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

2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês

10.1074/jbc.m605129200

ISSN

1083-351X

Autores

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

Tópico(s)

Enzyme-mediated dye degradation

Resumo

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 (fur4Δfui1Δ) 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. 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 (fur4Δfui1Δ) 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. Nucleoside transporters are integral membrane proteins that mediate the uptake and release of naturally occurring nucleosides and cytotoxic nucleoside analogs (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 2Cass C.E. Young J.D. Baldwin S.A. Cabrita M.A. Graham K.A. Griffiths M. Jennings L.L. Mackey J.R. Ng A.M.L. Ritzel M.W.L. Vickers M.F. Yao S.Y.M. Amidon G.L. Sadée W. Membrane Transporters as Drug Targets. 1st. Kluwer Academic/Plenum Publishers, New York1999Google Scholar, 3Landfear S.M. Ullman B. Carter N.S. Sanchez M.A. Eukaryot. Cell. 2004; 3: 245-254Crossref PubMed Scopus (94) Google Scholar, 4Pastor-Anglada M. Molina-Arcas M. Casado F.J. Bellosillo B. Colomer D. Gil J. Leukemia (Basingstoke). 2004; 18: 385-393Crossref PubMed Scopus (92) Google Scholar). Mammalian nucleoside transporters are classified into two structurally unrelated protein families, the concentrative (CNTs) 6The abbreviations used are: CNTs, concentrative nucleoside transporters; ENTs, equilibrative nucleoside transporters; TM, transmembrane; h, human; ORF, open reading frame; GFP, green fluorescent protein; CMM, complete minimal medium; FUrd, 5-fluorouridine; ChCl, choline chloride; MES, 4-morpholineethanesulfonic acid; CCCP, carbonyl cyanide m-chloro-phenylhydrazone; MeUrd, methyluridine; ddUrd, dideoxyuridine.6The abbreviations used are: CNTs, concentrative nucleoside transporters; ENTs, equilibrative nucleoside transporters; TM, transmembrane; h, human; ORF, open reading frame; GFP, green fluorescent protein; CMM, complete minimal medium; FUrd, 5-fluorouridine; ChCl, choline chloride; MES, 4-morpholineethanesulfonic acid; CCCP, carbonyl cyanide m-chloro-phenylhydrazone; MeUrd, methyluridine; ddUrd, dideoxyuridine. and equilibrative (ENTs) nucleoside transporters (1Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 2Cass C.E. Young J.D. Baldwin S.A. Cabrita M.A. Graham K.A. Griffiths M. Jennings L.L. Mackey J.R. Ng A.M.L. Ritzel M.W.L. Vickers M.F. Yao S.Y.M. Amidon G.L. Sadée W. Membrane Transporters as Drug Targets. 1st. Kluwer Academic/Plenum Publishers, New York1999Google Scholar, 5Vickers M.F. Young J.D. Baldwin S.A. Cass C.E. Emerg. Ther. Targets. 2000; 4: 515-539Crossref Scopus (14) Google Scholar). 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 (6Vickers M.F. Yao S.Y.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; 275: 25931-25938Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Wagner R. de Montigny J. de Wergifosse P. Souciet J.L. Potier S. FEMS Microbiol. Lett. 1998; 159: 69-75Crossref PubMed Google Scholar). S. cerevisiae cells also 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 (8Visser F. Zhang J. Raborn R.T. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2005; 67: 1291-1298Crossref PubMed Scopus (36) Google Scholar). Although considerable information is available for the Fur4p and Fyc2p nucleobase transporters of S. cerevisiae (9Volland C. Garnier C. Haguenauer-Tsapis R. J. Biol. Chem. 1992; 267: 23767-23771Abstract Full Text PDF PubMed Google Scholar, 10Seron K. Blondel M.O. Haguenauer-Tsapis R. Volland C. J. Bacteriol. 1999; 181: 1793-1800Crossref PubMed Google Scholar, 11Volland C. Urban-Grimal D. Geraud G. Haguenauer-Tsapis R. J. Biol. Chem. 1994; 269: 9833-9841Abstract Full Text PDF PubMed Google Scholar, 12Pinson B. Pillois X. Brethes D. Chevallier J. Napias C. Eur. J. Biochem. 1996; 239: 439-444Crossref PubMed Scopus (18) Google Scholar, 13Pinson B. Napias C. Chevallier J. Van den Broek P.J. Brethes D. J. Biol. Chem. 1997; 272: 28918-28924Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 14Pinson B. Chevallier J. Urban-Grimal D. Biochem. J. 1999; 339: 37-42Crossref PubMed Google Scholar, 15Eddy A.A. Hopkins P. Biochem. J. 1998; 336: 125-130Crossref PubMed Scopus (5) Google Scholar, 16Marchal C. Haguenauer-Tsapis R. Urban-Grimal D. J. Biol. Chem. 2000; 275: 23608-23614Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17Garnier C. Blondel M.O. Haguenauer-Tsapis R. Mol. Microbiol. 1996; 21: 1061-1073Crossref PubMed Scopus (23) Google Scholar), relatively little is known about Fui1p. Fui1p belongs to the uracil/allantoin permease family (Fur family) of yeast, which also includes Fur4p, Thi10p (thiamine permease), and Dal4p (allantoin permease). Fui1p (629 amino acids, 72 kDa) shares high amino acid identity (50-60%) with the other family members. The predicated topology of Fur4p consists of 10 transmembrane (TM) segments with long N- and C-terminal tails, which have been shown to be intracellular (17Garnier C. Blondel M.O. Haguenauer-Tsapis R. Mol. Microbiol. 1996; 21: 1061-1073Crossref PubMed Scopus (23) Google Scholar). It is believed that the two-dimensional Fur4p structural model could be extended to all members of the yeast uracil/allantoin permease family (14Pinson B. Chevallier J. Urban-Grimal D. Biochem. J. 1999; 339: 37-42Crossref PubMed Google Scholar). The similarity of amino acid sequences is greatest in the putative TM segments of the four proteins. 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. Charged amino acid residues in the membrane-spanning regions of transporters are known to play important roles in permeant binding (18Unkles S.E. Rouch D.A. Wang Y. Siddiqi M.Y. Glass A.D. Kinghorn J.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17549-17554Crossref PubMed Scopus (37) Google Scholar, 19Muth T.R. Schuldiner S. EMBO J. 2000; 19: 234-240Crossref PubMed Scopus (164) Google Scholar), proton coupling (20Grewer C. Watzke N. Rauen T. Bicho A. J. Biol. Chem. 2003; 278: 2585-2592Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), transporter stability and activity (21Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5539-5543Crossref PubMed Scopus (74) Google Scholar), and plasma membrane targeting (22Arastu-Kapur S. Ford E. Ullman B. Carter N.S. J. Biol. Chem. 2003; 278: 33327-33333Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Although Fur4p contains three charged amino acid residues in TM regions, only the one located in TM segment 4 (Lys272), which is highly conserved in the uracil/allantoin permease family, was identified as a critical residue involved in uracil binding and translocation (14Pinson B. Chevallier J. Urban-Grimal D. Biochem. J. 1999; 339: 37-42Crossref PubMed Google Scholar). The functional importance of the corresponding lysine residue of Fui1p (Lys288) in TM segment 4 and of the only two other charged TM residues (in TM segments 3 and 7) was investigated in this study. In vivo labeling of DNA using nucleosides and nucleoside analogs such as thymidine and 5-bromo-2′-dUrd has long been a cornerstone of replication studies. S. cerevisiae has been used extensively as a model organism in defining the genetic elements required for DNA replication. In the absence of the introduction of heterogeneous nucleoside transporters (e.g. human (h) ENT1) (23Vernis L. Piskur J. Diffley J.F. Nucleic Acids Res. 2003; 31: e120Crossref PubMed Scopus (46) Google Scholar), Fui1p is the dominant route that allows entry of nucleosides and nucleoside analogs into S. cerevisiae. Among the nucleoside transporters identified so far from bacteria to higher eukaryotes, only S. cerevisiae Fui1p mediates transport of Urd but not that of other naturally occurring pyrimidine and purine nucleosides, implying a specialized function for Urd in S. cerevisiae. The abundance of Fur4p is determined by extracellular uracil availability by regulation of the efficiency of its ubiquitylation (24Blondel M.O. Morvan J. Dupre S. Urban-Grimal D. Haguenauer-Tsapis R. Volland C. Mol. Biol. Cell. 2004; 15: 883-895Crossref PubMed Scopus (99) Google Scholar). Fui1p has also been shown to be sorted for early vacuolar degradation in cells exposed to toxic levels of Urd, indicating that extracellular Urd controls Fui1p trafficking and prevents harmful Urd uptake that results in a decrease in growth rate (24Blondel M.O. Morvan J. Dupre S. Urban-Grimal D. Haguenauer-Tsapis R. Volland C. Mol. Biol. Cell. 2004; 15: 883-895Crossref PubMed Scopus (99) Google Scholar). 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. Fui1p orthologs of Candida albicans and Candida glabrata with high sequence identities to Fui1p of S. cerevisiae (>70%) were revealed from contigs (groups of overlapping clones) of the Stanford C. albicans genome sequence data bank and the assembled open reading frame (ORF) data bank of the C. glabrata genome (GenBank™ GI:50287475) (25Dujon B. Sherman D. Fischer G. Durrens P. Casaregola S. Lafontaine I. de Montigny J. Marck C. Neuveglise C. Talla E. Goffard N. Frangeul L. Aigle M. Anthouard V. Babour A. Barbe V. Barnay S. Blanchin S. Beckerich J.M. Beyne E. Bleykasten C. Boisrame A. Boyer J. Cattolico L. Confanioleri F. De Daruvar A. Despons L. Fabre E. Fairhead C. Ferry-Dumazet H. Groppi A. Hantraye F. Hennequin C. Jauniaux N. Joyet P. Kachouri R. Kerrest A. Koszul R. Lemaire M. Lesur I. Ma L. Muller H. Nicaud J.M. Nikolski M. Oztas S. Ozier-Kalogeropoulos O. Pellenz S. Potier S. Richard G.F. Straub M.L. Suleau A. Swennen D. Tekaia F. Wesolowski-Louvel M. Westhof E. Wirth B. Zeniou-Meyer M. Zivanovic I. Bolotin-Fukuhara M. Thierry A. Bouchier C. Caudron B. Scarpelli C. Gaillardin C. Weissenbach J. Wincker P. Souciet J.L. Nature. 2004; 430: 35-44Crossref PubMed Scopus (1234) Google Scholar), respectively. One of the most commonly encountered human pathogens, C. albicans causes a wide variety of infections, ranging from superficial disorders in generally healthy individuals to invasive, rapidly fatal systemic infections in individuals with impaired immunity. C. glabrata has emerged as the second causative agent of human candidiasis worldwide and is more resistant to drug therapy than C. albicans. Few classes of drugs are effective against these fungal infections, and drug efficacy is limited by toxicity and side effects. Nucleoside antibiotics such as the nikkomycins and neopolyoxins have been considered as candidate inhibitors of opportunistic candidal infections in AIDS and organ transplant patients (26Kimura K. Bugg T.D. Nat. Prod. Rep. 2003; 20: 252-273Crossref PubMed Scopus (184) Google Scholar). Efforts to develop more effective nucleoside analogs are under way (27Rapp R.P. Pharmacotherapy. 2004; 24 (quiz 29S-32S): 4S-28SCrossref PubMed Scopus (97) Google Scholar). We report here the functional characterization of Fui1p in a double-permease knock-out yeast strain (fur4Δfui1Δ) that enabled us to analyze Fui1p-mediated Urd uptake in an otherwise nucleoside transport-free background. Fui1p transported Urd into yeast with high affinity and high capacity in a protondependent manner. The roles of three charged amino acid residues (Glu259, Lys288, and Asp474) in putative TM segments 3, 4, and 7, as well as the cellular location of the mutant transporters, were investigated using site-directed mutagenesis and green fluorescent protein (GFP) and c-Myc tags. Of the three charged residues, only Lys288 was important for the transport capacity of Fui1p. A quantitative inhibitor sensitivity assay was used to gain an understanding of the structural regions of Urd that interact with Fui1p. Because transportability is a potential determinant of the cytotoxic efficacy of nucleoside analog drugs, knowledge of the Urd binding profile of Fui1p will guide the design of novel antifungal nucleoside analogs that may selectively target Fui1p orthologs in pathogenic fungi. Strains and Media—BY4742-YBR021W (MATα, his3, leu2, lys2, ura3, fur4Δ), which contains a disruption in FUR4, the gene encoding the endogenous uracil permease, was purchased from the American Type Culture Collection (Manassas, VA) and used as the parental yeast strain to generate the double-permease knock-out strain fur4Δfui1Δ (previously named fui1::HIS3) by deleting FUI1 using the PCR-mediated one-step gene disruption method as described previously (28Zhang J. Smith K.M. Tackaberry T. Visser F. Robins M.J. Nielsen L.P. Nowak I. Karpinski E. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2005; 68: 830-839Crossref PubMed Scopus (35) Google Scholar). Other strains were generated by transformation of the yeast-Escherichia coli shuttle vector pYPGE15 (29Brunelli J.P. Pall M.L. Yeast. 1993; 9: 1309-1318Crossref PubMed Scopus (92) Google Scholar) into fur4Δfui1Δ using a standard lithium acetate method (30Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Yeast strains were maintained in complete minimal medium (CMM) containing 0.67% yeast nitrogen base (Difco), amino acids (as required to maintain auxotrophic selection), and 2% glucose (CMM/Glc). Agar plates contained CMM with various supplements and 2% agar (Difco). Plasmids were propagated in E. coli strain TOP10F′ (Invitrogen) and maintained in Luria broth with ampicillin (100 μg/ml). Plasmid Construction—All oligonucleotide primers were synthesized by Invitrogen. For S. cerevisiae expression, the FUI1 ORFs were amplified from vector pYSE2-FUI1 (6Vickers M.F. Yao S.Y.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; 275: 25931-25938Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) by PCR methodology using primers 5′-XbaI-FUI1 (5′-CTG TCT AGA ATG CCG GTA TCT GAT TCT GGA TTC-3′, with the restriction site underlined) and 3′-XhoI-FUI1 (5′-CGA CTC GAG TTA GAT ATA TCG TAT CTT TTC ATA GC-3′). For construction of c-Myc-tagged Fui1p, the c-Myc tag (CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC) was introduced into primer 3′-XhoI-FUI1. For Xenopus oocyte expression, primers 5′-BamHI-FUI1 (5′-GTC GGA TCC ATG CCG GTA TCT GAT TCT GGA TTC-3′) and 3′-XbaI-FUI1 (5′-CGA TCT AGA TTA GAT ATA TCG TAT CTT TTC ATA G-3′) were used. To construct GFP-tagged Fui1p, the ORF of FUI1 without a stop codon was first amplified using forward primer 5′-XbaI-FUI1 and a reverse primer containing (3′ to 5′) 21 bases with homology to FUI1 and a unique tag sequence complementary to the first 50 nucleotides of the ORF of GFP. C-terminally GFP-tagged Fui1p was obtained by overlapping PCR using the product of the first run PCR and the pGFPuv vector (Promega, Madison, WI) as templates and 5′-XbaI-FUI1 and 3′-KpnI-GFP (5′-CTG GGT ACC CTA TTT GTA GAG CTC ATC CAT GCC) as primers. GFP-ORF was also amplified by PCR using forward primer 5′-XbaI-GFP (5′-CGT TCT AGA ATG GCC AGC AAA GGA GAA CTT-3′) and reverse primer 3′-EcoRI-GFP (5′-CGT GAA TTC CTA TTT GTA GAG CTC ATC CAT GCC). The amplified ORFs were inserted into pYPGE15 (a high copy number episomal yeast vector that expresses the inserted DNA constitutively under the transcriptional control of the phosphoglycerate kinase promoter) to generate pYPFUI1, pYP-FUI1-GFP, and pYPGFP or into the Xenopus expression vector pGEM-HE to generate pGEFUI1. pYPFUI1-K288A, pYPFUI1-K288E, pYPFUI1-K288R, pYPFUI1-E259A, pYPFUI1-D474A, and pYPFUI1-E259A,D474A and the corresponding GFP- and c-Myc-tagged versions were generated using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The PCRs were performed using Pwo polymerase (Roche Applied Science), and all constructs were verified by DNA sequencing using an ABI PRISM 310 sequence detection system (PerkinElmer Life Sciences). Nucleoside Transport in S. cerevisiae—The uptake of [3H]Urd or 5-[3H]fluorouridine (FUrd; Moravek Biochemicals Inc., Brea, CA) into logarithmically proliferating yeast cells was measured using a cell harvester as described previously (31Zhang J. Visser F. Vickers M.F. Lang T. Robins M.J. Nielsen L.P. Nowak I. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2003; 64: 1512-1520Crossref PubMed Scopus (56) Google Scholar, 32Visser F. Baldwin S.A. Isaac R.E. Young J.D. Cass C.E. J. Biol. Chem. 2005; 280: 11025-11034Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Yeast cells containing pYPFUI1, pYPFUI1-GFP, or individual mutant transporters were grown in CMM/Glc to A600 = 0.7-0.9, washed twice with fresh medium, and resuspended to A600 = 4.0. Uptake assays were performed at room temperature at pH 4.5 unless specified otherwise by adding 50-μl portions of yeast suspensions to 50-μl portions of twice concentrated 3H-labeled nucleoside in CMM/Glc in 96-well microtiter plates. Yeast cells were collected on filter mats using a Micro96 cell harvester (Skatron Instruments, Lier, Norway) and rapidly washed with deionized water. The individual filter circles corresponding to wells of the microtiter plates were removed from filter mats and transferred to vials for scintillation counting. The binding of Urd and its analogs to recombinant Fui1p was assessed by measuring their abilities to inhibit inward transport of 1 μm [3H]Urd in an "inhibitor sensitivity" assay as follows. Yeast cells producing Fui1p were incubated with 1 μm [3H]Urd for 30 s in the absence or presence of graded concentrations of Urd or Urd analogs. The 30-s exposures to [3H]Urd were shown in time course experiments to be sufficient to provide true initial rates of uptake into yeast cells, thereby providing rates of transport across plasma membranes rather than rates of intracellular metabolism (see Fig. 1, upper panel). Each experiment was repeated at least three times. Nonspecific radioactivity was determined in the presence of 10 mm nonradioactive Urd, and these values were subtracted from total uptake values. Data were subjected to nonlinear regression analysis using GraphPad Prism software (Version 3.0; GraphPad Software, Inc., San Diego, CA) to obtain IC50 values (concentrations that inhibited reactions by 50%) for Urd and Urd analogs. Ki (inhibitory constant) values were determined from the Cheng-Prusoff equation (33Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12132) Google Scholar) and the Km values for Urd. Gibbs free energy (ΔG°) was calculated from ΔG° =-RT ln(Ki), where R is the gas constant and T is the absolute temperature. The thermodynamic stability of transporter-inhibitor complexes was quantitatively estimated from ΔG° as described (34De Koning H.P. Jarvis S.M. Acta Trop. 2001; 80: 245-250Crossref PubMed Scopus (56) Google Scholar). Measurements of Fui1p-induced H+ Currents and H+/Urd Coupling Ratios—pGEFUI1 was linearized with NheI and transcribed with T7 polymerase using the mMESSAGE mMACHI-NE™ transcription system (Ambion, Inc., Austin, TX). In vitro synthesized transcripts were injected into isolated mature stage VI oocytes from Xenopus laevis as described previously (35Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.-Z. Karpinski E. Young J.D. J. Physiol. 2004; 558: 807-823Crossref PubMed Scopus (79) Google Scholar). Mock-injected oocytes were injected with water alone. Electrophysiological studies used transport medium in which choline was substituted for sodium, i.e. 100 mm choline chloride (ChCl), 2 mm KCl, 1 mm CaCl2, 1mm MgCl2, and 10 mm HEPES (for pH values >6.5) or 10 mm MES (for pH values ≤6.5). Proton dependence was tested in ChCl-containing transport medium at pH 4.5-8.5. Urd-induced membrane currents were measured in Fui1p-producing oocytes at room temperature (20 °C) using a GeneClamp 500B oocyte clamp (Axon Instruments, Foster City, CA) in the two-electrode voltage-clamp mode as described previously (35Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.-Z. Karpinski E. Young J.D. J. Physiol. 2004; 558: 807-823Crossref PubMed Scopus (79) Google Scholar) and interfaced to an IBM-compatible PC via a Digidata 1200A/D converter and controlled by pCLAMP software (Version 8.0; Axon Instruments). The microelectrodes were filled with 3 m KCl and had resistances that ranged from 0.5 to 2.5 mΩ megaohms. Oocytes were penetrated with the microelectrodes, and their membrane potentials were monitored for periods of 10-15 min. Oocytes were discarded when membrane potentials were unstable or more positive than -30 mV. The oocyte membrane potential was clamped at a holding potential of -50 mV, and Urd was added in the appropriate transport medium. Current signals were filtered at 20 Hz (four-pole Bessel filter) and sampled at intervals of 20 ms. For data presentation, the signals were further filtered at 0.5 Hz by the pCLAMP program suite. The H+/Urd coupling ratio for Fui1p was determined by simultaneously measuring H+ currents and uptake of [14C]Urd (200 μm, 1 μCi/ml; Amersham Biosciences) under voltage-clamp conditions. Individual Fui1p-producing oocytes were placed in a perfusion chamber and voltage-clamped at a holding potential of -50 mV in sodium-free (100 mm ChCl) and nucleoside-free medium (pH 5.5) for a 10-min period to monitor base-line currents. When the base line was stable, the nucleoside-free medium was exchanged with medium of the same composition containing [14C]Urd. Current was measured for 2 min, and uptake was terminated by washing the oocyte with nucleoside-free medium until the current returned to the base line. The oocyte was then transferred to a scintillation vial and solubilized with 1% (w/v) SDS for quantitation of oocyte-associated radioactivity. Urd-induced current was calculated as the difference between base-line current and total inward current. The total charge translocated into the oocyte during the uptake period was calculated from the current-time integral and correlated with the measured radiolabeled flux for each oocyte to determine the charge/uptake ratio. Basal [14C]Urd uptake was determined in control water-injected oocytes (from the same donor frog) under equivalent conditions and used to correct for endogenous non-mediated nucleoside uptake over the same incubation period. Coupling ratios (±S.E.) were calculated from slopes of least-squares fits of Urd-dependent charge versus Urd accumulation in oocytes. Confocal Microscopy of Yeast—Logarithmically growing yeast cells transformed with a GFP-tagged vector (10 μm, A600 = 0.7-0.9) were mixed with 30 μl of anti-fading mounting medium, smeared on a glass slide, and checked for green fluorescence using an excitation wavelength of 488 nm. Confocal images were collected using a Zeiss LSM510 confocal laser scanning microscope with a 63 × 1.4 objective (Plan-Apochromat) using a frame size of 512 × 512 pixels with a pixel resolution of 0.08 μm and a pixel depth of 8 bits. Isolation of Plasma Membranes and Immunoblotting—Yeast membranes were fractionated on sucrose gradients as described (36Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1997Google Scholar). Briefly, 1 liter of yeast cells at A600 = 1 was collected, washed with sucrose breaking buffer (0.4 m sucrose, 1 mm EDTA, and 10 mm Tris (pH 7.4)) containing additional protease inhibitors (Complete protease inhibitor mixture, Roche Applied Science), and lysed by vortexing in the presence of glass beads (425-600 μm; Sigma) for 15 min at 4 °C. Unbroken cells and glass beads were removed from lysates by centrifugation at 500 × g for 20 min at 4 °C, and membrane fractions were obtained by centrifugation of lysates at 21,000 × g for 40 min at 4 °C. The resulting crude membrane pellets were resuspended in sucrose breaking buffer containing protease inhibitors. The crude membranes were layered onto a stepwise sucrose gradient (0.4, 1.1, 1.65, and 2.25 m sucrose) containing 10 mm Tris, 1 mm EDTA (pH 7.4), and protein inhibitor mixture. After centrifugation at 80,000 × g (Beckman SW 41 Ti rotor) for 14 h at 4 °C, fractions of band 3 from the top, which contained enriched plasma membranes, were collected and resuspended with sucrose breaking buffer containing protease inhibitors. After centrifugation at 21,000 × g for 90 min at 4 °C, the pellets were dissolved with sucrose breaking buffer, and the proteins present in the membranes were separated electrophoretically and analyzed by immunoblotting as described previously (31Zhang J. Visser F. Vickers M.F. Lang T. Robins M.J. Nielsen L.P. Nowak I. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2003; 64: 1512-1520Crossref PubMed Scopus (56) Google Scholar). The primary antibodies used in immunoblotting were monoclonal antibodies against the c-Myc epitope tag (9E10; BAbCo, Richmond, CA), against Pma1 (plasma membrane marker; Abcam, Cambridge, MA), against Dpm1p (dolichol phosphate mannose synthase endoplasmic reticulum membrane marker; Invitrogen), and against the V-ATPase 100-kDa subunit (vacuole membrane marker; Invitrogen). The proteins were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences) and autoradiography on a film. The film was scanned, and the quantities of the proteins were evaluated using ImageQuant software (Version 5.2; GE Healthcare). Urd Analogs—The structures of Urd and its analogs were given previously (31Zhang J. Visser F. Vickers M.F. Lang T. Robins M.J. Nielsen L.P. Nowak I. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2003; 64: 1512-1520Crossref PubMed Scopus (56) Google Scholar). The Urd analogs used in this study were either obtained from R.I. Chemical, Inc. (Orange, CA) or synthesized as described previously (31Zhang J. Visser F. Vickers M.F. Lang T. Robins M.J. Nielsen L.P. Nowak I. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2003; 64: 1512-1520Crossref PubMed Scopus (56) Google Scholar). Stock solutions of test compounds were prepared in water or Me2SO (Sigma), and the final concentration of Me2SO in transport reactions was 0.1% when Me2SO was used as a solvent. The fur4Δfui1Δ yeast strain with or without pYPGE15 has no active uptake of Urd as described previously (28Zhang J. Smith K.M. Tackaberry T. Visser F. Robins M.J. Nielsen L.P. Nowak I. Karpinski E. Baldwin S.A. Young J.D. Cass C.E. Mol. Pharmacol. 2005; 68: 830-839Crossref PubMed Scopus (35) Google Scholar). At pH 4.5, the uptake of 1 μm [3H]Urd into fur4Δfui1Δ yeast containing pYPFUI1 was rapid and linear over 90 s, with a mean rate (±S.E.) of 285 ± 5 pmol/mg of protein/s (Fig. 1, upper panel). This rate was reduced to 0.3 ± 0.1 pmol/mg of protein/s in the presence of 10 mm nonradioactive Urd, indicating the presence of functional Fui1p in yeast plasma membranes. Urd uptake rates were determined for all subsequent e

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