The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression

Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis ve...

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Bibliographic Details
Published in:The Journal of biological chemistry 1999-12, Vol.274 (49), p.35255-35261
Main Authors: Chiang, C W, Carter, N, Sullivan, Jr, W J, Donald, R G, Roos, D S, Naguib, F N, el Kouni, M H, Ullman, B, Wilson, C M
Format: Article
Language:English
Subjects:
Aak gene
adenine arabinoside
adenosine
Adenosine - genetics
Adenosine - pharmacokinetics
Adenosine Kinase - genetics
adenosine transporter
ak gene
Amino Acid Sequence
Animals
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Membrane - metabolism
Cloning, Molecular
DNA, Complementary - metabolism
Molecular Sequence Data
Mutagenesis, Insertional
Nucleic Acid Hybridization
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Substrate Specificity
TgAT gene
Time Factors
Toxoplasma - genetics
Toxoplasma - metabolism
Toxoplasma gondii
Transcription, Genetic
Xenopus
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Summary:Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A-resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 microM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.49.35255