Proteolysis Of Cell Cycle Regulators Research Articles

A Phosphosignaling Adaptor Primes the AAA+ Protease ClpXP to Drive Cell Cycle-Regulated Proteolysis

The response regulator CpdR couples phosphorylation events in Caulobacter crescentus with the AAA+ protease ClpXP to provide punctuated degradation of crucial substrates involved in cell cycle regulation. CpdR functions like an adaptor to alter substrate choice by ClpXP; however, it remains unclear how CpdR influences its multiple targets. Here we show that, unlike canonical ClpXP adaptors, CpdR alone does not strongly bind its substrate. Instead, CpdR binds the N-terminal domain of ClpX and prepares (primes) the unfoldase for substrate engagement. This priming creates a recruitment interface that docks multiple substrates and additional adaptor components. We show that adaptor-dependent priming of ClpX avoids concentration-dependent inhibition that limits traditional scaffolding adaptors. Phosphosignaling disrupts the adaptor-protease interaction, and mutations in CpdR that impact ClpX binding tune adaptor activity and biological function. Together, these results reveal how a single adaptor can command global changes in proteome composition through priming of a protease.

Zoledronic acid induces S-phase arrest via a DNA damage response in normal human oral keratinocytes

ObjectiveThis study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct. DesignChanges in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10μM ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (γ)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2. ResultsZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27KIP1, Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27KIP1. Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of γ-H2A.X was also seen. ConclusionsThese results indicate that a 10-μM ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiquitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover.

Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.

Spindle Checkpoint Insufficiency and Unscheduled APC-Dependent Proteolysis in Acute Myeloid Leukemia.

Genetic instability including aneuploidy is frequent in most cancers. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism responsible for accurate chromosome segregation. Unattached chromosomes or lack of spindle tension are sensed by the SAC. The activated SAC inhibits the ubiquitin-ligase anaphase-promoting complex (APC), which prevents the proteolysis of cell cycle regulators in order to delay progression through mitosis and allow cells to recover from defective mitotic spindle attachment. Spindle checkpoint malfunction proved to favor the generation of aneuploidy. In our recent work we investigated the roles of essential SAC proteins in acute myeloid leukemia (AML). We found the SAC-protein Bub1 to be posttranscriptionally downregulated in all investigated AML cell lines. As a consequence, after exposure to the microtubule disrupting agent nocodazole we observed a defective mitotic delay mechanism in comparison to SAC-competent cell lines and increased apoptosis consistent with the effects of Bub1 downregulation by RNA interference. At the molecular level we found a dramatic decline in mitotic regulator levels such as cyclin B1 and securin despite lasting spindle disruption. Additional data showed that the levels of these regulator proteins can be efficiently restored by exposure to the proteasome inhibitor MG-132 indicating that APC-dependent proteolysis is directly involved in SAC insufficiency. Thus, continuous activation of the APC triggers degradation of essential regulator proteins even in leukemic cells faced to mitotic stress such as complete spindle disruption. Such defects can lead to establishment of aneuploidy in vivo. Our findings emphasize a role of SAC insufficiency and unscheduled proteolysis in rise and progression of AML with complex karyotype.

The Balance between Cell Division and Endoreplication Depends on E2FC-DPB, Transcription Factors Regulated by the Ubiquitin-SCFSKP2A Pathway inArabidopsis

The balance between cell proliferation, cell cycle arrest, and differentiation needed to maintain the organogenetic program depends on the coordination of gene expression, posttranslational modification, and specific proteolysis of cell cycle regulators. The G1/S and G2/M transitions are critical checkpoints controlled, in part, by cyclin-dependent kinases in the retinoblastoma (RBR)/E2F/DP pathway. Arabidopsis thaliana DPB is regulated by phosphorylation and targeted to proteasome-mediated proteolysis by the SCF(SKP2A) complex. In addition, DPB interacts in vivo with E2FC, because ectopic coexpression of E2FC and DPB produces severe developmental defects. To understand E2FC/DPB heterodimer function, we analyzed the effect of reducing E2FC mRNA levels with RNA interference. The e2fc-R plants developed organs with more but smaller cells and showed increased cell cycle marker gene expression and increased proliferative activity in developing leaves, meristems, and pericycle cells. This last feature produces plants with more lateral roots, consistent with an E2FC role in restricting lateral root initiation. The e2fc-R plants also show marked reductions in ploidy levels of mature leaves. These results indicate that the transition from cell division to the endocycle is sensitive to different pathways, E2FC/DPB being one of them. Our results show that E2FC/DPB is a key factor in controlling the balance between cell proliferation and the switch to the endocycle program.

Downregulation of Bub1 and a Defective Spindle Checkpoint in Acute Myeloid Leukemia.

Chromosomal aberrations are common in leukemia. The spindle assembly checkpoint (SAC) is a surveillance mechanism responsible to ensure proper chromosome segregation. Unattached chromosomes to the mitotic spindle or lack of spindle tension are sensed by the SAC leading to metaphase arrest. The activated SAC inhibits the ordered proteolysis of regulatory proteins and thereby prevents mitotic progression providing time for the repair of spindle defects. Ubiquitin-dependent proteolysis of cell cycle regulators is a central mechanism of cell cycle control. The anaphase-promoting complex (APC) is an essential ubiquitin-ligase responsible for mitotic progression. The activated SAC inhibits the APC preventing degradation of cyclin B1 and the anaphase inhibitor securin and thereby delaying chromosome separation. We found this mitotic delay mechanism to be defective in acute myeloid leukemia (AML). Leukemia cells that were exposed to the microtubule disrupting agent nocodazole were not capable of preventing anaphase onset. This is indicated by a low mitotic index and degradation of the APC targets securin and cyclin B1 in nocodazole treated Kasumi-1 cells compared to the SAC competent burkitt lymphoma cell line DG-75. Thus, unattached chromosomes were unable to induce prolonged metaphase arrest in these cells. They undergo apoptosis instead due to the complete absence of the mitotic spindle. Similar results with defective mitotic arrest and enhanced apoptosis were obtained for several other leukemia cell lines. In vivo SAC malfunction in AML can favor the generation of aneuploidy, consistent with the numerical aberrations observed in these cells. In order to identify the underlying defect we screened for mutations or altered expression of checkpoint proteins and identified the SAC component Bub1 to be posttranscriptionally downregulated in all tested AML cell lines. Our studies suggest that impaired SAC function is an important mechanism to create genetic instability and thus may contribute to rise and progression of AML.

Anaphase-promoting complex-dependent proteolysis of cell cycle regulators and genomic instability of cancer cells

Genomic instability can be found in most cancer cells. Cell proliferation is under tight control to ensure accurate DNA replication and chromosome segregation. Cyclin-dependent kinases (Cdks) and their activating subunits, the cyclins, are the driving forces of the cell division cycle. Regulation of cyclin oscillation by ubiquitin-dependent proteolysis thereby has a central role in cell cycle regulation. The anaphase-promoting complex (APC) is a specific ubiquitin ligase and is essential for chromosome segregation, exit from mitosis and a stable subsequent G1 phase allowing cell differentiation or accurate DNA replication in the following S phase. The APC is activated by the regulatory subunits Cdc20 (APC(Cdc20)) and Cdh1 (APC(Cdh1)) to target securin, mitotic cyclins and other cell cycle regulatory proteins for proteasomal degradation. This review is focused on the role of APC-dependent proteolysis in cell cycle regulation and how its deregulation may lead to genomic instability of cancer cells.

Speriolin Is a Novel Spermatogenic Cell-specific Centrosomal Protein Associated with the Seventh WD Motif of Cdc20

The fundamental mechanisms of mitosis are conserved throughout evolution in eukaryotes, including ubiquitin-mediated proteolysis of cell cycle regulators by the anaphase-promoting complex/cyclosome. The spindle checkpoint protein Cdc20 activates the anaphase-promoting complex/cyclosome in a substrate-specific manner. It is present in the cytoplasm and concentrated in the centrosomes throughout the cell cycle, accumulates at the kinetochores in metaphase, and is no longer detected following anaphase. However, it is unknown whether Cdc20 has the same activities and distribution during meiosis in male germ cells. We found that in mice, Cdc20 accumulates in the cytoplasm of pachytene spermatocytes during meiosis I, is distributed throughout spermatocytes undergoing meiotic division, and is present in the cytoplasm of postmeiotic spermatids. Several proteins bind to and regulate the function of Cdc20 during mitosis. We identified speriolin and determined that it is a novel spermatogenic cell-specific Cdc20-binding protein, is present in the cytoplasm, and is concentrated at the centrosomes of spermatocytes and spermatids and that a leucine zipper domain is required to target speriolin to the centrosome. The seven tandem WD motifs of Cdc20 probably fold into a seven-blade beta-propeller structure, and we determined that they are required for speriolin binding and for localization of Cdc20 to the centrosomes and nucleus, suggesting that speriolin might regulate or stabilize the folding of Cdc20 during meiosis in spermatogenic cells.

Defining the Pathway to Bacterial Polarity

Caulobacter crescentus is a flagellated bacterium that undergoes a transition from a motile swarmer cell to a stalked cell during cell division, which produces a new swarmer cell and the stalked cell. After division, the stalked cell can initiate DNA replication immediately, but the new swarmer cell must undergo the transition to a stalked cell in order to divide. During the swarmer-to-stalked cell transition (which corresponds to G 1 -to-S phase transition) and just before cell separation (predivisional cells), the response regulator and transcriptional regulator CtrA, which represses chromosome replication among other functions, must be localized to the stalked cell pole and degraded. Ryan et al. investigated the mechanism whereby CtrA polarity is established. Expression of various mutants fused to the green fluorescent protein variant (YFP) followed by microscopic analysis showed that neither phosphorylation nor proteolysis was required for proper polar localization of CtrA. The signal for polarization was located in the receiver domain. The authors also analyzed CtrA localization in cells carrying mutations in kinases (DivJ and PleC) responsible for regulating the phosphorylation status of response regulator DivK, which is necessary for CtrA proteolysis. Although DivK is localized to the stalked pole, a site of CtrA accumulation, it also localizes to the swarmer cell pole, a site that does not accumulate CtrA. Analysis of the divK , divJ , and pleC mutant strains indicated that polar localization of DivK was not required for stalked pole localization of CtrA--some cells maintained polar accumulation of CtrA, but aberrations in localization or timing of the polarity were also observed--but that these three components participate in a pathway that allows the phosphorylation state of DivK to dictate the activity of DivK and the asymmetric proteolysis of CtrA. Holtzendorff et al. have identified a global cell cycle regulator, GcrA, in Caulobacter crescentus , which forms an oscillatory circuit with the CtrA. GcrA depletion is lethal. Phosphorylated CtrA repressed the gcrA gene, and the abundance of GcrA was in inverse proportion to that of CtrA, such that in stalked cells, in which CtrA is proteolyzed, GcrA accumulated. GcrA in turn stimulated ctrA expression and was a direct transcriptional regulator that bound to the ctrA promoter. Microarray experiments indicated that GcrA regulated 125 genes, including those encoding PleC, DivK, and CtrA. Thus, GcrA and CtrA directly regulate each other so that their concentrations oscillate out of phase temporally and spatially during the cell cycle to drive cell cycle progression and expression of polar differentiation factors. K. R. Ryan, S. Huntwork, L. Shapiro, Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis. Proc. Natl. Acad. Sci. U.S.A. 101 , 7415-7420 (2004). [Abstract] [Full Text] J. Holtzendorff, D. Hung, P. Brende, A. Reisenauer, P. H. Viollier, H. H. McAdams, L. Shapiro, Oscillating global regulators control the genetic circuit driving a bacterial cell cycle. Science 304 , 983-987 (2004). [Abstract] [Full Text]

Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis.

The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Analysis of the requirements for CtrA polar localization and CtrA proteolysis revealed that both processes require a motif within amino acids 1-56 of the CtrA receiver domain, and neither process requires CtrA phosphorylation. These results strongly suggest that CtrA polar localization is coupled to its cell cycle-regulated proteolysis. The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Mutations in the divJ and pleC histidine kinases perturb the characteristic asymmetry of CtrA localization and proteolysis in the predivisional cell. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address.

Alterations of anaphase-promoting complex genes in human colon cancer cells.

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.

D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p.

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation. We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation.

Chemical inhibitors: a tool for plant cell cycle studies

Synchrony provides a large number of cells at defined points of the cell cycle. Highly synchronised cells are powerful and effective tools for molecular analyses and for studying the biochemical events of the cell cycle in plants. Usually, plant cell suspensions can be synchronised by chemical agents, which arrest the cell cycle by acting on the driving forces of the cell cycle engine such as cyclin-dependent kinase activity, enzymes involved in DNA synthesis or proteolysis of cell cycle regulators or by acting on the cell cycle apparatus (mitotic spindle). The specificity, reversibility and efficiency of each type of cell cycle inhibitor are described and related to their mode of action.

Proteolysis of the McpA chemoreceptor does not require the Caulobacter major chemotaxis operon.

The degradation of the McpA chemoreceptor in Caulobacter crescentus accompanies the swarmer cell to the stalked-cell differentiation event. To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the mcpA gene and in the mcpA chemotaxis operon. Internal deletions of the mcpA gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis. The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.

Feedback control of a master bacterial cell-cycle regulator.

The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.

The anaphase-promoting complex: new subunits and regulators.

Ubiquitin-mediated proteolysis of cell cycle regulators is a crucial process during the cell cycle. The anaphase-promoting complex (APC) is a large, multiprotein complex whose E3-ubiquitin ligase activity is required for the ubiquitination of mitotic cyclins and other regulatory proteins that are targeted for destruction during cell division. The recent identification of new APC subunits and regulatory proteins has begun to reveal some of the intricate mechanisms that govern APC regulation. One mechanism is the use of specificity factors to impose temporal control over substrate degradation. A second mechanism is the APC-mediated proteolysis of specific APC regulators. Finally, components of both the APC and the SCF E3 ubiquitin-ligase complex contain several conserved sequence motifs, including WD-40 repeats and cullin homology domains, which suggest that both complexes may use a similar mechanism for substrate ubiquitination.

The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1.

DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.

The yeast nuclear import receptor is required for mitosis.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.

The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae.

The Drosophila cell cycle gene fizzy (fzy) is required for normal execution of the metaphase-anaphase transition. We have cloned fzy, and confirmed this by P-element mediated germline transformation rescue. Sequence analysis predicts that fzy encodes a protein of 526 amino acids, the carboxy half of which has significant homology to the Saccharomyces cerevisiae cell cycle gene CDC20. A monoclonal antibody against fzy detects a single protein of the expected size, 59 kD, in embryonic extracts. In early embryos fzy is expressed in all proliferating tissues; in late embryos fzy expression declines in a tissue-specific manner correlated with cessation of cell division. During interphase fzy protein is present in the cytoplasm; while in mitosis fzy becomes ubiquitously distributed throughout the cell except for the area occupied by the chromosomes. The metaphase arrest phenotype caused by fzy mutations is associated with failure to degrade both mitotic cyclins A and B, and an enrichment of spindle microtubules at the expense of astral microtubules. Our data suggest that fzy function is required for normal cell cycle-regulated proteolysis that is necessary for successful progress through mitosis.

Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle

It is thought that DNA replication and mitosis in yeast are triggered by oscillations in the level of G1-specific (CLN1 and CLN2) and G2-specific (CLB1-CLB4) cyclins, which determine the substrate specificity of the CDC28 protein kinase. It is not understood how the time and order of appearance of different cyclin types are determined. We show here that CLB2 proteolysis, which is important for transition from mitosis to G1, is not confined to a narrow window at the end of mitosis as previously thought but continues until reactivation of CDC28 by CLN cyclins toward the end of the subsequent G1 period. Thus, cell cycle-regulated proteolysis prevents accumulation of G2-specific CLB cyclins during G1 and thereby ensures that the CLN-associated forms of the CDC28 kinase are activated without interference from CLB cylins. Accumulation of CLN cyclins leads to inactivation of CLB cyclin proteolysis, which is a precondition for subsequent activation of G2-specific B-type cyclins.