scholarly journals Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

2015 ◽  
Vol 209 (4) ◽  
pp. 519-527 ◽  
Author(s):  
Yang Yang ◽  
Dai Tsuchiya ◽  
Soni Lacefield
Keyword(s):  
Chromosome Segregation ◽  
Unknown Function ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Impaired Binding

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.

scholarly journals Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

2008 ◽  
Vol 182 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Kenji Kitamura ◽  
Daisuke Hihara ◽  
Yukinobu Hirose ◽  
Satoshi Katsuyama ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Related Factor ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Yeast Meiosis ◽  
Meiosis I

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

scholarly journals Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Atasi Poddar ◽  
P. Todd Stukenberg ◽  
Daniel J. Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
In Cells

ABSTRACT Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

scholarly journals Mad2-independent Spindle Assembly Checkpoint Activation and Controlled Metaphase–Anaphase Transition inDrosophilaS2 Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 850-863 ◽  
Author(s):  
Bernardo Orr ◽  
Hassan Bousbaa ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Progression ◽  
Experimental Conditions ◽  
Checkpoint Activation ◽  
Nuclear Envelope Breakdown ◽  
Chromatin Bridges ◽  
Delay Progression

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase–anaphase transition and normal patterns of chromosome segregation.

scholarly journals Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31comet

2011 ◽  
Vol 22 (22) ◽  
pp. 4227-4235 ◽  
Author(s):  
Luying Jia ◽  
Bing Li ◽  
Ross T. Warrington ◽  
Xing Hao ◽  
Shixuan Wang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Checkpoint ◽  
Functional Redundancy ◽  
Mitotic Checkpoint ◽  
Human Cells ◽  
Unresolved Issue ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Checkpoint Complex

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase–anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31comet delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31comet and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.

scholarly journals Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55

2013 ◽  
Vol 202 (5) ◽  
pp. 765-778 ◽  
Author(s):  
Claudio Vernieri ◽  
Elena Chiroli ◽  
Valentina Francia ◽  
Fridolin Gross ◽  
Andrea Ciliberto
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Feedback Loop ◽  
Spindle Checkpoint ◽  
Place Cells ◽  
Double Negative ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Mitotic Kinase ◽  
Mitotic Cyclin

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2ACdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2ACdc55 and APC/CCdc20 (i.e., a positive feedback loop) that controls APC/CCdc20 activity. The circuit could guarantee sustained APC/CCdc20 activity after Clb2 starts to be degraded.

scholarly journals A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhejian Ji ◽  
Haishan Gao ◽  
Luying Jia ◽  
Bing Li ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Phosphorylation Cascade ◽  
Mitotic Checkpoint Complex

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract 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/G1 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 cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ 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 mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

scholarly journals Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

2020 ◽  
Vol 219 (4) ◽  
Author(s):  
Gisela Cairo ◽  
Anne M. MacKenzie ◽  
Soni Lacefield
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Mitotic Chromosome ◽  
Budding Yeast ◽  
Meiotic Chromosome ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Chromosome Missegregation ◽  
Anaphase Onset ◽  
Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

scholarly journals The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

2018 ◽  
Vol 217 (3) ◽  
pp. 861-876 ◽  
Author(s):  
Eleni Petsalaki ◽  
Maria Dandoulaki ◽  
George Zachos
Keyword(s):  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Membrane Remodeling ◽  
Mitotic Progression ◽  
Mitotic Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Endosomal Sorting ◽  
Anaphase Onset

The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod–ZW10–Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore–microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport–dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.

scholarly journals The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

2008 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
Author(s):  
Evan C. Osmundson ◽  
Dipankar Ray ◽  
Finola E. Moore ◽  
Qingshen Gao ◽  
Gerald H. Thomsen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
E3 Ligase ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Carboxyl Terminus ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Anaphase Onset

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

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