This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protein interactions that can be used to identify small ubiquitin-related modifier (SUMO) interacting proteins and to determine the nature of the SUMO-protein interactions that occur. SUMO binds to a protein in two different ways: covalently and noncovalently. In a covalent interaction an isopeptide bond forms between the glycine residue at the C terminus of the mature SUMO and a lysine side-chain on the substrate protein. Alternatively, SUMO can interact noncovalently with another protein, usually via insertion of a beta strand from a substrate SUMO-interacting motif (SIM) into a hydrophobic groove next to the SUMO beta2 strand. By mutating either the C-terminal diglycine motif or amino acids within the beta2 strand of SUMO, these respective interactions can be abolished. The expression of the two-hybrid SUMO constructs with either of these mutations can help distinguish the type of interaction that occurs between a SUMO and a given protein. Sumoylation can be verified by independent methods, such as a SUMO mobility shift assay. Finally, the chapter will compare the two-hybrid approach with mass spectrometric analysis as a means of identifying SUMO-interacting proteins.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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