Using a coimmunoprecipitation strategy, we showed that the Cdc34 ubiquitin (Ub)-conjugating enzyme from Saccharomyces cerevisiae self-associates in cell lysates, thereby indicating an in vivo interaction. The ability of Cdc34 to interact with itself is not dependent on its association with the ubiquitin ligase Skp1-Cdc53/Cul1-Hrt1-F-box complex. Rather, this interaction depends upon the integrity of the Cdc34-Ub thiolester. Furthermore, several principal determinants within the Cdc34 catalytic domain, including the active-site cysteine, amino acid residues S73 and S97, and its catalytic domain insertion, also play a role in self-association. Mutational studies have shown that these determinants are functionally important in vivo and operate at the levels of both Cdc34-Ub thiolester formation and Cdc34-mediated multi-Ub chain assembly. These determinants are spatially situated in a region that is close to the active site, corresponding closely to the previously identified E2-Ub interface. These observations indicate that the formation of the Cdc34-Ub thiolester is important for Cdc34 self-association and that the interaction of Cdc34-Ub thiolesters is in turn a prerequisite for both multi-Ub chain assembly and Cdc34's essential function(s). A conclusion from these findings is that the placement of ubiquitin on the Cdc34 surface is a structurally important feature of Cdc34's function.
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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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