Only one of the charged amino acids located in membrane-spanning regions is important for the function of the Saccharomyces cerevisiae uracil permease
| Autor: | Pinson, B, Chevallier, J, Urban-Grimal, D |
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| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Sequence Homology Amino Acid Lysine Cell Membrane Molecular Sequence Data Membrane Proteins Membrane Transport Proteins Biological Transport Saccharomyces cerevisiae Nucleotide Transport Proteins Mutagenesis Site-Directed Amino Acid Sequence Uracil Research Article Protein Binding |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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