title: The fluorouridine insensitive 1 (fur1) mutant is defective in equilibrative nucleoside transporter 3 (ENT3), and thus represents an important pyrimidine nucleoside uptake system in Arabidopsis thaliana.
abstract: The fluorouridine insensitive 1 (fur1) locus in Arabidopsis thaliana (L.) Heynh. has previously been identified in a screen for growth resistance towards the toxic compound fluorouridine. Mutation of this locus by ethylmethane sulfonate (EMS) allows mutants to grow on this uridine analogue. We identified that the A. thaliana equilibrative nucleoside transporter (AtENT3) was encoded by the fur1 locus. T-DNA insertional mutant plants for AtENT3 resemble the fur1 mutant phenotype: i.e. they grow on fluorouridine, and seedlings as well as leaf discs exhibit a markedly reduced uptake capacity for uridine and cytidine, but a less pronounced reduced uptake for adenosine and guanosine. These results indicate that AtENT3 is an important pyrimidine nucleoside transporter in Arabidopsis. In addition, we identified the mutation in fur1 as a single base-pair exchange, guanine --> adenine, leading to an amino acid exchange G --> R at position 281. Furthermore, we showed that this mutation is indeed responsible for the observed alterations in nucleoside transport in the fur1-1 line, because the introduction of this mutation in AtENT3 promoted fluorouridine resistance in yeast cells expressing this mutated protein. The biochemical characterization of AtENT3 expressed in Xenopus oocytes identified a proton-coupled concentrative mode of nucleoside transport, although this carrier possesses structural features characteristic for equilibrative nucleoside carriers.
authors.affiliation: Abteilung Pflanzenphysiologie, Fachbereich Biologie, Technische Universitat Kaiserslautern, PO Box 3049, D-67653 Kaiserslautern, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Traub
authors.forename: Martin
authors.initials: M
authors.lastname: Flörchinger
authors.forename: Jennifer
authors.initials: J
authors.lastname: Piecuch
authors.forename: Hans-Henning
authors.initials: HH
authors.lastname: Kunz
authors.forename: Alexandra
authors.initials: A
authors.lastname: Weise-Steinmetz
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Deitmer
authors.forename: H
authors.initials: H
authors.lastname: Ekkehard Neuhaus
authors.forename: Torsten
authors.initials: T
authors.lastname: Möhlmann
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D004594:Electrophysiology; D033705:Equilibrative Nucleoside Transport Proteins / Q000235:genetics*; D005260:Female; D018506:Gene Expression Regulation, Plant; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D014981:Xenopus
chemical_list: D033705:Equilibrative Nucleoside Transport Proteins; C001943:5-fluorouridine; D014529:Uridine