title: Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.
abstract: While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.
authors.affiliation: Institute of Biomedical and Life Sciences, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
authors.forename: Richard J S
authors.initials: RJ
authors.lastname: Burchmore
authors.forename: Lynsey J M
authors.initials: LJ
authors.lastname: Wallace
authors.forename: Denise
authors.initials: D
authors.lastname: Candlish
authors.forename: Mohammed I
authors.initials: MI
authors.lastname: Al-Salabi
authors.forename: Paul R
authors.initials: PR
authors.lastname: Beal
authors.forename: Michael P
authors.initials: MP
authors.lastname: Barrett
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: Harry P
authors.initials: HP
authors.lastname: de Koning
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003001:Cloning, Molecular; D019271:Hypoxanthine / Q000378:metabolism; D008969:Molecular Sequence Data; D033702:Nucleobase Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011687:Purines / Q000378:metabolism; D013379:Substrate Specificity; D014170:Transformation, Genetic; D014346:Trypanosoma brucei brucei / Q000737:chemistry; D014981:Xenopus; D015003:Yeasts / Q000378:metabolism
chemical_list: C476344:NBT1 protein, Trypanosoma brucei; D033702:Nucleobase Transport Proteins; D015800:Protozoan Proteins; D011687:Purines; D019271:Hypoxanthine