Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).     

The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae.

Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the passage of cells through anaphase. Destruction of the anaphase inhibitor Pds1p is necessary for separation of sister chromatids, whereas destruction of the mitotic cyclin Clb2p is important for disassembly of the mitotic spindle, cytokinesis and re-replication of the genome. Pds1p proteolysis precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their genome before they have separated sister chromatids at the previous mitosis. What triggers Pds1p proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the APC, these two proteolytic events differ in their dependence on other cofactors. Pds1p proteolysis depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another, related WD protein called Hct1/Cdh1p. On the other hand, destruction of Clb2p, but not that of Pds1p, depends on the Polo-like kinase, Cdc5p. Cdc20p is essential for separation of sister chromatids, whereas Cdc5p is not. We show that both Cdc5p and Cdc20p are unstable proteins whose proteolysis is regulated by the APC. Both proteins accumulate during late G2/M phase and disappear at a late stage of anaphase. Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis.     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.     

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.     

Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1.     

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).     

Cyclin destruction in mitosis: a crucial task of Cdc20

Proteolytic destruction of cyclins is a fundamental process for cell division. At the end of mitosis, degradation of mitotic cyclins results in the inactivation of cyclin-dependent kinases. Cyclin proteolysis is triggered by the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit complex which contains ubiquitin ligase activity. Recent data in yeast demonstrated that a partial degradation of the mitotic cyclin Clb2, mediated by APC/C and its activator protein Cdc20, is essential and sufficient for the mitotic exit. Remarkably, a complete inactivation of cyclin-dependent kinases seems to be not essential. This review discusses recent novel insights into cyclin destruction and its implications for the mitotic exit.     

The yeast mitotic cyclin Clb2 cannot substitute for S phase cyclins in replication origin firing

Cyclin-dependent kinases (CDKs) drive the cell cycle, central to which is the accurate control of chromosome replication. In Saccharomyces cerevisiae, six closely related B-type cyclins (Clb1–6) drive the events of S phase and mitosis. Either Clb5 or Clb6 can activate early-firing replication origins, whereas only Clb5 can activate late origins. Clb1–4 are expressed later in the cell cycle. Whether Clb cyclins differ only in timing of expression, or else impart different kinase specificities is under ongoing investigation. This study shows that the expression of Clb2 during S phase in cells lacking Clb5 failed to rescue late origin activation. Early expression of Clb2 in cells lacking both Clb5 and Clb6 did not activate early origins on schedule to restore the correct S phase entry time. Therefore, Clb2 cannot drive timely activation of either early or late replication origins, demonstrating that Clb2-directed CDK has a specificity distinct from that driven by Clb5 and Clb6.     

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.     

S-phase cyclin-dependent kinases promote sister chromatid cohesion in budding yeast

Genome stability depends on faithful chromosome segregation, which relies on maintenance of chromatid cohesion during S phase. In eukaryotes, Pds1/securin is the only known inhibitor that can prevent loss of cohesion. However, pds1Δ yeast cells and securin-null mice are viable. We sought to identify redundant mechanisms that promote cohesion within S phase in the absence of Pds1 and found that cells lacking the S-phase cyclins Clb5 and Clb6 have a cohesion defect under conditions of replication stress. Similar to the phenotype of pds1Δ cells, loss of cohesion in cells lacking Clb5 and Clb6 is dependent on Esp1. However, Pds1 phosphorylation by Cdk-cyclin is not required for cohesion. Moreover, cells lacking Clb5, Clb6, and Pds1 are inviable and lose cohesion during an unperturbed S phase, indicating that Pds1 and specific B-type cyclins promote cohesion independently of one another. Consistent with this, we find that Mcd1/Scc1 is less abundant on chromosomes in cells lacking Clb5 and Clb6 during replication stress. However, clb5Δ clb6Δ cells do accumulate Mcd1/Scc1 at centromeres upon mitotic arrest, suggesting that the cyclin-dependent mechanism is S phase specific. These data indicate that Clb5 and Clb6 promote cohesion which is then protected by Pds1 and that both mechanisms are required during replication stress.     

Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex.

Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.     

Control of Mitotic Events by Nap1 and the Gin4 Kinase

Little is known about the pathways used by cyclins and cyclin-dependent kinases to induce the events of the cell cycle. In budding yeast, a protein called Nap1 binds to the mitotic cyclin Clb2, and Nap1 is required for the ability of Clb2 to induce specific mitotic events, but the role played by Nap1 is unclear. We have used genetic and biochemical approaches to identify additional proteins that function with Nap1 in the control of mitotic events. These approaches have both identified a protein kinase called Gin4 that is required for the ability of Clb2 and Nap1 to promote the switch from polar to isotropic bud growth that normally occurs during mitosis. Gin4 is also required for the ability of Clb2 and Nap1 to promote normal progression through mitosis. The Gin4 protein becomes phosphorylated as cells enter mitosis, resulting in the activation of Gin4 kinase activity, and the phosphorylation of Gin4 is dependent upon Nap1 and Clb2 in vivo. Affinity chromatography experiments demonstrate that Gin4 binds tightly to Nap1, indicating that the functions of these two proteins are closely tied within the cell. These results demonstrate that the activation of Gin4 is under the control of Clb2 and Nap1, and they provide an important step towards elucidating the molecular pathways that link cyclin-dependent kinases to the events they control.     

The role of Cdh1p in maintaining genomic stability in budding yeast

Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G(1) transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1delta and mad2delta single mutants, the mad2delta cdh1delta double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2delta cdh1delta and pds1delta cdh1dDelta strains were rescued by overexpressing Swe1p, a G(2)/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1delta mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Spatially distinct functions of Clb2 in the DNA damage response

In budding yeast four mitotic cyclins (Clb1–4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G₂/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.