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

Jing Zhang,Kyla M Smith,Tracey Tackaberry,Xuejun Sun,Pat Carpenter,Melissa D Slugoski,Morris J Robins,Lars P.C Nielsen,Ireneusz Nowak,Stephen A Baldwin,James D Young,Carol E Cass

doi:10.1074/jbc.m605129200

Abstract

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 (fur4Deltafui1Delta) 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 Lys(288) 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.

Highlights

Sides and cytotoxic nucleoside analogs [1– 4]
Fui1p orthologs of Candida albicans and Candida glabrata with high sequence identities to Fui1p of S. cerevisiae (Ͼ70%) were revealed from contigs of the Stanford C. albicans genome sequence data bank and the assembled open reading frame (ORF) data bank of the C. glabrata genome (GenBankTM GI:50287475) [25], respectively
We report here the functional characterization of Fui1p in a double-permease knock-out yeast strain that enabled us to analyze Fui1p-mediated Urd uptake in an otherwise nucleoside transport-free background

Summary

Introduction

Sides and cytotoxic nucleoside analogs [1– 4]. Mammalian nucleoside transporters are classified into two structurally unrelated protein families, the concentrative (CNTs) and equilibrative (ENTs) nucleoside transporters [1, 2, 5]. Nucleoside permeation into Saccharomyces cerevisiae is mediated by Fui1p, a permease with high specificity for Urd and with no sequence similarities to any of the mammalian nucleoside transporters [6, 7]. S. cerevisiae cells salvage nucleobases through Fur4p (uracil permease) and Fyc2p (purine-cytosine permease), but they appear to lack the capacity to transport thymidine and purine nucleosides across plasma membranes [8]. Based on the high sequence identity of Fur4p and Fui1p, we hypothesized that these two transporters might have similar transport mechanisms and that Fui1p might operate as an electrogenic proton/permeant symporter. Knowledge of the transport mechanism and permeant selectivities of Fui1p will contribute to an understanding of its physiological significance in the budding yeast S. cerevisiae, one of the most important model organisms for DNA replication and repair studies. Efforts to develop more effective nucleoside analogs are under way [27]

Methods

Results

Conclusion