Figure 6

Overexpression of mitochondrially localized TbPth4 abates the dKO TbMrf1 phenotype. (a) RT-qPCR analysis of TbPth4 transcript levels in dKO TbMrf1 1wk, 7wk and dKO TbMrf1 + V5 TbPth4 cells compared to BF 427 trypanosomes. The relative changes in transcript abundance were plotted on a log scale. Transcript levels of β-tubulin were used as internal controls. (b) Growth curves spanning 14 days were generated for tetracycline induced (+tet) and noninduced (-tet) dKO TbMrf1 + V5 TbPth4 cells. The figure is representative of three independent experimental tetracycline inductions. Inset: Whole cell lysates from cultures either noninduced (-tet) or induced ( + tet) with tetracycline for 48 hours were probed with a specific anti-V5 antibody. (c) Plot of doubling times calculated from the linear growth of BF 427, dKO TbMrf1 7wk and tetracycline induced (+tet) and noninduced (-tet) dKO TbMrf1 + V5 TbPth4 cells. (d) & (e) Alamar Blue assays determined either the oligomycin (c) or carboxyatractyloside (d) sensitivity of induced (+tet) and noninduced (-tet) dKO TbMrf1 + V5 TbPth4 cells compared to BF 427 cells. Error bars represent the standard deviations calculated from three independent experimental replicates. (f) Immunofluorescence assays verify that V5-tagged TbPth4 is targeted to the mitochondrion in BF dKO TbMrf1 + V5 TbPth4 cells induced for 48 hours (+Tet). The ectopic TbPth4 was visualized with a Texas Red-conjugated secondary antibody that recognizes a primary monoclonal anti-V5 antibody. Noninduced (-Tet) BF BF dKO TbMrf1 + V5 TbPth4 cells were included as a control, while the single tubular mitochondrion was visualized with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody that recognizes a polyclonal primary antibody detecting F₁-ATPase subunit β. The DNA contents were visualized using DAPI (4,6-diamidino-2-phenylindole). The overall cell morphology is depicted in the differential interference contrast (DIC) microscopy images. (g) Subcellular localization of TbPth4 was determined in dKO TbMrf1 cells expressing V5-tagged TbPth4. Harvested parasites (Tot) were lysed under hypotonic conditions to obtain cytosolic (Cyt) and mitochondrial (Mt) fractions. Mt pellets treated with Na₂CO₃ underwent differential centrifugation to produce matrix (Mx) and mt membrane (Mm) fractions. Purified fractions were analyzed by Western blot with the following antibodies: anti-V5 (V5-TbPth4), anti-MRP1 (mt matrix), subunit β (mt matrix and membranes), anti-AAC (mt membranes), anti-enolase (cytosol).