In a survey of yeast genomic sequences encoding calcium- and phospholipid-binding C2 domains, three homologous genes were identified that encode proteins that each have three C2 domains and an apparent transmembrane domain near the N terminus. The name tricalbins is suggested for these proteins, corresponding to the open reading frames YOR086c (TCB1), YNL087w (TCB2), and YML072c (TCB3). An antiserum was raised against the C-terminal portion of tricalbin 2 and used on Western blots to demonstrate that the corresponding protein is expressed in yeast and appears as a high-molecular-weight band at 130 kDa with smaller fragments at 39 kDa and 46 kDa. A fusion protein consisting of full length tricalbin 2 fused to the green fluorescent protein was expressed in cells and found to traffic from the cell surface to intracellular vesicles near the vacuole. A two-hybrid interaction screen with the C-terminal portion of tricalbin 2 indicated that tricalbin 2 binds the C-terminal portions of tricalbins 1 and 3 suggesting that the tricalbins may form heterodimers in vivo. Tricalbin 2 also interacted with the activation domain of the pleiotropic drug resistance transcription factor Pdr1p. Combinatorial disruptions of the tricalbin genes revealed that tcb2 single mutants or tcb1, tcb3 double mutants have an altered vacuole morphology and are hypersensitive to cycloheximide. A screen for single-copy suppressors of the cycloheximide sensitivity of tricalbin mutants yielded RSP5, which encodes a C2-domain-containing, ubiquitin-conjugating ligase essential for receptor-mediated and fluid phase endocytosis. The results suggest that the tricalbins function as multimers in membrane-trafficking events and may provide insights into the roles of multi-C2-domain proteins, such as the synaptotagmins, in other organisms.
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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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