A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [H-3]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent K-m of 0.46 +/- 0.09 mu M and a V-max of 0.65 +/- 0.08 pmol.(10(7) cells)(-1) s(-1). [H-3]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (K-i value of 48 +/- 15 mu M). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (K-i = 3.2 +/- 0.4 mu M). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [H-3]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.