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J. Biol. Chem., Vol. 281, Issue 38, 28210-28221, September 22, 2006

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Characterization of the Transport Mechanism and Permeant Binding Profile of the Uridine Permease Fui1p of Saccharomyces cerevisiae^*

Jing Zhang^¶1, Kyla M. Smith^||, Tracey Tackaberry^¶, Xuejun Sun^¶, Pat Carpenter^¶, Melissa D. Slugoski^||, Morris J. Robins^**2, Lars P. C. Nielsen^**3, Ireneusz Nowak^**, Stephen A. Baldwin, James D. Young^||4, and Carol E. Cass^¶5

From the Membrane Protein Research Group and the Departments of Oncology and ^||Physiology, University of Alberta, and the ^¶Cross Cancer Institute, Edmonton, Alberta T6H 1Z2, Canada, the ^**Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, and the Astbury Centre for Structural Molecular Biology, Institute for Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

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 knock-out strain (fur4fui1) 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 (Glu²⁵⁹, Lys²⁸⁸, and Asp⁴⁷⁴ 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-3 or inversions of configuration at C-2' and C-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.

Received for publication, May 30, 2006 , and in revised form, July 17, 2006.

^* This work was supported in part by Canadian Cancer Society grants from the National Cancer Institute of Canada (to C. E. C. and J. D. Y.), the Canadian Institutes of Health Research (to C. E. C. and J. D. Y.), and pharmaceutical company unrestricted gift funds (to M. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by studentships from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research and Department of Oncology Endowed Studentship.

² J. Rex Goates Professor of Chemistry.

³ Supported by a Brigham Young University undergraduate research fellowship.

⁴ Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

⁵ Canada Research Chair in Oncology. To whom correspondence should be addressed: Dept. of Oncology, Cross Cancer Inst., 11540 University Ave., Edmonton, Alberta T6H 1Z2, Canada. Tel.: 780-432-8320; Fax: 780-432-8425; E-mail: carol.cass{at}cancerboard.ab.ca.

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