Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 38, Pages 28210-28221Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M605129200
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The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knockout strain (fur4 Delta fui1 Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p- mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry. Mutagenesis analysis of three charged amino acids (Glu(259), Lys(288), and Asp(474) in putative transmembrane segments 3, 4, and 7, respectively) revealed that only Lys288 was required for maintaining high Urd transport efficiency. Analysis of binding energies between Fui1p and different Urd analogs indicated that Fuip1 interacted with C( 3')-OH, C(2')-OH, C(5)-H, and N(3)-H of Urd. Fui1p- mediated transport of Urd was inhibited by analogs with modifications at C-5', but was not inhibited significantly by analogs with modifications at C-3', C-5, and N-3orinversionsofconfigurationatC-2' andC-3'. This characterization of Fui1p contributes to the emerging knowledge of the structure and function of the Fur family of permeases, including the Fui1p orthologs of pathogenic fungi.
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