without nocodazole (A) or with nocodazole (B). Samples were collected for Western blot analysis every 25 min. {alpha}-Factor was added back to the media after cells had budded to restrict the analysis to a single cell cycle. Budding morphology is presented as the percentage of large-budded cells from time of release from {alpha}-factor (C and D). Quantification of Western blots (from A and B) is shown in E. The intensity ratio was calculated by determining the intensity of the Clb5 or Pds1 band over intensity of the corresponding band in the loading control (tubulin). Quantification was done using ImageQuant.

 
Our data show that APCCdc20 actively degrades Clb5 but not Pds1 during mitotic arrest induced by the SAC, suggesting that APCCdc20 inhibition is substrate specific. In higher eukaryotes, cyclin A turnover by APCCdc20 occurs early in mitosis, while other APCCdc20 substrates, such as securin, are present in the cell but not degraded (GELEY et al. 2001). Cyclin A turnover is also independent of SAC activation (GELEY et al. 2001). One interpretation is that cyclin A is a better substrate than securin for the APCCdc20. Recent work demonstrated that processive ubiquitylation by the APC/C causes rapid turnover of substrates in vitro. Interestingly, cyclin A is ubiquitylated in a distributive manner and is a relatively poor substrate for APCCdc20 (RAPE et al. 2006). The authors propose that cyclin A possesses additional motifs that allow APC/C recognition in the presence of SAC activation, which also may be the case for the B-type cyclin Clb5. The undetermined roles of individual APC/C subunits may also play some role in substrate recognition.

Clb5 turnover was more rapid with high levels of Cdc20 (compare Figures 1A and 2A), supporting the hypothesis that Clb5 is a good substrate of APCCdc20. Interestingly, the kinetics of Pds1 turnover were unaffected by the levels of Cdc20. The preferred proteolysis of Clb5 probably helps to restrict DNA replication to once per cell cycle and to prevent re-replication in SAC-arrested cells (JACOBSON et al. 2000). In addition, a small pool of active Cdc20 may also be important for recovery from a SAC arrest. Others have reported that excess Cdc20 abrogates the SAC (SCHOTT and HOYT 1998; PAN and CHEN 2004). However, we found that Pds1 was mostly stabilized when the SAC was activated in the presence of excess Cdc20 (Figure 1C), suggesting that APCCdc20 regulation is more complicated than stoichiometric inhibition by SAC proteins. We did observe slight turnover in Pds1 at late time points, indicating perhaps that the SAC may be unable to inhibit the onset of anaphase at such high levels of Cdc20. This study suggests a more complex model of APC/C regulation where inhibiting APCCdc20 by the SAC is substrate specific. Further studies are needed to determine the mechanism by which the SAC inhibits the APC/C.


   LITERATURE CITED
TOP
 ABSTRACT
 LITERATURE CITED
 

ACQUAVIVA, C., and J. PINES, 2006 The anaphase-promoting complex/cyclosome: APC/C. J. Cell Sci. 119: 2401–2404.[Free Full Text]

EPSTEIN, C. B., and F. R. CROSS, 1992 CLB5: a novel B cyclin from budding yeast with a role in S phase. Genes Dev. 6: 1695–1706.[Abstract/Free Full Text]

GELEY, S., E. KRAMER, C. GIEFFERS, J. GANNON, J. M. PETERS et al., 2001 Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153: 137–148.[Abstract/Free Full Text]

HWANG, L. H., L. F. LAU, D. L. SMITH, C. A. MISTROT, K. G. HARDWICK et al., 1998 Budding yeast Cdc20: a target of the spindle checkpoint. Science 279: 1041–1044.[Abstract/Free Full Text]

JACOBSON, M. D., S. GRAY, M. YUSTE-ROJAS and F. R. CROSS, 2000 Testing cyclin specificity in the exit from mitosis. Mol. Cell. Biol. 20: 4483–4493.[Abstract/Free Full Text]

LEW, D. J., and D. J. BURKE, 2003 The spindle assembly and spindle position checkpoints. Annu. Rev. Genet. 37: 251–282.[CrossRef][Medline]

PAN, J., and R. H. CHEN, 2004 Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae. Genes Dev. 18: 1439–1451.[Abstract/Free Full Text]

PETERS, J. M., 2006 The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell Biol. 7: 644–656.[CrossRef][Medline]

PINES, J., 2006 Mitosis: a matter of getting rid of the right protein at the right time. Trends Cell Biol. 16: 55–63.[CrossRef][Medline]

RAPE, M., S. K. REDDY and M. W. KIRSCHNER, 2006 The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124: 89–103.[CrossRef][Medline]

REED, S. I., 2006 The ubiquitin-proteasome pathway in cell cycle control. Results Probl. Cell Differ. 42: 147–181.[Medline]

SCHOTT, E. J., and M. A. HOYT, 1998 Dominant alleles of Saccharomyces cerevisiae CDC20 reveal its role in promoting anaphase. Genetics 148: 599–610.[Abstract/Free Full Text]

SCHWOB, E., and K. NASMYTH, 1993 CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev. 7: 1160–1175.[Abstract/Free Full Text]

SHIRAYAMA, M., A. TOTH, M. GALOVA and K. NASMYTH, 1999 APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5. Nature 402: 203–207.[CrossRef][Medline]

VISINTIN, R., S. PRINZ and A. AMON, 1997 CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 278: 460–463.[Abstract/Free Full Text]

YELLMAN, C. M., and D. J. BURKE, 2004 Assaying the spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Methods Mol. Biol. 280: 275–290.[Medline]

YELLMAN, C. M., and D. J. BURKE, 2006 The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae. Mol. Biol. Cell 17: 658–666.[Abstract/Free Full Text]

Communicating editor: N. HOLLINGSWORTH





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