Identification and characterization of the three members of the CLC family of anion transport proteins in Trypanosoma brucei

Abstract

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.

Fig 1. Radial phylogram of TbVCLs and related proteins.

Protein sequences of TbVCLs were aligned to related proteins from other kinetoplastids, human, S. cerevisiae and A. thaliana by using MUSCLE [14]. The resulting alignment was used to grow a minimum evolution tree. Scale bar indicates nucleotide substitutions per site.

Fig 2. Sensitivity of gef1∆ mutant S. cerevisiae expressing TbVCLs to iron deprivation.

Gef1∆ mutant (Y16838) transformed with vector (pDR), TbVCL1, TbVCL2 or TbVCL3 were grown in complete SC medium in the presence of 15 μM (A) or various concentrations (B) of the iron chelator bathophenanthrolinedisulfonic acid (BPS) for 25 h. Vector-transformed parental strain (BY4742) was used as a control. A) Time-dependent growth of transformants. B) Growth after 25 h incubation in the presence of various concentrations of BPS. Data points are normalized as follows: optical density (O.D.₆₀₀) values of two replicates of a representative experiment in which the maximal growth (O.D.₆₀₀ ~0.7) was adjusted to 100% and minimal growth (O.D.₆₀₀ ~0.05) to 0%. Half inhibitory concentrations (IC₅₀) values were calculated from three independent experiments ± SD.

Fig 3. Electrophysiological characterization of TbVCL2 expressed in Xenopus laevis oocytes.

The 2-electrode voltage-clamp was used to study currents in oocytes injected with cRNA coding for TbVCL2 or water. All recordings were done by applying the voltage-step protocol depicted in (F). The holding potential was -40 mV. With a frequency of 1 Hz, voltage-steps of 300 ms duration were applied from -100 mV to +80 mV in 10 mV intervals. A representative example of current traces recorded from an oocyte expressing TbVCL2 in media (A) with chloride as major anion (ClME), (B) with gluconate as major anion (GlucME) and (C) inhibition of chloride currents by 300 μM DIDS. For comparison, currents recorded from water-injected control oocytes in media with chloride as major anion (ClME) and gluconate as major anion (GlucME) are shown in (D) and (E). (G) averaged I/V-relationships obtained using the voltage-step protocol shown in panel F from TbVCL2-expressing oocytes (mean ± SEM, n = 19–29). Substitution of chloride by gluconate (GlucME, open squares) leads to a substantial reduction of the outward currents observed during pulses to positive potentials compared to medium containing chloride as major anion (ClME, closed circles). Addition of 300 μM DIDS (open circles) to the medium containing chloride as major anion led to a similar reduction of the outward currents. (H) Concentration-dependent inhibition of the current observed at +80 mV of TbVCL2-expressing oocytes in ClME with increasing concentrations of DIDS (mean ± SEM, n = 6). The inhibition was fitted with an IC₅₀ of 30 ± 3 μM.

Fig 4. Determination of the relative anion selectivity of TbVCL1, TbVCL2 and TbVCL3.

(A) Representative current traces of an oocyte expressing TbVCL2 in chloride medium (ClME; upper panel) and nitrate medium (NO₃ME; lower panel). Currents of water-injected control oocytes recorded in the different media were averaged and then subtracted from the values obtained with cRNA-injected oocytes. These corrected currents were then normalized to the averaged current amplitude observed at +80 mV in chloride medium (2.65 ± 0.16 μA for TbVCL1; 2.74 ± 0.13 μA for TbVCL2 and 2.20 ± 0.35 μA for TbVCL3). The current-voltage relationships recorded from TbVCL1-, TbVCL2- and TbVCL3-expressing oocytes are shown in (B), (C) and (D), respectively (all values are mean ± SEM, n = 6–9 for TbVCL1; mean ± SEM, n = 6–10 for TbVCL2 and mean ± SD, n = 3 for TbVCL3). The major anion in the tested media are chloride (circles), bromide (triangles), iodide (diamonds) and nitrate (squares). For detailed media composition see section materials and methods.

Fig 5. Localization of TbVCL1, TbVCL2 and TbVCL3 in T. brucei procyclic forms.

C-terminally HA-tagged TbVCL1 and N-terminally tagged TbVCL2 and TbVCL3 were co-localized with markers for organelles: CatL, lysosome; GRASP, Golgi; BiP, endoplasmic reticulum. TbVCL1 shows a unique localization within the group of TbVCLs, localizing to a few puncta in proximity of the nucleus. For TbVCL2 and TbVCL3, co-staining with the endoplasmic reticulum marker is shown. Cells were counterstained with DAPI, shown in blue, visualizing the nuclear and kinetoplast DNA. DIC, differential interference contrast. Scale bars indicate 10 μm.

Fig 6. Down-regulation of TbVCL1, TbVCL2 and TbVCL3 by tetracycline-induced RNAi and its effect on parasite growth.

Growth of procyclic parasites in absence (open circles) and presence (squares) of 1μg / mL tetracycline was monitored for 10 days. Induction of RNAi against TbVCL1 (A), TbVCL2 (B) or TbVCL3 (C) showed no growth retardation. All data points represent means from three independent experiments. (D) mRNA levels of TbVCL1, TbVCL2 and TbVCL3 were determined by qRT-PCR 48 h post induction. All values were normalized to mRNA level of the respective RNAi-target in non-induced cells (n = 3; mean ± SD). Horizontal striped bars represent the results using TbVCL1-specific primers, diagonal striped bars represent the results using TbVCL2-specific primers and dotted bars represent the results using TbVCL3-specific primers.