The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.
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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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