PMC

Quantification of upregulation of the NT1 permease in the Δnt3 and Δnt2Δnt3 null mutants. Membrane fractions from wild type (20 µg), Δnt3 (A) and Δnt2Δnt3 (B) null mutants (0.1–20 µg) were separated by SDS-PAGE and analyzed by immunoblotting as in .

Effect on Ado uptake of growing the Δnt2 and Δnt2Δnt3 null mutants in medium supplemented with 100 µM Guo or Xan. Parasites were grown in medium containing 20% dialyzed FBS and supplemented with 100 µM Guo (A) or Xan (B) for 5 days, and uptake of 1µM [³H]Ado was measured.

Effect on Ado uptake and NT1 expression of supplementing the growth medium with Guo or Xan. A. The Δnt3 null mutant grown in M199 medium supplemented with 100 µM Xan were pelleted, washed, and transferred to fresh medium containing either 100 µM Guo or Xan, and uptake of 1 µM Ado was measured over a 24 h time course. B. Experiment as in part A, except that membrane fractions (20 µg) were monitored by immunoblotting. C. Wild type parasites and the Δnt2Δnt3 null mutant were grown for 24 h in medium supplemented with 100 µM Guo or Xan, and uptake of Ado was measured. D. Lysates from parasites in part C were analyzed by immunoblotting.

Upregulation of Ado and Urd uptake and of the NT1 adenosine/pyrimidine nucleoside transporter in Δnt3 and Δnt2Δnt3 purine transporter null mutants. A. Uptake of 1 µM [³H]Ado and 10 µM [³H]Urd was measured (1 min time point), and the rates of uptake were plotted for wild type parasites (WT), the Δnt3 and Δnt2Δnt3 purine transporter null mutants, and the null mutants complemented with the NT3 purine nucleobase transporter gene maintained on an episomal expression vector ([pNT3]). Uptake data in this and subsequent figures represent the average and standard deviations of 3 replicate measurements. B. Immunoblot of membrane fractions (20 µg protein) probed with the anti-NT1 antibody (NT1) and anti-α-tubulin antibody (Tubulin). The left panel displays an immunoblot employing WT and WT[pNT1] cells to reveal the position of the ~43–45 kDa bands representing NT1, and the right panel displays an immunoblot of the same cell lines analyzed in part A. The numbers at the left (43 and 55) represent the positions of molecular weight markers, designated in kilodaltons.

Effect of purine starvation on uptake of Ado and NT1 expression in wild type L. major promastigotes. A. Parasites grown in M199 medium supplemented with 100 µM Xan (early log phase, 4 × 10⁶ cells ml⁻¹) were pelleted, washed, and resuspended (at 0.2–5 × 10⁶ cell ml⁻¹ with samples to be assayed earliest resuspended at higher densities than those assayed later) in medium containing 20% dialyzed fetal bovine serum and supplemented with either 100 µM Ado (squares, 100 µM Ado) or no purine (triangles, 0 µM Ado). Uptake of 1 µM [³H]Ado was measured in triplicate at various time points from 0 to 48 h (cells at ~5 × 10⁶ cell ml⁻¹ at time of uptake assay). The level of induction for adenosine transport at 48 h was ~60-fold for purine-starved compared to purine-replete cells. B. Parasites grown in purine deficient medium for 48 h (4 × 10⁶ cell ml⁻¹) were pelleted, washed, and resuspended as in part A. Uptake of 1 µM [³H]Ado was measured as in part A. The level of decrease for Ado transport at 48 h was ~47-fold for purine-replete versus purine-starved cells. C. Membrane fractions (20 µg) from parasites cultured for 24 h in medium containing 0 or 100 µM Ado were analyzed by immunoblotting. D. Increase in rate of uptake of Ado, Ino, and Ade for purine-starved versus purine-replete cells. Uptake of [³H]Ado, [³H]Ino, and [³H]Ade was measured for parasites cultured for 24 h in the presence of 0 or 100 µM Ado. Values plotted on the y-axis represent the relative uptake (fold increase) of each purine in purine-starved compared to purine-replete parasites.

Translational control of NT1 expression by purine limitation. A. Inhibition of translation with cycloheximide (CHX) prevents upregulation of Ado uptake in purine-starved parasites. Promastigotes were incubated for 24 h in medium containing either 0 or 100 µM Ado in the absence (−) or presence (+) of 10 µg ml⁻¹ cycloheximide. Uptake of 1 µM [³H]Ado was measured as in . Cycloheximide inhibits upregulation of the NT1 protein by purine starvation. Parasites cultured as in part A were analyzed by immunoblotting. C. Pulse labeling demonstrates that biosynthesis of NT1 protein is induced in Δnt2Δnt3 parasites. Wild type (WT) promastigotes, parasites overexpressing the NT1 protein from an episomal expression vector ([pNT1]), and Δnt2Δnt3 parasites were labeled with a mixture of [³⁵S]methionine and [³⁵S]cysteine for 2 h followed by immunoprecipitation with anti-NT1 antibody. The band at ~43 kDa represents newly synthesized NT1 protein, and the band at ~55 kD is a non-specifically precipitated protein, probably abundantly expressed tubulins. D. Turnover of adenosine transporters for wild type and Δnt2Δnt3 null mutants. Parasites were incubated in medium containing 10 µg ml⁻¹ cycloheximide to inhibit protein synthesis, and the decay of Ado transport activity was measured by quantifying uptake of 1 µM [³H]Ado from 0–75 h after addition of drug. For each cell line, the level of uptake at t = 0 was set to a value of 1.0, and the relative uptake values for later time points were normalized to this value. The absolute uptake value at t = 0 was 3.58 ± 0.078 pmol min⁻¹ per 10⁷ cells for wild type and 115 ± 1.8 pmol min⁻¹ per 10⁷ cells for Δnt2Δnt3 cells (triplicate measurements).