The transport of uracil into the yeast Saccharomyces cerevisiae is mediated by uracil permease, a specific co-transporter encoded by the FUR4 gene. Uracil permease is a multispan membrane protein that is delivered to the plasma membrane via the secretory pathway. Experimental results led to the proposal of a two-dimensional model of the protein's topology. According to this model, the membrane domain of Fur4p contains three charged amino acid residues (Glu-243, Lys-272 and Glu-539) that are conserved in the members of the FUR family of yeast transporters. We have previously shown that a mis-sense mutation leading to the replacement of Lys-272 by Glu severely impairs the function of uracil permease. In the present paper, the role of the three charged residues present in the membrane-spanning regions of Fur4p was further investigated by using site-directed mutagenesis. The variant permeases were correctly targeted to the plasma membrane and their stabilities were similar to that of the wild-type permease. The effect of the mutations was studied by measuring the uptake constants for uracil on whole cells and equilibrium binding parameters on plasma membrane-enriched fractions. We found no evidence for ionic interaction between either of the glutamic residues in transmembrane segments 3 and 9 and the lysine residue in transmembrane segment 4. Of the three charged residues, only Lys-272 was important for the transport activity of the transporter. Its replacement by Ala, Glu or even Arg strongly impaired both the binding and the translocation of uracil.
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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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