In recent years, a growing number of proteins have been shown to be localized in more than one subcellular location, although encoded from a single gene. Fundamental aspects in the research of such dual-distributed proteins involve determination of their subcellular localization and their location-specific functions. The lack of sensitive and suitable tools to address these issues has led us to develop a novel tool for functional detection of cytosolic/nuclear isoproteins in the cell, which we term location-specific depletion or subcellular knockout. The depletion of the protein occurs post-translationally via degradation by the ubiquitin-proteasome system, which operates only in the cytosol and the nucleus. As an example, we fused the yeast tricarboxylic acid (TCA) cycle enzyme aconitase to a degron sequence (SL17) recognizable by the ubiquitin-proteasome system. This fusion resulted in the degradation of the cytosolic enzyme, specifically eliminating its activity within the cytosolic glyoxylate shunt without disrupting the protein's activity within the mitochondrial TCA cycle. We show that the degradation of the fusion protein can be attributed specifically to the ubiquitin-proteasome system and that inhibition of this degradation restores its cytosolic activity. This novel tool can be used to detect small subpopulations of dual-targeted proteins, thereby revealing isoproteins that were considered to be confined to a single compartment. The particular advantage of this specific subcellular depletion is that it can reveal the functions of the cytosolic/nuclear isoproteins.
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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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