GO:0000408 EKC/KEOPS complex: Components, Assembly and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0000408 describes the EKC/KEOPS complex, an evolutionarily conserved protein complex required for the t6A modification of tRNA.
• The complex is built from subunits such as Bud32p, Kae1p, Gon7p, Cgi121p, and Pcc1p in Saccharomyces cerevisiae, with human orthologs including PRPK, TPRKB, LAGE3, OSGEP, and GON7.
• Its best-established molecular role is catalyzing threonylcarbamoyladenosine (t6A) formation on ANN-decoding tRNAs, which supports translation fidelity and protein synthesis homeostasis.
• Loss of EKC/KEOPS function causes pleiotropic defects in cell polarity, centromere regulation, and growth control in model organisms.
• The human complex interacts with Cullin2 ubiquitin ligases through the tumor antigen PRAME, linking it to cancer biology.
• CRISPR knockout, point-mutation, knock-in, and overexpression models are powerful tools for dissecting EKC/KEOPS subunit function in disease and development.
Description
The EKC/KEOPS complex (GO:0000408) is a conserved cellular component defined by its role in the t6A tRNA modification. It was initially identified through genetic and biochemical studies in yeast and has since been found in archaea, Drosophila, and humans. The complex is named after its founding subunits: endopeptidase-like kinase chromatin-associated protein (EKC) and kinase, putative endopeptidase and other proteins of small size (KEOPS). Researchers study this complex because it sits at the intersection of translation, genome stability, and cell growth control. The core biochemical function of the EKC/KEOPS complex is the synthesis of threonylcarbamoyladenosine (t6A), a universal modification at position 37 of tRNAs that decode ANN codons. This modification is essential for efficient and accurate protein synthesis, and its loss leads to widespread cellular defects. In Saccharomyces cerevisiae, the complex includes Bud32p, Kae1p, Gon7p, Cgi121p, and Pcc1p, while metazoans have orthologous subunits such as PRPK, TPRKB, LAGE3, OSGEP, and GON7. Beyond tRNA modification, the EKC/KEOPS complex has been implicated in diverse processes, including cell polarity, centromere function, and mRNA stability. In humans, it is recruited to Cullin2 ubiquitin ligases by the tumor antigen PRAME, suggesting a role in cancer-associated pathways. The Drosophila complex regulates protein synthesis homeostasis and animal growth, highlighting its importance in development. These findings make GO:0000408 a compelling target for functional genomics and disease research.
EKC/KEOPS complex At A Glance
| GO ID | GO:0000408 |
|---|---|
| GO term | EKC/KEOPS complex |
| Ontology | cellular_component |
| Synonym | endopeptidase-like kinase chromatin-associated protein complex; KEOPS/EKC complex; kinase, putative endopeptidase and other proteins of small size protein complex; TCTC; threonyl-carbamoly transferase complex |
| Major function | t6A tRNA modification and translation fidelity |
| Subunits (yeast) | Bud32p, Kae1p, Gon7p, Cgi121p, Pcc1p |
| Subunits (human) | PRPK, TPRKB, LAGE3, OSGEP, GON7 |
| Conservation | Archaea to eukaryotes |
| Associated processes | Protein synthesis homeostasis, cell polarity, centromere regulation, mRNA stability |
What Is GO:0000408?
GO:0000408, the EKC/KEOPS complex, is a protein complex involved in the t6A modification of tRNA. In Saccharomyces cerevisiae, the complex contains Bud32p, Kae1p, Gon7p, Cgi121p, and Pcc1p. The term is also known by synonyms such as endopeptidase-like kinase chromatin-associated protein complex, KEOPS/EKC complex, kinase, putative endopeptidase and other proteins of small size protein complex, TCTC, and threonyl-carbamoly transferase complex. It is annotated as a cellular component and is conserved across archaea and eukaryotes.
Why Is EKC/KEOPS complex Important in Cell Biology?
The EKC/KEOPS complex is essential for the t6A modification of tRNA, a universal chemical mark that ensures accurate decoding of ANN codons during translation. Disruption of this complex leads to translation defects, growth arrest, and pleiotropic phenotypes in model organisms, underscoring its fundamental role in cell physiology. In humans, mutations or dysregulation of EKC/KEOPS subunits have been linked to cancer through interactions with PRAME and Cullin2 ubiquitin ligases. The complex also influences cell polarity and centromere function, connecting it to genome stability and cell division. Therefore, studying GO:0000408 provides insights into basic translation mechanisms and human disease.
• Required for t6A tRNA modification, which is essential for translation fidelity and protein synthesis homeostasis.
• Conserved from archaea to humans, making it a model for evolutionary cell biology.
• Dysregulation of human subunits is implicated in cancer through PRAME-mediated recruitment to Cullin2 ligases.
• Loss of function causes cell polarity defects in yeast, linking it to cytoskeletal organization.
• Modulates centromere complex stability via mRNA decay pathways in fission yeast.
• Affects animal growth and development in Drosophila.
• Provides a target for CRISPR screens to identify synthetic lethal interactions in cancer.
• Serves as a paradigm for studying tRNA modification enzymes and their disease relevance.
EKC/KEOPS complex: Biological Process, Structure, and Molecular Mechanism
t6A tRNA Modification
In simple terms: The complex adds a chemical tag to tRNA so proteins are built correctly.
The primary biological process of the EKC/KEOPS complex is the synthesis of threonylcarbamoyladenosine (t6A) at position 37 of tRNAs that decode ANN codons. This modification is required for efficient and accurate translation, and its loss leads to misreading and ribosomal pausing. In Saccharomyces cerevisiae, Gcn4 misregulation revealed a direct role for the complex in t6A modification. The modification is conserved across all domains of life.
Protein Synthesis Homeostasis
In simple terms: The complex helps keep protein production balanced.
In Drosophila, the EKC/KEOPS complex is required for protein synthesis homeostasis and animal growth. Loss of complex subunits leads to reduced translation and developmental defects. This suggests that the complex couples tRNA modification to organismal growth control.
Cell Polarity and Centromere Regulation
In simple terms: The complex also helps cells organize their shape and divide correctly.
In Saccharomyces cerevisiae, proper localization of the Bud9 landmark protein depends on the EKC/KEOPS complex, linking it to cell polarity. In Schizosaccharomyces pombe, the complex negatively regulates the Mis17-Mis6 centromere complex through mRNA decay pathways. These findings expand the functional repertoire of the complex beyond tRNA modification.
Structure and Composition
In simple terms: The complex is made of several proteins that fit together like a machine.
The EKC/KEOPS complex is composed of multiple subunits. In Saccharomyces cerevisiae, it contains Bud32p, Kae1p, Gon7p, Cgi121p, and Pcc1p. The archaeal Kae1/Bud32 fusion protein MJ1130 provides a structural model for the eukaryotic subcomplex. The human complex includes PRPK (Bud32 ortholog), TPRKB (Cgi121 ortholog), LAGE3, OSGEP (Kae1 ortholog), and GON7. Crystal structures of the human PRPK-TPRKB complex reveal key interaction interfaces.
Molecular Mechanism and Regulation
In simple terms: The complex uses energy and partner proteins to modify tRNA and interact with other cellular machines.
The catalytic core of the complex is formed by Kae1 and Bud32, which together catalyze t6A formation. Bud32 is a kinase-like protein, and its activity may regulate complex function. The human complex is recruited to Cullin2 ubiquitin ligases by the tumor antigen PRAME, suggesting a role in ubiquitination pathways. LAGE3, a human subunit, stabilizes VEGFA mRNA and promotes angiogenesis in hepatocellular carcinoma. These interactions indicate that the complex integrates tRNA modification with signaling and RNA stability.
Key Genes Involved in GO:0000408 EKC/KEOPS complex
The following genes and proteins are core components or interactors of the EKC/KEOPS complex, with established roles in tRNA modification, translation, and disease.
| Gene | Major Role | Research Relevance |
|---|---|---|
| KAE1 (OSGEP) | Catalytic subunit for t6A modification | Essential for tRNA modification and translation fidelity |
| BUD32 (PRPK) | Kinase-like subunit, structural core | Regulates complex assembly and function |
| CGI121 (TPRKB) | Accessory subunit, enhances catalysis | Structural component of the complex |
| GON7 | Small subunit, stabilizes complex | Required for t6A modification in yeast and humans |
| PCC1 | Subunit in yeast, links to chromatin | Involved in complex integrity |
| LAGE3 | Human subunit, mRNA stabilization | Promotes angiogenesis in hepatocellular carcinoma |
| PRAME | Recruits complex to Cullin2 ligases | Tumor antigen, cancer-associated |
| CUL2 | Ubiquitin ligase scaffold | Interacts with complex via PRAME |
| MIS17 | Centromere protein | Regulated by EKC/KEOPS in fission yeast |
| MIS6 | Centromere protein | Regulated by EKC/KEOPS in fission yeast |
| BUD9 | Cell polarity landmark | Localization depends on EKC/KEOPS |
| GCN4 | Translation regulator | Misregulated upon EKC/KEOPS loss |
| VEGFA | Angiogenic factor | mRNA stabilized by LAGE3 |
| MJ1130 | Archaeal Kae1/Bud32 fusion | Structural model for complex |
| PRPK | Human Bud32 ortholog | Kinase subunit, drug target |
| TPRKB | Human Cgi121 ortholog | Part of human complex |
| OSGEP | Human Kae1 ortholog | Catalytic subunit in humans |
| GON7 | Human Gon7 ortholog | Complex stability and function |
How Is EKC/KEOPS complex Regulated?
The EKC/KEOPS complex is regulated at multiple levels. In Saccharomyces cerevisiae, its function in tRNA modification is directly tied to translation demand, as loss of t6A leads to Gcn4 misregulation. In Schizosaccharomyces pombe, the complex negatively regulates the Mis17-Mis6 centromere complex through mRNA decay pathways, indicating post-transcriptional control. In humans, the complex is recruited to Cullin2 ubiquitin ligases by the tumor antigen PRAME, linking its activity to ubiquitin-mediated protein degradation. Additionally, LAGE3 stabilizes VEGFA mRNA, suggesting that complex subunits can modulate RNA stability. These regulatory mechanisms integrate the complex into broader cellular signaling networks.
EKC/KEOPS complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| LAGE3 | Hepatocellular carcinoma angiogenesis | Knockout in HepG2 cells, VEGFA mRNA stability assay |
| PRAME | Cancer, ubiquitination | Overexpression in HEK293T, Cullin2 interaction |
| OSGEP | Translation-related growth defects | Point mutation in yeast, t6A assay |
| TPRKB | Complex assembly defects | Knockout in human cell lines, proteomics |
| GON7 | t6A modification deficiency | Knockout in yeast, tRNA modification analysis |
Cancer and Tumorigenesis
The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the tumor antigen PRAME, implicating it in cancer-associated ubiquitination pathways. LAGE3, a subunit of the complex, promotes angiogenesis in hepatocellular carcinoma by stabilizing VEGFA mRNA. These findings suggest that EKC/KEOPS components can contribute to tumor progression and angiogenesis.
Developmental and Growth Disorders
In Drosophila, the EKC/KEOPS complex is required for protein synthesis homeostasis and animal growth. Loss of complex function leads to developmental defects, suggesting that mutations in human orthologs could cause growth-related disorders. The essential role of t6A modification in translation supports this link.
Genome Stability and Centromere Function
In Schizosaccharomyces pombe, the EKC/KEOPS complex negatively regulates the Mis17-Mis6 centromere complex, which is critical for chromosome segregation. Defects in this regulation could lead to genome instability, a hallmark of cancer and developmental diseases. This connection highlights the complex's role beyond tRNA modification.
From EKC/KEOPS complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of EKC/KEOPS subunit affect tRNA modification? | CRISPR knockout of OSGEP or GON7 in human cells, followed by tRNA sequencing |
| How does PRPK kinase activity regulate complex function? | Point mutation of catalytic residues in PRPK, kinase assays |
| Can LAGE3 stabilize VEGFA mRNA? | Knock-in of tagged LAGE3, RNA immunoprecipitation |
| What is the role of PRAME in recruiting complex to Cullin2? | Overexpression of PRAME, co-immunoprecipitation |
| Does EKC/KEOPS regulate centromere function? | Knockout in S. pombe, chromosome segregation assays |
| How does complex loss affect cell polarity? | Knockout in S. cerevisiae, Bud9 localization imaging |
How to Study the EKC/KEOPS complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| tRNA sequencing | t6A modification levels | Assess complex activity |
| Ribo-seq | Translation efficiency and pausing | Link complex to protein synthesis |
| Mass spectrometry | Protein interactions and modifications | Identify subunits and interactors |
| Co-immunoprecipitation | Protein-protein interactions | Study PRAME-Cullin2 recruitment |
| Fluorescence microscopy | Subcellular localization | Analyze cell polarity and centromere |
| RNA immunoprecipitation | RNA-protein binding | LAGE3-VEGFA mRNA interaction |
| CRISPR screening | Gene essentiality and synthetic lethality | Identify complex dependencies |
tRNA Modification Analysis
To directly assess EKC/KEOPS function, researchers use tRNA sequencing and mass spectrometry to quantify t6A levels. These methods can detect changes in modification status upon knockout or point mutation of complex subunits.
Ribosome Profiling (Ribo-seq)
Ribo-seq measures translation efficiency and codon-specific pausing. Loss of t6A modification leads to ribosome pausing at ANN codons, which can be detected by Ribo-seq. This method is ideal for linking complex function to protein synthesis homeostasis.
Proteomics and Interactomics
Affinity purification coupled with mass spectrometry can identify complex subunits and interactors, such as Cullin2 and PRAME. Proteomics also reveals changes in protein abundance upon complex disruption.
Imaging and Localization Studies
Fluorescence microscopy can visualize the localization of complex subunits and their effect on cell polarity landmarks like Bud9. Live-cell imaging in yeast and human cells provides spatial insights.
How CRISPR Can Be Used to Study GO:0000408 EKC/KEOPS complex
Knockout
CRISPR knockout of EKC/KEOPS subunits such as OSGEP, TPRKB, or GON7 in human cell lines can abolish t6A modification and cause translation defects. These models are useful for studying the essentiality of the complex and identifying compensatory pathways.
Point Mutation
Introducing point mutations in catalytic residues of PRPK or KAE1 allows researchers to dissect kinase activity versus structural roles. Such models can separate t6A modification from other functions.
Knock-in
Knock-in of epitope-tagged subunits (e.g., FLAG-LAGE3) enables affinity purification and localization studies. Tagged knock-in models are valuable for interactomics and live-cell imaging.
Overexpression
Overexpression of PRAME or LAGE3 can mimic cancer-associated upregulation and reveal interactions with Cullin2 or VEGFA mRNA. These models help study gain-of-function effects in tumorigenesis.
How EDITGENE Supports EKC/KEOPS complex Research
Researchers studying EKC/KEOPS complex-related genes often need to determine whether a candidate gene is causally involved in tRNA modification, translation, or disease. EDITGENE provides comprehensive CRISPR services to generate precisely engineered cell models for functional validation.
Contact EDITGENE today to design your custom CRISPR model for EKC/KEOPS complex research.
Frequently Asked Questions About EKC/KEOPS complex
What is the EKC/KEOPS complex?
The EKC/KEOPS complex (GO:0000408) is a protein complex involved in the t6A modification of tRNA, conserved from archaea to humans.
What genes are involved in the EKC/KEOPS complex?
In yeast, the complex includes BUD32, KAE1, GON7, CGI121, and PCC1; human orthologs include PRPK, OSGEP, GON7, TPRKB, and LAGE3.
What is the function of GO:0000408?
Its primary function is catalyzing threonylcarbamoyladenosine (t6A) modification on tRNAs, which ensures translation fidelity.
How is the EKC/KEOPS complex linked to cancer?
The human complex is recruited to Cullin2 ubiquitin ligases by the tumor antigen PRAME, and LAGE3 promotes angiogenesis in hepatocellular carcinoma.
What diseases are associated with EKC/KEOPS complex mutations?
Dysregulation is linked to cancer and developmental defects, though direct human mutations are still being characterized.
How can I study the EKC/KEOPS complex using CRISPR?
CRISPR knockout, point mutation, knock-in, and overexpression models can be used to dissect subunit function and tRNA modification.
What model organisms are used for EKC/KEOPS research?
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, and human cell lines are commonly used.
What is t6A modification?
t6A is a chemical modification at position 37 of tRNAs that decode ANN codons, essential for accurate translation.
Does the EKC/KEOPS complex affect cell polarity?
Yes, in Saccharomyces cerevisiae, the complex is required for proper localization of the Bud9 landmark protein.
What methods are used to measure EKC/KEOPS activity?
tRNA sequencing, Ribo-seq, mass spectrometry, and imaging are standard methods.
Conclusion
The EKC/KEOPS complex (GO:0000408) is a conserved cellular machine essential for t6A tRNA modification and translation fidelity. Its subunits are implicated in diverse processes, from cell polarity and centromere regulation to cancer-associated pathways. Understanding its molecular mechanism and disease relevance requires precise genetic models, which CRISPR technologies can provide. Future research will likely uncover additional roles for this complex in human health and disease.
References
- 1. Xu X et al.. 2019. Negative Regulation of the Mis17-Mis6 Centromere Complex by mRNA Decay Pathway and EKC/KEOPS Complex in Schizosaccharomyces pombe.. G3 (Bethesda) 9(6):1815-1823 PMID: 30967422
- 2. Rojas-Benítez D et al.. 2013. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.. Fly (Austin) 7(3):168-72 PMID: 23823807
- 3. Costessi A et al.. 2012. The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the human tumour antigen PRAME.. PLoS One 7(8):e42822 PMID: 22912744
- 4. Kato Y et al.. 2011. Cell polarity in Saccharomyces cerevisiae depends on proper localization of the Bud9 landmark protein by the EKC/KEOPS complex.. Genetics 188(4):871-82 PMID: 21625000
- 5. Daugeron MC et al.. 2011. Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.. Nucleic Acids Res 39(14):6148-60 PMID: 21459853
- 6. Hecker A et al.. 2008. Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex.. EMBO J 27(17):2340-51 PMID: 19172740
- 7. Li J et al.. 2021. Crystal structure of the human PRPK-TPRKB complex.. Commun Biol 4(1):167 PMID: 33547416
- 8. Cong Z et al.. 2024. LAGE3 promotes angiogenesis on hepatocellular carcinoma by stabilizing VEGFA mRNA.. Biochim Biophys Acta Mol Basis Dis 1870(5):167196 PMID: 38653358
