A, Isd11 is localized in the mitochondrion of the procyclic (PF) and bloodstream stages (BF). Cytosolic and mitochondrial fractions were obtained from both developmental stages by fractionation using digitonin. Upon resolution by SDS-PAGE the lysates were blotted and immunoprobed with α-Isd11 antibodies. Antibodies against enolase and GAP1 proteins were used as cytosolic and mitochondrial markers, respectively. Total cell lysates (T), cytosolic (C), and mitochondrial (M) fractions were screened in both stages. Positions of the protein markers are indicated on the right. B, tagged Isd11 and IscU were visualized by fluorescence microscopy using polyclonal anti-c-Myc antiserum coupled with fluorescein isothiocyanate-conjugated secondary antibody. Co-localization immunofluorescence was performed with monoclonal antibody mAb61 against the mitochondrial MRP1/2 complex (30). The first column (phase) shows phase-contrast light microscopy of the T. brucei procyclic cells; the second column (Dapi) shows staining of nuclear and mitochondrial (=kinetoplast) DNA with 4,6-diamidino-2-phenylindole (DAPI); the third column (c-Myc) shows localization of the TAP-tagged proteins using the polyclonal α-Myc antibody; the fourth column (mAb61) shows co-localization of the mitochondrial MRP1/2 complex using monoclonal α-mAb61 antibody; the last column (merge) documents merged fluorescence images. C, proteins were identified by mass spectrometry analysis of TAP tags from cells expressing TAP-tagged Isd11, IscU, or frataxin. Only proteins identified in both IscU and Isd11 TAP tag purifications and at least by two unique peptides are shown. The number indicates the total number of unique peptides identified.

Silencing of Isd11 affects stability of other Fe/S cluster assembly proteins in procyclic T. brucei. Expression level of Isd11, cysteine desulfurase (Nfs), scaffold protein (IscU), and frataxin as determined by immunoblot analysis of whole cell lysates of the parental 29-13 cell line (wt), non-induced cells (−), and cells 3, 5, and 7 days of RNAi induction. Enolase was used as a cytosolic loading control. Positions of the protein markers are indicated on the right.

Isd11 is essential for cytosolic tRNA thiolation. RNA isolated from non-induced (−) and RNAi-induced Isd11 cells (+) was separated on an APM gel followed by Northern blot analysis (A). Bands corresponding to thiolated and non-thiolated tRNAs are indicated. H₂O₂ is used as a control to show complete oxidation of thiolation, eliminating the mobility shift. The ratio of total tRNA isolated from RNAi-induced (+) to non-induced (−) cells after standardizing with non-thiolated tRNAs (B) is shown below each panel, where a ratio of 1 would indicate no change, less than 1 indicates lower steady-state levels of a particular tRNA.

Isd11 and Mtu1 are essential for mitochondrial thiolation of tRNA_CCA^Trp. Cytosolic and mitochondrial RNA from wild type cells (WT) and RNAi-induced cells was separated by APM acrylamide gel electrophoresis. A and B, Northern blot analysis probing for tRNA^Trp and tRNA^Glu. Bands corresponding to thiolated and non-thiolated tRNAs are indicated. H₂O₂ is used as a control to show complete oxidation of thiolation, eliminating the mobility shift. The level of tRNA thiolation is shown below the panel and calculated as described previously. The tRNA^Phe (C) is used as a loading control and ethidium bromide shows the purity of mitochondrial fractions (D).

A, effects of Isd11 RNAi on the growth of the parasite. Growth curves of non-induced (TET−; triangles) and induced (TET+; squares) knockdown for Isd11. The y axis is labeled by a log scale and represents the products of the measured cell densities and total dilutions. Cell densities were measured using the Beckman Z2 cell counter. The arrow indicates the sampling time point for the latter experiments. B, Isd11 mRNA levels were analyzed by blotting total RNA extracted from non-induced cells (−) and cells harvested 3 days of RNAi induction against Isd11 (+). The position of the targeted mRNA and double-stranded RNA synthesis following induction are indicated with open and closed arrows, respectively. As a loading control, the gel was stained with ethidium bromide to visualize rRNA bands. WT, wild type.

Activity of fumarase and aconitase (Fe/S cluster-containing proteins) is significantly reduced after down-regulation of Isd11. The percentage of specific aconitase and fumarase activity in total cell lysate (total), cytosol (cyto), and mitochondrion (mito) in the non-induced and RNAi-induced cells is shown. Values for the non-induced cells were considered as 100% activity. The mean ± S.D. values of six independent induction experiments are shown. The purity of cellular fractions obtained by digitonin fractionation from the non-induced (−) and RNAi-induced cells (+) was controlled with the α-enolase (cytosolic marker) and α-Gap1 antibodies (mitochondrial marker).

Silencing of Mtu1 affects neither growth phenotype nor cytosolic tRNA thiolation. A, growth curves of non-induced (TET−; triangles) and RNAi-induced (TET+; squares) knockdown for Mtu1 are presented as in Fig. 2A. Dotted and solid arrows indicate time points for RNA isolation and mitochondrial membrane potential measurements, respectively. B, Mtu1 mRNA levels were analyzed by blotting total RNA extracted from the non-induced cells (−) and cells harvested 3 days of RNAi induction against Mtu1 (+). Northern blot and the stained gel are as described in the legend to Fig. 2B. Mtu1 probe 1 was prepared from a fragment of the gene other than the one used for RNAi. Mtu1 probe 2 corresponds precisely to the gene fragment cloned into the RNAi vector. C, RNA isolated from the non-induced (−) and RNAi-induced Mtu1 cells (+) was separated on an APM gel followed by Northern blot analysis. Bands corresponding to thiolated and non-thiolated tRNAs are indicated. H₂O₂ was used as a control to show complete oxidation of thiolation, eliminating the mobility shift. The level of tRNA thiolation is shown below the panel and calculated as described previously. D, the tRNA^Gly and tRNA^Ile were used as loading controls.

Effect of RNAi against either Isd11 or Mtu1 on mitochondrial membrane potential. Mitochondrial membrane potential was measured in non-induced cells (thick black line), as well as in cells with down-regulated Isd11 (A) or Mtu1 (B) (thin gray line) that were, after incubation with 0.4% tetramethylrhodamine ethyl ester (TMRE), analyzed using flow cytometry. Ablated cell lines were analyzed 3 days of RNAi induction in the case of Isd11 and 3, 6, 9, and 12 days in case of Mtu1, respectively. The distribution of fluorescence was plotted as frequency histogram.