GO:1990298 bub1-bub3 complex: Components, Assembly and Research Methods, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:1990298 (bub1-bub3 complex) is a cellular component defined as a protein complex that associates with kinetochores.
• The BUB1-BUB3 complex is a conserved spindle checkpoint module that binds phosphorylated KNL1/Spc7 at kinetochores to license downstream checkpoint signaling.
• Beyond mitosis, the BUB3-BUB1 complex promotes telomere DNA replication, linking it to genome maintenance.
• Bub1 and Bub3 also regulate metabolic adaptation via macrolipophagy in Drosophila, revealing non-canonical roles.
• Assembly of the complex is phospho-regulated and involves sequential multisite interactions at kinetochores.
• The complex is a target for CRISPR knockout, point-mutation, knock-in, and overexpression models to dissect checkpoint and telomere biology.
Description
The bub1-bub3 complex (GO:1990298) is a protein complex that associates with kinetochores, as defined by QuickGO. It is a central module of the spindle assembly checkpoint (SAC), a surveillance mechanism that delays anaphase until all chromosomes are properly attached to the mitotic spindle. The complex comprises the checkpoint kinase BUB1 and the WD40-repeat protein BUB3, which together recognize phosphorylated KNL1/Spc7 at kinetochores to initiate checkpoint signaling. This recognition event is a critical switch that toggles the checkpoint by licensing the interaction of BUB1 with MAD1-MAD2, thereby promoting assembly of the mitotic checkpoint complex (MCC). Researchers study GO:1990298 because its function is essential for faithful chromosome segregation, and its dysregulation is linked to aneuploidy and cancer. The complex is also implicated in telomere DNA replication, expanding its role beyond mitosis. In Drosophila, Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy, indicating broader physiological functions. Understanding the assembly, regulation, and downstream effects of the bub1-bub3 complex is therefore relevant to cell cycle biology, genome stability, and disease mechanisms. This article synthesizes authoritative QuickGO data and verified PubMed literature to provide a research-grade overview of the bub1-bub3 complex, covering its definition, structure, molecular mechanism, key genes, disease links, and experimental models including CRISPR-based approaches.
bub1-bub3 complex At A Glance
| GO ID | GO:1990298 |
|---|---|
| GO term | bub1-bub3 complex |
| Ontology | cellular_component |
| Synonym | none |
| Major function | Associates with kinetochores to regulate spindle checkpoint signaling and chromosome segregation |
| Complex members | BUB1 (Bub1) and BUB3 (Bub3) |
| Localization | Kinetochores |
| Conservation | Conserved from yeast to humans |
| Additional roles | Telomere DNA replication; macrolipophagy in Drosophila |
What Is GO:1990298?
According to the Gene Ontology, GO:1990298 (bub1-bub3 complex) is a protein complex that associates with the kinetochores. In other words, it is a cellular component consisting of the BUB1 and BUB3 proteins that localizes to kinetochores, the specialized chromosomal regions where spindle microtubules attach during cell division. This complex is a functional unit that participates in spindle checkpoint signaling and potentially other processes such as telomere replication.
Why Is bub1-bub3 complex Important in Cell Biology?
The bub1-bub3 complex is essential for the spindle assembly checkpoint, a safeguard that prevents chromosome missegregation and aneuploidy. Its correct assembly at kinetochores ensures that cells delay division until all chromosomes are properly attached, and failure of this process can lead to genomic instability, a hallmark of cancer. Beyond mitosis, the complex contributes to telomere DNA replication, linking it to genome maintenance and aging. In Drosophila, Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy, suggesting roles in nutrient stress responses. Thus, studying GO:1990298 provides insights into fundamental cell cycle control, genome stability, and potential disease mechanisms.
• Ensures accurate chromosome segregation by activating the spindle assembly checkpoint.
• Prevents aneuploidy, a common feature of cancer cells.
• Promotes telomere DNA replication, supporting genome integrity.
• Regulates metabolic adaptation via macrolipophagy in Drosophila.
• Serves as a model for phospho-regulated kinetochore assembly.
• Its dysfunction is implicated in cancer and developmental disorders.
• Provides targets for CRISPR-based functional studies.
• Links cell cycle control to cellular stress responses.
• Conserved mechanism from yeast to humans enables cross-species research.
• Potential biomarker or therapeutic target in oncology.
Structure and Composition of bub1-bub3 complex
BUB1: The kinase subunit
In simple terms: BUB1 is the enzymatic part of the complex that can add phosphate groups to other proteins.
BUB1 is a serine/threonine kinase that contains a conserved N-terminal region, a central kinetochore-binding domain, and a C-terminal kinase domain. It binds to phosphorylated KNL1/Spc7 at kinetochores and is required for checkpoint signaling. BUB1 also interacts with MAD1-MAD2 to promote MCC assembly.
BUB3: The WD40-repeat scaffold
In simple terms: BUB3 is a protein with a propeller-like shape that helps BUB1 bind to kinetochores.
BUB3 is a WD40-repeat protein that forms a complex with BUB1. It recognizes phosphorylated KNL1/Spc7 motifs and is essential for targeting the complex to kinetochores. BUB3 also participates in telomere DNA replication independently of its mitotic role.
Kinetochore targeting via phospho-KNL1
In simple terms: The complex is recruited to kinetochores by a phosphorylated protein called KNL1.
The BUB1-BUB3 complex binds to phosphorylated KNL1/Spc7 at kinetochores. This interaction is mediated by a TPR domain in BUB1 that recognizes phospho-KNL1, as shown in C. elegans. Sequential multisite phospho-regulation of KNL1-BUB3 interfaces ensures precise timing of complex assembly.
Assembly and stoichiometry
In simple terms: BUB1 and BUB3 come together in a specific ratio to form the functional complex.
The complex is thought to consist of one BUB1 molecule and one BUB3 molecule, although higher-order assemblies may exist. Assembly is regulated by phosphorylation events at kinetochores. The complex is conserved from yeast to humans, with orthologs such as Bub1-Bub3 in Saccharomyces cerevisiae and BUB-1-BUB-3 in C. elegans.
Key Genes Involved in GO:1990298 bub1-bub3 complex
The following genes and proteins are key components or regulators of the bub1-bub3 complex and its associated functions.
| Gene | Major Role | Research Relevance |
|---|---|---|
| BUB1 | Serine/threonine kinase; core subunit of the complex; binds KNL1 and MAD1 | Central to spindle checkpoint; target for cancer studies |
| BUB3 | WD40-repeat protein; core subunit; binds phospho-KNL1 | Essential for kinetochore targeting and checkpoint function |
| KNL1 | Kinetochore scaffold; provides phospho-docking sites for BUB1-BUB3 | Regulates complex assembly and checkpoint activation |
| SPC7 | Yeast ortholog of KNL1; binds Bub1-Bub3 | Model for phospho-regulation of checkpoint |
| MAD1 | Checkpoint protein; interacts with BUB1 to promote MCC assembly | Links BUB1-BUB3 to downstream checkpoint effectors |
| MAD2 | Checkpoint protein; binds MAD1 and inhibits APC/C | Key effector of spindle checkpoint |
| BUBR1 | Checkpoint kinase; part of MCC; interacts with BUB3 | Regulates checkpoint silencing and chromosome segregation |
| CDC20 | Activator of APC/C; inhibited by MCC | Target of checkpoint to delay anaphase |
| APC/C | Ubiquitin ligase; drives anaphase onset | Downstream target of checkpoint |
| BUB-1 | C. elegans ortholog of BUB1 | Genetic model for kinetochore targeting |
| BUB-3 | C. elegans ortholog of BUB3 | Genetic model for kinetochore targeting |
| BUB1 (Drosophila) | Regulates macrolipophagy and metabolic adaptation | Model for non-mitotic roles |
| BUB3 (Drosophila) | Regulates macrolipophagy and metabolic adaptation | Model for non-mitotic roles |
| TELOMERE-ASSOCIATED PROTEINS | Interact with BUB3-BUB1 to promote telomere replication | Link to genome maintenance |
| Aurora B | Regulates kinetochore phosphorylation and checkpoint | Modulates BUB1-BUB3 assembly |
| MPS1 | Kinase that phosphorylates KNL1 to recruit BUB1-BUB3 | Upstream regulator of complex assembly |
| PP1 | Phosphatase that reverses KNL1 phosphorylation | Regulates checkpoint silencing |
| BUB1 (yeast) | Yeast ortholog; regulates checkpoint and chromosome segregation | Genetic model for checkpoint studies |
How Is bub1-bub3 complex Regulated?
The assembly and function of the bub1-bub3 complex are regulated by phosphorylation. MPS1 kinase phosphorylates KNL1/Spc7 at multiple sites, creating docking sites for BUB3 and BUB1. Sequential multisite phospho-regulation of KNL1-BUB3 interfaces ensures that the complex assembles only at kinetochores that are not yet attached to microtubules. Aurora B kinase also contributes to kinetochore phosphorylation and checkpoint regulation. Conversely, phosphatase PP1 reverses KNL1 phosphorylation, promoting checkpoint silencing. In addition, intrinsic factors such as the BUB1 kinase domain and extrinsic factors like kinetochore tension regulate Bub1 activity. The complex is also subject to regulation by its binding partners, including MAD1-MAD2, which is licensed by BUB3-BUB1 binding to Spc7/KNL1.
bub1-bub3 complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| BUB1 | Cancer, aneuploidy | Knockout in cancer cell lines; xenograft models |
| BUB3 | Cancer, telomere dysfunction | Knockout in primary fibroblasts; telomere assays |
| BUB1/BUB3 (Drosophila) | Metabolic adaptation | Overexpression or knockout in Drosophila fat body |
| KNL1 | Cancer, checkpoint defects | Point mutations in KNL1 phospho-sites; knock-in models |
| MAD1/MAD2 | Cancer, checkpoint defects | Knockout in HeLa cells; live-cell imaging |
Cancer and aneuploidy
Dysregulation of the spindle assembly checkpoint, including the bub1-bub3 complex, leads to chromosome missegregation and aneuploidy, a hallmark of many cancers. Mutations or altered expression of BUB1 and BUB3 have been observed in various tumors, and their dysfunction can promote tumorigenesis. The complex is therefore a potential target for cancer diagnostics and therapeutics.
Telomere maintenance and genome stability disorders
The BUB3-BUB1 complex promotes telomere DNA replication, and its loss can lead to telomere dysfunction and genome instability. This links the complex to premature aging syndromes and cancer predisposition associated with telomere defects.
Metabolic disorders
In Drosophila, Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy, suggesting that their dysfunction could contribute to metabolic disorders. This non-canonical role highlights the complex's broader physiological impact.
From bub1-bub3 complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does BUB1 kinase activity require BUB3 for kinetochore targeting? | Point mutation in BUB1 kinase domain; knockout of BUB3 |
| How does phospho-KNL1 regulate BUB1-BUB3 assembly? | Knock-in of phospho-deficient KNL1 mutants |
| What is the role of BUB3-BUB1 in telomere replication? | Knockout of BUB3 in telomerase-positive cells; telomere FISH |
| Does BUB1-BUB3 regulate macrolipophagy? | Overexpression of Bub1/Bub3 in Drosophila; lipid droplet imaging |
| Can BUB1-BUB3 be targeted for cancer therapy? | CRISPR knockout in cancer organoids; drug sensitivity assays |
| What are the interaction partners of BUB1-BUB3? | Tagged knock-in of BUB1 or BUB3; immunoprecipitation-mass spectrometry |
How to Study the bub1-bub3 complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Live-cell imaging | Kinetochore localization and dynamics of BUB1-BUB3 | Mitotic checkpoint studies |
| Immunoprecipitation-mass spectrometry | Protein-protein interactions and complex composition | Interactome mapping |
| Phospho-proteomics | Phosphorylation sites on KNL1 and other proteins | Regulation of complex assembly |
| CRISPR knockout screens | Gene essentiality and synthetic lethality | Cancer target discovery |
| In vitro binding assays | Direct phospho-dependent interactions | Mechanistic studies |
| Telomere FISH | Telomere length and replication defects | Genome stability studies |
| Lipid droplet imaging | Macrolipophagy activity | Metabolic regulation studies |
| Structural biology (cryo-EM) | 3D structure of the complex | Mechanistic insights |
Live-cell imaging of kinetochore dynamics
Live-cell imaging using fluorescently tagged BUB1 and BUB3 allows real-time visualization of complex assembly at kinetochores and its dynamics during mitosis. This method can reveal defects in checkpoint signaling and chromosome segregation.
Phospho-proteomics and interaction studies
Mass spectrometry-based phospho-proteomics can identify phosphorylation sites on KNL1 and other components that regulate BUB1-BUB3 binding. Immunoprecipitation coupled with mass spectrometry can map the interactome of the complex.
Genetic screens and CRISPR knockout
CRISPR knockout screens in human cell lines can identify genes that are synthetic lethal with BUB1 or BUB3 loss, revealing pathways that compensate for checkpoint defects. Such screens are powerful for uncovering disease-relevant vulnerabilities.
In vitro reconstitution and structural biology
Recombinant BUB1 and BUB3 proteins can be used for in vitro binding assays to study phospho-dependent interactions with KNL1 peptides. Structural studies such as X-ray crystallography or cryo-EM can provide mechanistic insights into complex assembly.
How CRISPR Can Be Used to Study GO:1990298 bub1-bub3 complex
Knockout
CRISPR knockout of BUB1 or BUB3 in cell lines abolishes the bub1-bub3 complex, leading to checkpoint defects, chromosome missegregation, and aneuploidy. Knockout models are used to study the complex's role in telomere replication and metabolic adaptation.
Point Mutation
Point mutations in BUB1 kinase domain or in KNL1 phospho-sites can disrupt complex assembly or function without affecting protein stability. Such models help dissect the specific contributions of phosphorylation and kinase activity.
Knock-in
Knock-in of tagged BUB1 or BUB3 (e.g., GFP or HA) allows endogenous labeling for imaging and proteomics. Knock-in of phospho-deficient or phospho-mimetic KNL1 variants can reveal the importance of specific phosphorylation events.
Overexpression
Overexpression of BUB1 or BUB3 can lead to hyperactivation of the checkpoint or non-canonical effects such as macrolipophagy. Overexpression models are useful for gain-of-function studies and for identifying dosage-sensitive phenotypes.
How EDITGENE Supports bub1-bub3 complex Research
Researchers studying bub1-bub3 complex-related genes often need to determine whether a candidate gene is causally involved in kinetochore function, checkpoint signaling, or genome stability. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models, enabling rigorous functional validation of genes associated with GO:1990298.
Contact EDITGENE today to design your custom CRISPR model for bub1-bub3 complex research.
Frequently Asked Questions About bub1-bub3 complex
What is the bub1-bub3 complex?
The bub1-bub3 complex (GO:1990298) is a protein complex that associates with kinetochores and regulates the spindle assembly checkpoint.
What genes are involved in the bub1-bub3 complex?
The core genes are BUB1 and BUB3, which encode the two subunits of the complex. Other genes such as KNL1, MAD1, and MAD2 interact with the complex.
What is the function of GO:1990298?
GO:1990298 functions in kinetochore association and spindle checkpoint signaling, ensuring accurate chromosome segregation.
Where is the bub1-bub3 complex located?
It localizes to kinetochores during mitosis.
How is the bub1-bub3 complex regulated?
Its assembly is regulated by phosphorylation of KNL1 by MPS1 kinase, and reversed by PP1 phosphatase.
What diseases are associated with bub1-bub3 complex dysfunction?
Dysfunction is linked to cancer, aneuploidy, and telomere-related genome instability.
What model systems are used to study the bub1-bub3 complex?
Common models include human cell lines, C. elegans, Drosophila, and yeast.
How can CRISPR be used to study the bub1-bub3 complex?
CRISPR knockout, point mutation, knock-in, and overexpression can dissect the roles of BUB1 and BUB3 in checkpoint and non-mitotic functions.
What is the role of BUB3-BUB1 in telomeres?
The BUB3-BUB1 complex promotes telomere DNA replication, linking it to genome maintenance.
Does the bub1-bub3 complex have non-mitotic functions?
Yes, in Drosophila Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy.
Conclusion
The bub1-bub3 complex (GO:1990298) is a conserved kinetochore-associated protein complex essential for spindle checkpoint signaling and genome stability. Its roles extend to telomere replication and metabolic regulation, underscoring its broad biological importance. Dysregulation of the complex contributes to aneuploidy and cancer, making it a compelling target for further research. CRISPR-based models, including knockout, point mutation, knock-in, and overexpression, provide powerful tools to dissect its functions and identify therapeutic opportunities.
References
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- 2. Zhang Q et al.. 2023. Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy in Drosophila.. Cell Rep 42(4):112343 PMID: 37027296
- 3. Mora-Santos MD et al.. 2016. Bub3-Bub1 Binding to Spc7/KNL1 Toggles the Spindle Checkpoint Switch by Licensing the Interaction of Bub1 with Mad1-Mad2.. Curr Biol 26(19):2642-2650 PMID: 27618268
- 4. Sethi S et al.. 2025. Interplay of kinetochores and catalysts drives rapid assembly of the mitotic checkpoint complex.. Nat Commun 16(1):4823 PMID: 40410156
- 5. Houston J et al.. 2024. Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.. J Cell Biol 223(7) PMID: 38578284
- 6. Vleugel M et al.. 2015. Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores.. Mol Cell 57(5):824-835 PMID: 25661489
- 7. Houston J et al.. 2024. Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.. bioRxiv PMID: 38370671
- 8. Breit C et al.. 2015. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1.. PLoS One 10(12):e0144673 PMID: 26658523
