Article Figures & Data
Figures
- FIG 1
Nuclear localization signal is present in Nfs from T. brucei and other eukaryotes. Multiple sequence alignment of the Nfs proteins from distantly related eukaryotes. The alignment was generated using ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Identical residues have a black background, and conserved residues have a gray background. The nuclear localization signal (NLS) identified in yeast is boxed (38). The following sequences were used: Trypanosoma brucei Tb927.11.1670, Homo sapiens NP_001185918.1, Saccharomyces cerevisiae NP_009912.2, Arabidopsis thaliana NP_201373.1, Leishmania mexicana XP_003876680.1, Plasmodium falciparum XP_001349169.1, Blastocystis hominis CBK24185.2, Encephalitozoon cuniculi NP_586483.1, and Entamoeba histolytica XP_655257.1.
- FIG 2
Immunofluorescence analysis of tagged proteins in T. brucei BS and PS. The cells were stained with monoclonal anti-v5 and anti-HA antibodies (green), MitoTracker (red), and DAPI (blue). All target proteins were detected in mitochondria, as confirmed by colocalization with the MitoTracker signal. Identical results were obtained for the BS and PS cells and for the v5 and HA tags.
- FIG 3
Nfs and Isu proteins are present in the mitochondrion and in another subcellular compartment. Six samples were prepared from each cell line, incubated with increasing amount of digitonin, and centrifuged, and the supernatants obtained were used for Western blot analyses. Monoclonal anti-v5 and anti-HA antibodies were used for detection of the tagged proteins, and polyclonal anti-Nfs antibody was used to detect the endosomal protein. The mitochondrial membrane channel VDAC, the intermembrane protein Erv1, and the mitochondrial matrix protein MRP2 were used as mitochondrial controls; TIM424, the La protein, and enolase were used as glycosomal, nuclear, and cytosolic controls, respectively. These controls determined that mitochondrial proteins are present only in the last two fractions, but Nfs was released by lower concentrations of digitonin in both the BS and PS samples. Isu-v5 was detected in both the monomeric (25 kDa) and dimeric (50 kDa) forms. Again, the fraction of the signal was extramitochondrial. In the PS, the bulk of both proteins is present in the mitochondrion. The HA-tagged frataxin was detected solely in the mitochondrion-containing fractions.
- FIG 4
T. brucei cells overexpressing v5-tagged Nfs or Isu were labeled with anti-v5 antibody. Immunolocalization of Nfs (A to C, G, and H) and Isu (D to F) in ultrathin cryosections of PS (A to F and I) and BS (G and H). The gold particles were detected in the mitochondrion (A and D), often associated with kDNA (B, F, and G), and in the nucleolus (C, E, and H). Ferredoxin B (I) was localized only in the mitochondrion of PS. Mt, mitochondrion; Nc, nucleolus; N, nucleus; k, kDNA. Bar, 500 nm (A to E and H) and 200 nm (F, G, and I).
- FIG 5
Statistical analysis of immunogold labeling on cryosections of PS. Nfs is present in the nucleolus and the mitochondrion, where half of the protein is associated with the kDNA. Similarly, Isu is present in the nucleolus and the mitochondrion, but with somewhat lower affinity for the kDNA. Ferredoxins A and B are present solely in the mitochondrion.
- FIG 6
Fe-S cluster assembly is diminished but essential in BS. (A) Samples containing the same numbers of cells were prepared from the PS and BS cells and analyzed by Western blotting. Nfs and Isu are expressed at 45% and 22%, respectively, in the BS compared to the PS. Enolase was used as a loading control. (B) Growth of the BS Isu RNAi cells in the absence (solid line) or presence (dashed line) of tetracycline was examined. After the induction, a significant growth defect was observed. The efficiency of RNAi was verified by Western blotting using anti-Isu antibody and enolase as a loading control (inset).
- FIG 7
Cytosolic thiolation in the BS (A) or PS (B) depleted for Isu. RNA isolated from different cell lines was separated on an APM gel followed by Northern blotting. Bands corresponding to thiolated (+S2U) and nonthiolated (−S2U) tRNAs are indicated. H2O2 was used in s as a control of complete oxidation of thiolation, eliminating the mobility shift. The number of days after induction of RNAi by tetracycline is indicated above the lanes. −, noninduced RNAi.
- FIG 8
Enzymatic activities of fumarase (A) and aconitase (B) in the PS and BS. Aconitase was measured in cytosolic and mitochondrial fractions; fumarase was measured only in the cytosol. Activities of both Fe-S-dependent enzymes were measured in the wild-type PS and BS flagellates and in the BS with inducible overexpression (↑) of the target proteins. The activity obtained in the PS was set as 100%. Whereas in the wild-type BS, the activities are almost undetectable, levels comparable to those in the PS were reached after overexpression of each enzyme. These results confirm that functional Fe-S clusters are produced even in the BS in both cytosolic and mitochondrial compartments. Values are means and standard deviations from three independent experiments.
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