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J Biol Chem, Vol. 274, Issue 49, 35255-35261, December 3, 1999

The Adenosine Transporter of Toxoplasma gondii
IDENTIFICATION BY INSERTIONAL MUTAGENESIS, CLONING, AND RECOMBINANT EXPRESSION

Chi-Wu Chiang, Nicola Carter^¶, William J. Sullivan Jr., Robert G. K. Donald, David S. Roos, Fardos N. M. Naguib, Mahmoud H. el Kouni, Buddy Ullman^¶, and Craig M. Wilson

From the  Department of Biology and Division of Geographic Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, the ^¶ Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, the  Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the  Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294

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 µM. 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.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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