Structure of yeast Bub3

Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal. These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids. To explore the molecular interactions mediated by Bub3, the crystal structure of the Saccharomyces cerevisiae protein Bub3p was attained at 2.35 Å resolution. Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members. Several loops are substantially elongated, but extra domains or insertions are not present at the termini. In particular, two extended loops project from the top face of the propeller, forming a cleft. Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins. It is proposed that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1. Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces. Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding (Larsen, 2004).

Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and beta-transducin repeats), short sequence motifs appearing in clusters of 4-16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. The crystal structure of Bub3p from Saccharomyces cerevisiae was determined at 1.1 Å and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure (Wilson, 2004).

Association of the spindle checkpoint protein Bub3p with altered kinetochores and with a unique defective centromere

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). Green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, a model was tested in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of the model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], it was determined that Bub3-GFP can associate with a single defective kinetochore. These studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae (Kerscher, 2003).

Yeast Bub3 interacts with Bub1 and the molecular checkpoint function of Bub1 requires Bub3

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, a screen was carried out for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. BUB1 gene and its product have been characterized. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrate a physical association between the two. Sequence analysis of BUB1 reveals a domain with similarity to protein kinases. In vitro experiments confirm that Bub1 possesses kinase activity; Bub1 is able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus (Roberts, 1994).

A feedback control mechanism, or cell cycle checkpoint, delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. The murine homolog of Bub1 is not only required for checkpoint response to spindle damage, but also restrains progression through a normal mitosis. A human homolog of Bub3 has been characterized. It consists of a 37-kD protein with four WD repeats. Like Bub1, Bub3 localizes to kinetochores before chromosome alignment. In addition, Bub3 and Bub1 interact in mammalian cells. Deletion mapping was used to identify the domain of Bub1 required for binding Bub3. Significantly, this same domain is required for kinetochore localization of Bub1, suggesting that the role of Bub3 is to localize Bub1 to the kinetochore, thereby activating the checkpoint in response to unattached kinetochores. The identification of a human Mad3/Bub1-related protein kinase, hBubR1, which can also bind Bub3 in mammalian cells, is described. Ectopically expressed hBubR1 also localizes to kinetochores during prometaphase, but only when hBub3 is overexpressed. The implications of the common interaction between Bub1 and hBubR1 with hBub3 for checkpoint control are discussed (Taylor, 1998).

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. Known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Saccharomyces Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage (Warren, 2002).

Yeast Bub1/BubR1 is required kinetochore association of Bub3

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. A Xenopus homolog of the spindle checkpoint component Bub1 has been isolated, and its role in the spindle checkpoint has been investigated in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. These studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts (Sharp-Baker, 2001).

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores (Chen, 2002).

During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation. A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1. The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p. Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: (1) cell cycle arrest induced by SAC activation; (2) kinetochore accumulation of Bub1p in checkpoint-activated cells; (3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells, and (4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells. Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus. These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p (Kadura, 2005).

A Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. Addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, this anaphase delay was shown to require the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. It is proposed that this checkpoint ensures spindles are properly oriented before anaphase takes place (Tournier, 2004).

Spindle checkpoint function of the yeast Mad1p-Bub1p-Bub3p complex

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. A dominantly acting BUB1 allele has been identified that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 does not cause detectable spindle damage, yet it delays yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possess a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein do not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay is dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. The mitotic delay caused by BUB1-5 or MPS1 overexpression is interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal (Farr, 1998).

MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46% and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p and two-hybrid assays and coimmunoprecipitation experiments were used to show that Mad3p can also bind to Bub3p. In addition, Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. The two regions of homology between Mad3p and Bub1p are crucial for these interactions and loss of function mutations within each domain of Mad3p were identified. The roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint, are discussed (Hardwick, 2000).

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus. The Mad and Bub proteins are key components of the spindle checkpoint through budding yeast genetics and are highly conserved. Most of the spindle checkpoint proteins have been localized to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p, and has been shown to recruit Mad2p to kinetochores. Similarly, Bub3p binds to Bub1p and may target it to kinetochores. Budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and this complex is found at significantly higher levels once the spindle checkpoint is activated. Formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, a conserved motif has been identified within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism (Brady, 2000).

The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. It is suggested that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation (Fraschini, 2001).

The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Fission yeast devoid of mad3(+) are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory 'wait anaphase' signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, the dependency of Mad3/BubR1 protein recruitment to kinetochores has been systematically studied. Mad3-GFP kinetochore localization is dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. The implications of these findings are discussed in the context of the current understanding of spindle checkpoint function (Millband, 2002).

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. It is proposed that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways (Gillett, 2004).

Yeast spindle checkpoint protein dynamics at kinetochores

To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, GFP fusions and live-cell imaging were used to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. These data support catalytic models of checkpoint activation (Howell, 2004).

The highly conserved Ndc80 kinetochore complex, which assembles independently of the checkpoint complex, is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity

The Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. The spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. It is proposed that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling (McCleland, 2003).

Generation of a spindle checkpoint arrest from synthetic signaling assemblies

The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex (see Drosophila Bub3). This leads to dynamic kinetochore recruitment of Mad proteins, a conformational change in Mad2, and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2: see Drosophila Cdc20). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. This study took a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest in fission yeast. A Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, it is concluded that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization (Yuan, 2016).

Reconstitution of the checkpoint complex in Xenopus

The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and antibodies to Xenopus Bub3 (XBub3) have been used to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. Two forms of XBub3 are detected in egg extracts and both are found to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells, and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. The mechanism by which these alpha-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway are discussed (Campbell, 2003).

The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. While the first three processes are relatively well understood, details of Ran's role in mitotic progression remain obscure. Elevated levels of Ran's exchange factor (RCC1) have been found to abrogate the spindle assembly checkpoint in Xenopus egg extracts, restore APC/C activity, and disrupt the kinetochore localization of checkpoint regulators, including Mad2, CENP-E, Bub1, and Bub3. Depletion of Ran's GTPase activating protein (RanGAP1) and its accessory factor (RanBP1) similarly abrogates checkpoint arrest. By contrast, the addition of RanGAP1 and RanBP1 to extracts with exogenous RCC1 restores the spindle checkpoint. Together, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Finally, a clear wave of RCC1 association to mitotic chromosomes has been observed at the metaphase-anaphase transition in normal cycling extracts, suggesting that this mechanism has an important role in unperturbed cell cycles (Arnaoutov, 2003).

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway was studied. The results show that the first regulatory step of this cascade is defined by Aurora B/INCENP (see Drosophila Aurora B) complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other (Vigneron, 2004).

Bub3 in mammals

Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases. Genes required for this delay first were identified in budding yeast (the MAD and BUB genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest. This study reports the isolation and analysis of a murine homologue of BUB3, a gene whose deletion abolishes mitotic checkpoint function in Saccharomyces cerevisiae. mBub3 belongs to a small gene family that has been highly conserved through evolution. By expressing recombinant proteins in insect cells, it was shown that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity. During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores. High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment. Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole (Martinez-Exposito, 1999).

Bub3 is a conserved component of the mitotic spindle assembly complex. The protein is essential for early development in Bub3 gene-disrupted mice, evident from their failure to survive beyond day 6.5-7.5 postcoitus (pc). Bub3 null embryos appear normal up to day 3.5 pc but accumulate mitotic errors from days 4.5-6.5 pc in the form of micronuclei, chromatin bridging, lagging chromosomes, and irregular nuclear morphology. Null embryos treated with a spindle-depolymerising agent fail to arrest in metaphase and show an increase in mitotic disarray. The results confirm Bub3 as a component of the essential spindle checkpoint pathway that operates during early embryogenesis (Kalitsis, 2000).

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. The mechanism by which the checkpoint inhibits the APC/C was examined by purifying an APC/C inhibitory factor from HeLa cells. The mitotic checkpoint complex (MCC) consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, since it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells is sensitive to inhibition by MCC. The majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes do not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. It is proposed that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC (Sudakin, 2001).

The mRNA export factor RAE1 (also called GLE2) and the mitotic checkpoint protein BUB3 share extensive sequence homology in yeast as well as higher eukaryotes, although the biological relevance of their similarity is unclear. Previous work in HeLa cells has shown that human (h)RAE1 binds the nuclear pore complex protein hNUP98 via a short NUP98 motif called GLEBS (for GLE2p-binding sequence). The two known binding partners of hBUB3, the mitotic checkpoint proteins hBUB1 and hBUBR1, both carry a region with remarkable similarity to the GLEBS motif of hNUP98. GLEBS-like motifs of mouse (m)BUB1 and mBUBR1 are sufficient for mBUB3 binding. mBUB3 lacks affinity for the hNUP98 GLEBS, demonstrating its binding specificity for GLEBS motifs of mitotic checkpoint proteins. Interestingly, mRAE1 does not exclusively bind to the GLEBS motif of hNUP98 and can cross-interact with the mBUB1 GLEBS. Full-length RAE1 and BUB1 proteins interact in mammalian cells and accumulate both at the kinetochores of prometaphase chromosomes. These findings demonstrate that GLEBS motifs reside in mammalian nucleoporins and mitotic checkpoint proteins and apparently serve as specific binding sites for either BUB3, RAE1, or both (Wang, 2001).

Mutation of mammalian Bub3

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, knockout mice were generated that have these genes deleted individually or in combination. Haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. Overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice have no detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, it was shown that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, these data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint (Babu, 2003).

Mammalian Bub3 interacts with PARP-1 and is poly(ADP-ribosyl)ated following induction of DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA strand breaks during cellular genotoxic stress response and catalyzes poly(ADP-ribosyl)ation of acceptor proteins. These acceptor proteins include those involved in modulation of chromatin structure, DNA synthesis, DNA repair, transcription, and cell cycle control. Thus, PARP-1 is believed to play a pivotal role in maintaining genome integrity through modulation of protein-protein and protein-DNA interactions. PARP-1 associates with normal mammalian centromeres and human neocentromeres by affinity purification and immunofluorescence. This study investigated the interaction of this protein with, and poly(ADP-ribosyl)ation of, three constitutive centromere proteins, Cenpa, Cenpb, and Cenpc, and a spindle checkpoint protein, Bub3. Immunoprecipitation and Western blot analyses demonstrate that Cenpa, Cenpb, and Bub3, but not Cenpc, interact with PARP-1, and are poly(ADP-ribosyl)ated following induction of DNA damage. The results suggest a role of PARP-1 in centromere assembly/disassembly and checkpoint control. Demonstration of PARP-1-binding and poly(ADP-ribosyl)ation in three of the four proteins tested further suggests that many more centromere proteins may behave similarly and implicates PARP-1 as an important regulator of diverse centromere function (Saxena, 2002).

BubR1 promotes Bub3-dependent APC/C inhibition during spindle assembly checkpoint signaling

The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase (see Drosophila Mps1) promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 (see Drosophila Bub3) is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 (see Drosophila Bub1) and BubR1 (see Drosophila Bub1R). A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. This study asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade (Overlack, 2017).

Bub3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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