Steinmann ME , Schmidt RS , Macêdo JP , Kunz Renggli C , Bütikofer P , Rentsch D , Mäser P , Sigel E .

	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.

Accardi, Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. 2004, Pubmed

Accardi, Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. 2004, Pubmed
Bochud-Allemann, Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei. 2002, Pubmed
Braun, The yeast CLC protein counteracts vesicular acidification during iron starvation. 2010, Pubmed
Brenndörfer, Selection of reference genes for mRNA quantification in Trypanosoma brucei. 2010, Pubmed
Burchmore, Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan. 2003, Pubmed , Xenbase
Burkard, Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. 2007, Pubmed
Buyse, Expression of human pICln and ClC-6 in Xenopus oocytes induces an identical endogenous chloride conductance. 1997, Pubmed , Xenbase
Dean, TrypTag.org: A Trypanosome Genome-wide Protein Localisation Resource. 2017, Pubmed
De Angeli, The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. 2006, Pubmed
De Angeli, Review. CLC-mediated anion transport in plant cells. 2009, Pubmed
de Chaumont, Icy: an open bioimage informatics platform for extended reproducible research. 2012, Pubmed
Dohmen, An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. 1991, Pubmed
Dutzler, X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. 2002, Pubmed
Eddy, A new generation of homology search tools based on probabilistic inference. 2009, Pubmed
Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. 2004, Pubmed
Enz, Expression of the voltage-gated chloride channel ClC-2 in rod bipolar cells of the rat retina. 1999, Pubmed
Estévez, Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K+ secretion. 2001, Pubmed , Xenbase
Estévez, Functional and structural conservation of CBS domains from CLC chloride channels. 2004, Pubmed , Xenbase
Gaxiola, The yeast CLC chloride channel functions in cation homeostasis. 1998, Pubmed
Gonzalez-Salgado, myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei. 2012, Pubmed , Xenbase
Greene, The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth. 1993, Pubmed
Huson, Dendroscope: An interactive viewer for large phylogenetic trees. 2007, Pubmed
Jentsch, Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. 1990, Pubmed , Xenbase
Jentsch, Discovery of CLC transport proteins: cloning, structure, function and pathophysiology. 2015, Pubmed
Jeworutzki, GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary subunit. 2012, Pubmed , Xenbase
Kida, Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant. 2001, Pubmed
Lange, ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. 2006, Pubmed
Leisle, ClC-7 is a slowly voltage-gated 2Cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity. 2011, Pubmed
Livak, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 2001, Pubmed
Mäser, Drug transport and drug resistance in African trypanosomes. 2003, Pubmed
Mathieu, Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids. 2014, Pubmed , Xenbase
Matulef, Side-dependent inhibition of a prokaryotic ClC by DIDS. 2005, Pubmed
Neagoe, The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression. 2010, Pubmed , Xenbase
Needleman, A general method applicable to the search for similarities in the amino acid sequence of two proteins. 1970, Pubmed
Ortiz, Two novel nucleobase/pentamidine transporters from Trypanosoma brucei. 2009, Pubmed , Xenbase
Picollo, Molecular determinants of differential pore blocking of kidney CLC-K chloride channels. 2004, Pubmed
Redmond, RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. 2003, Pubmed
Rentsch, NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis. 1995, Pubmed
Schmidt, Autophagy in Trypanosoma brucei: amino acid requirement and regulation during different growth phases. 2014, Pubmed
Schwappach, Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. 1998, Pubmed
Serricchio, Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei. 2013, Pubmed
Sigel, The Xenopus oocyte: system for the study of functional expression and modulation of proteins. 2005, Pubmed , Xenbase
Steinmann, A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes. 2015, Pubmed , Xenbase
Tamura, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. 2013, Pubmed
Teste, Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. 2009, Pubmed
Untergasser, Primer3Plus, an enhanced web interface to Primer3. 2007, Pubmed
Wang, Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters. 2000, Pubmed
Weinreich, Pores formed by single subunits in mixed dimers of different CLC chloride channels. 2001, Pubmed
Wirtz, A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. 1999, Pubmed