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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
FEBS J. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2813251
NIHMSID: NIHMS167080

Functions and cellular localization of cysteine desulfurase and selenocysteine lyase in Trypanosoma brucei

Summary

Nfs-like proteins have cysteine desulfurase activity, which removes sulfur (S) from cysteine, and provides S for iron-sulfur cluster assembly and thiolation of tRNAs. These proteins also have selenocysteine lyase activity in vitro, and cleave selenocysteine into alanine and elemental selenium (Se). It was shown previously that the Nfs-like protein called Nfs from the parasitic protist Trypanosoma brucei is a genuine cysteine desulfurase. A second Nfs-like protein is encoded in the nuclear genome of T. brucei. We called this protein SCL because phylogenetic analysis reveals that it is monophyletic with known eukaryotic selenocysteine lyases. The Nfs protein is located in the mitochondrion, whereas the SCL protein seems to be present in the nucleus and cytoplasm. Unexpectedly, the down-regulation of either Nfs or SCL protein leads to a dramatic decrease of both cysteine desulfurase and selenocysteine lyase activities concurrently in the mitochondrion and the cytosolic fractions. Because loss of Nfs causes a growth phenotype but loss of SCL does not, we propose that Nfs can fully complement SCL, while SCL can only partially replace Nfs under our growth conditions.

Keywords: Trypanosoma, RNAi, mitochondrion, Fe-S cluster, selenoprotein

Introduction

Nfs-like proteins have cysteine desulfurase activity, and were first discovered in the nitrogen-fixing microbe Azotobacter vinelandii, where they are dedicated to the assembly of the iron-sulfur (Fe-S) clusters of nitrogenase [1]. These pyridoxal 5-phosphate-dependent proteins catalyze the conversion of the amino acid cysteine into alanine and elemental sulfur (S) [1]. All organisms studied to date encode homologues of Nfs (termed NifS, IscS, CsdA or SufS in bacteria depending on the gene clusters in which they are found and Nfs in mitochondria) that provide the S for Fe-S clusters. Eukaryotic Nfs proteins have a stably interacting partner Isd11, which is required for their function [24], and transiently interact with the scaffold protein IscU, upon which the clusters are formed [5]. Thus, the Nfs protein has a central and conserved function in the assembly of Fe-S clusters [6,7]. In every prokaryotic and eukaryotic cell, these ancient and omnipresent cofactors are subsequently incorporated into dozens of Fe-S proteins. These Fe-S proteins are best known for their vital role in the redox reactions during mitochondrial electron transport, but also have a similar function in photosynthesis [8], formation of biotin and thiamine, gene expression and other cellular processes [6,7].

Moreover, many organisms contain more than one Nfs-like protein. For example, Escherichia coli contains three distinct Nfs-like proteins (IscS, CsdA, and SufS). While the role of CsdA in E. coli is not fully understood, IscS seems to have a general housekeeping role, and SufS is thought to function during oxidative stress [9]. The model plant Arabidopsis thaliana also encodes three functionally distinct Nfs-like proteins localized to the chloroplast, mitochondria, and cytosol [10]. Two Nfs-like proteins have been identified in the apicomplexan protist Plasmodium [11], including one localized to the apicoplast, while the yeast and human genomes encode only a single Nfs-like protein. However, the human NFS1 gene contains an alternative start site, which provides dual localization of the protein to the mitochondria or the cytosol and nucleus [12]. In similar fashion, the yeast Nfs1 protein is predominantly found in the mitochondion, but is also localized to the nucleus in small amount, and was shown to be indispensable for survival [13,14]. Since yeast is not dependent on mitochondrial electron transport during anaerobic growth, it is likely that the yeast Nfs1 protein is essential because of the Fe-S cluster assembly for proteins localized in the cytosol and the nucleus. Moreover, yeast Nfs1 is also necessary for thiolation of tRNAs [15]. Indeed, mutation of the nuclear localization signal in the mature Nfs1 protein is also lethal in yeast, despite having no effect on mitochondrial Fe-S proteins. These results suggest that the yeast Nfs1 protein has an essential role in both nuclear and cytosolic Fe-S cluster assembly [15].

Interestingly, in addition to cysteine desulfurase activity, all Nfs-like proteins have selenocysteine lyase (SCL) activity, which cleaves selenocysteine into alanine and selenium. [16]. The SCL activity is essential for organisms that require selenium, as first documented in bacteria and later in mammals, both of which contain selenoproteins [17]. Single-celled organisms, such as the green algae Chlamydomonas reinhardtii and Emiliana huxleyi are also known to contain selenoproteins [18], although their set is smaller as compared to mammals [19].

The genome of Trypanosoma brucei, the causative agent of African sleeping sickness, encodes two Nfs-like proteins [20]. Downregulation of the Nfs protein, which is confined to the mitochondrion, impaired ATP production, cellular respiration and growth, suggesting that this protein is essential for the assembly of Fe-S clusters incorporated into the mitochondrial proteins [20]. More recently, it was discovered that in trypanosomes ablated for Nfs, tRNA thiolation is disrupted [21]. Moreover, in Saccharomyces cerevisiae and T. brucei, the mitochondrially located Nfs1 and Nfs proteins, respectively, are responsible for thiolation of tRNAs in both the mitochondria and cytoplasm [21,22]. Since T. brucei contains a set of selenoproteins [2325], as well as a complete machinery for the formation of Sec-tRNASec [26], we undertook functional characterization of cells with down-regulated Nfs-like protein of the selenocysteine type.

Results

Phylogenetic analysis

A genome wide search revealed that T. brucei and all other kinetoplastid flagellates, for which full genome sequences are available, contain two NfS-like proteins in their nuclear genome. Recent evidence suggests that one of them, called Nfs (formerly TbIscS2), exhibits cysteine desulfurase activity and has a function in Fe-S cluster assembly similar to other well studied homologues found in eukaryotes [20]. The second gene codes for a 451 amino acid-long protein with calculated molecular weight 48.9 kDa. It contains a highly conserved PLP-binding lysine 258, the active cysteine 393 responsible for desulfuration, as well as histidine 125, which initiates the release of sulfur by deprotonation of L-cysteine. In the sequence, however, the conserved serine 255 is replaced by cysteine, and a substantial part of the active site loop, as well as the C-terminal region known to mediate interaction with IscU, are lacking. A predicted nuclear localization signal (PPLKKLR) is located in the N-terminal region of the protein sequence.

We have performed an extensive phylogenetic analysis of Nfs-like genes from T. brucei using maximum likelihood, maximum parsimony and neighbor joining analyses (see Materials and Methods for details). An unrooted phylogenetic tree obtained from an alignment of amino acid sequences of the Nfs/IscS and SCL genes from 90 prokaryotes and 60 eukaryotes revealed a very distant position of both T. brucei genes (Fig. 1). The analysis did not recover a single clade containing solely prokaryotic sequences, but rather several paraphyletic clades. The eukaryotic genes are split into two large groups of different origin, interspersed with numerous prokaryotic NfS-like sequences. The early-branching group brings together all putative eukaryotic selenocysteine lyases, which probably represents the gene originating in the eukaryotic nucleus. Consequently, this phylogenetic analysis indicates that the T. brucei Nfs-like gene encodes a selenocysteine lyase, and will be henceforth labeled as such (SCL).

Figure 1
Maximum likelihood phylogenetic tree as inferred from 348 amino acid positions of the Nfs/IscS and related proteins (SUFs were not included). Numbers above branches indicate maximum likelihood (ML) (300 replicates)/neighbor-joining (NJ) (1000 replicates)/maximum ...

The second well-supported group of genes contains cysteine desulfurases including Nfs of T. brucei. (Fig. 1). However, although these Nfs genes are encoded in the eukaryotic nucleus, they likely originate from the ancestor of the mitochondrion, since α-proteobacteria constitute a robust sister group. The ancestry of the Nfs gene from the mitochondrion is thus well supported, while the origin of the SCL gene remains unclear. Consequently, these two genes have obviously acquired different yet overlapping functions in the eukaryotic cell (see below). .

RNAi knockdown of SCL

An RNAi cell line was prepared by introducing into the insect (procyclic) stage of T. brucei strain 29-13 a pZJMβ vector containing a 415 bp-long fragment of the SCL gene. The criterion for the selection of this fragment was the lowest possible sequence similarity to the Nfs gene. Transfection of the procyclics resulted in stable integration and phleomycin-resistant transfectants were obtained by limiting dilution. Induction of the double stranded RNA synthesis upon the addition of tetracycline indeed resulted in efficient elimination of the SCL mRNA in two selected clones within 24 hrs of induction (Fig. 2A). In order to rule out the possibility that cross-reactivity also induced the down-regulation of Nfs, which shares with SCL 33% and 52% identical and similar amino acids, respectively, a Northern blot was performed with a probe against the Nfs gene, which confirmed that the respective mRNA is not targeted by non-specific RNAi (Fig. 2B). Despite effective silencing, the growth of the cloned procyclic cells was not inhibited upon RNAi induction with tetracycline, even when it was followed for a prolonged period of two weeks (Fig. 2C).

Figure 2
Effect of SCL RNAi on mRNA levels. (A) SCL mRNA levels were analyzed by blotting total RNA extracted from the non-induced SCL cells (−) and SCL cells harvested 2, 4 and 6 days after RNAi induction. The position of the targeted mRNA and the double ...

Western blot analysis with polyclonal antibodies generated against the T. brucei cysteine desulfurase Nfs and the scaffold protein IscU revealed that the ablation of the target SCL protein did not result in a detectable loss of the above-mentioned proteins even after 6 days upon RNAi induction (Fig. 3A). We have also used anti-Nfs antibodies to verify the predicted mitochondrial localization of this protein in the procyclic T. brucei. Indeed, the protein seems to be confined to the organelle (Fig. 3B). The purity of cellular fractions was confirmed by antibodies against cytosolic enolase and mitochondrial prohibitin (PHB1).

Figure 3
Effect of SCL RNAi on protein levels and cellular localization of Nfs. (A) Nfs and IscU protein levels were analyzed by Western blot analysis in extracts from 29-13 procyclics, as well as from the non-induced SCL cells (−) and SCL cells harvested ...

Localization of SCL protein

We used a tagging strategy to analyze the intracellular localization of this protein. A hemagglutinin (HA3)-tag was attached to the C-terminus of the full-size SCL gene in a vector that allows inducible expression of the tagged protein driven by a strong procyclin promoter. The tag was placed on the C-terminus in order not to interfere with a predicted nuclear import signal usually located at the N-terminus. Subcellular fractions of the transfected procyclic cells have been obtained by digitonin treatment performed as described elsewhere [20]. As shown by Western blot analysis of the total cell lysate and the mitochondrial and cytosolic fractions, tagged protein is detected only in the cytosolic fraction, which is composed of nuclei and the cytosol (Fig. 4A). Polyclonal antibodies against enolase and guide RNA-binding protein 1 (GAP1) were used as cytosolic and mitochondrial loading controls, respectively.

Figure 4
Nuclear localization of inducibly expressed HA3-tagged and TAP-tagged SCL protein, respectively. (A) Immunoblot analysis of the HA3-tagged protein in total cell lysates (T), and cytosolic (C) and mitochondrial fractions (M) obtained from non-induced cells ...

This result was further corroborated by fluorescent microscopy of tetracycline-induced cells bearing the TAP-tagged SCL gene. The cells were stained by DAPI and prepared for immunocytochemistry using a polyclonal α-myc antibody. Interestingly, most of the signal was observed in nuclei with some signal also distributed throughout the cytoplasm, which may imply a dual localization of the Nfs-like protein, or its presence in cytoplasm due to its over-expression (Fig. 4B). As a control for staining of the mitochondria, the monoclonal antibody mAb56 against the mitochondrial MRP1/2 complex [36] was used (Fig. 4B).

Mass spectrometry analysis of the TAP-tagged purified SCL protein failed to identify any protein associated with it, indicating that the SCL protein has no strongly interacting partner (data not shown).

Measurement of enzymatic activities

Selenoproteins have previously been detected in the trypanosome proteome [2325]. Because selenoprotein synthesis would require the generation of elemental Se from selenocysteine, we analyzed the SCL activity in the procyclic cells. Moreover, since Nfs-like proteins can use cysteine and selenocysteine as substrate [39], we have tested whether the elimination of SCL resulted in a decrease or disruption of the SCL and cysteine desulfurase activities. Specific activities for the cysteine and selenocysteine substrates were measured in the non-induced and RNAi-induced knock-down cells for SCL characterized above, and also in the non-induced and Nfs RNAi-induced cells described earlier [20]. The measurements in total cell lysates showed that specific activities for both substrates are decreased in each of the knock-downs (data not shown). This experiment strongly supports the hypothesis that both proteins function as possible cysteine desulfurases and could also have selenocysteine lyase activity.

To determine if the SCL and cysteine desulfurase activities differed in cellular compartments, cytosolic and mitochondrial protein fractions were prepared and analyzed separately (Fig. 5). Wild type SCL specific activity was 2.5-fold higher in the cytosol than in the single reticulated mitochondrion. Four days upon RNAi induction, both cell lines with down-regulated SCL or Nfs had a decrease in the SCL specific activity in the cytosol, and to an even greater extent in the mitochondrion (Fig. 5C and 5D). Knock-downs for Nfs, which is the procyclic T. brucei confined to the mitochondrion [20], had a lower SCL specific activity compared to cells in which SCL was ablated. The measurement of the cysteine desulfurase activity indicated an even more pronounced decrease. Again, in wild type cells, this specific activity was approximately 2.5-fold higher in the cytosol as compared to the mitochondrion. Roughly 20% and 40% of the specific activity in the cytosolic fraction was retained in the SCL and Nfs RNAi cell lines, respectively (Fig. 5A). In contrast, cysteine desulfurase specific activity was virtually eliminated from the mitochondrion of these cell lines, with only 11% of it present in the Nfs knock-downs (Fig. 5B).

Figure 5
Measurement of Cys desulfurase (CysD) and selenocysteine lyase (SCL) specific activities Mitochondrial and cytosolic protein extracts were obtained from parental 29-13 cells, and the Nfs and SCL knockdowns 4 days of RNAi induction, as described in Materials ...

In both knock-down cells, the SCL and cysteine desulfurase activities began to increase on day 8 after RNAi induction. This general trend is expected, as it is well known that T. brucei can become resistant to RNAi usually after one week. Still, it is worth noting that the SCL activity bounces back slower in the SCL than in the Nfs knock-downs, and the same applies for the cysteine desulfurase activity in the respective cells (data not shown).

Discussion

Initially, the mitochondrion was considered the sole compartment in which Fe-S clusters are generated for the entire eukaryotic cell [40]. Soon afterwards, the localization of Nfs-like proteins to the nucleus and cytosol were discovered [7,41]. Studies in plants also revealed that an independent center of Fe-S cluster synthesis is present in the chloroplast [38], an observation not surprising given the evolutionary history of plant plastids and the requirement of an electron transport chain in both the mitochondrial and chloroplastic compartments. It is now becoming more apparent that the assembly of Fe-S clusters is not restricted to where the cysteine desulfurases are localized. This scenario was primarily supported by the observation that the Fe-S assembly in yeast appeared to depend on a mitochondrial membrane transporter [42]. An increasing amount of data now point towards the existence of a cytosolic iron-sulfur cluster assembly pathway termed CIA, which may serve the synthesis of Fe-S clusters assembled onto nuclear and cytosolic proteins [7].

As reported earlier, the down-regulation of Nfs dramatically lowers the activities of mitochondrial Fe-S cluster-containing enzymes, causing significant decrease of the growth rate of T. brucei procyclics [20]. Moreover, in trypanosomes as well as in yeasts, this protein was recently shown to be indispensable for the thiolation of cytosolic and mitochondrial tRNAs [21, 22, 43]. Importantly, analysis of the status of tRNA thiolation in cells depleted for the SCL protein did not reveal any changes demonstrating that this enzyme is not involved in tRNA metabolism [21]. As we show in this study, after silencing of SCL, the cysteine desulfurase activity drops by about 75% both in the mitochondrion and the cytosol. Almost the same decrease is observed in cells in which Nfs was targeted by RNAi, although in the mitochondrion of these knock-downs the cysteine desulfurase activity drops by 90% (Fig. 5). In analogy with other eukaryotes containing selenoproteins [44], T. brucei was supposed to be dependent upon the SCL activity for the formation of putatively essential selenoproteins. However, recent finding suggest that selenoproteins are not needed for the survival of trypanosomes at least under cultivation conditions [26], hinting that SCL may also be dispensable. We have confirmed this unexpected observation by experiments with auranofin, a highly specific inhibitor of selenoenzymes [23], as the down-regulation of SCL did not influence cell’s sensitivity to the drug as compared to its wild type counterparts (data not shown). Many selenoproteins are involved in alleviating oxidative stress or have redox properties such as for example the glutathione peroxidases [10]. Perhaps selenoproteins in T. brucei are only expressed after infection of their mammalian host, as a way to survive an oxidative burst.

All Nfs-like proteins are known to contain both cysteine desulfurase and SCL activities [39]. Group I Nfs-like proteins (Nfs1, IscS, and Nfs in this study) typically have ~ 8 fold higher activity towards selenocysteine than cysteine. The preference for selenocysteine is much greater in Group II Nfs-like proteins (CpNifS, SufS, and SCL in this study), where the activity can be up to 3,000 fold higher towards selenocysteine [16,19]. Therefore, the interchangeable activities of SCL and Nfs of T. brucei are not surprising. It is worth noting that while in the T. brucei procyclics the down-regulation of Nfs leads to the concomitant decrease of its binding partner IscU (Changmai P. and J.L., unpubl. results), the level of IscU is not altered in cells depleted for SCL, indicating that there is no mutual dependence between these two proteins. Using specific antibodies against Nfs and α-TAP antibodies against the tagged SCL, we have shown that the former protein is confined to the mitochondrion, while the latter is, quite surprisingly, present mostly in the nucleus and cytoplasm. Thus, we have anticipated that upon down-regulation of one of these enzymes, the cysteine desulfurase and SCL activities will decrease only in the compartment where the ablated protein resides. However, the down-regulation of SCL leads to the decrease of both activities in the cytosol and the mitochondrion, and a similar result was found for cells in which Nfs was targeted. Since the selected RNAi strategy and Northern analysis ruled out possible off-target RNAi silencing, another explanation has to be put forward. It is possible that despite their immunoreactivity in only a single compartment, both proteins are also present at amounts undetectable with available antibodies in the other cellular compartment, namely SCL in the mitochondrion and Nfs in the cytosol. Such a dual localization is known for Nfs1 in yeast, where the bulk of the enzyme resides in the organelle, but a small amount is also active in the nucleus [15]. Due to only a faint signal [45], human cysteine desulfurase was initially overlooked in the nucleus. Recent identification of its binding partner Isd11 in this compartment, as well as in the mitochondrion, speaks in favor of a dual (or even multiple) localization of numerous Fe-S cluster assembly proteins in the eukaryotic cell [46].

Indeed, to explain the measured activities and their down-regulation in respective RNAi knock-downs of T. brucei, such a dual localization of cysteine desulfurase and SCL can be invoked. However, the amounts of both proteins in the “other” compartment have to be very small, since neither the polyclonal antibody against Nfs, nor tagging of SCL allowed the detection of respective proteins in the cytosol and mitochondrion, respectively. Alternatively, indirect secondary effects may explain the observed activity profiles. Nfs down-regulation leads to a strong pleiotropic phenotype which could in turn result in a reduction of cytosolic SCL and cysteine desulfurase activities. In contrast, down-regulation of SCL does not lead to an observable phenotype and the effects on mitochondrial enzyme activities are not as pronounced as the effect of Nfs ablation on cytosolic activities. The simultaneous loss of activities in both cytosolic and mitochondrial compartments may also be a reflection of some kind of coordination between cytosolic and mitochondrial Fe-S assembly machineries. RNAi-induced knockdown of either SCL or Nfs decreases both activities in the T. brucei procyclics. One important difference between these RNAi cell lines is that while knockdown of SCL shows no growth phenotype, the down-regulation of Nfs substantially slows the growth of T. brucei, suggesting that it is the main Nfs protein in these flagellates. However, based on the available data, the growth phenotype of the Nfs knockdown can be ascribed to another function of this protein. The absence of Nfs disrupts Fe-S cluster assembly, monitored by the drop of the activities of the Fe-S cluster-containing proteins, such as the cytosolic and mitochondrial aconitases [20]. At the same time, a general decrease of tRNA thiolation affects their stability and surprisingly acts as a negative determinant for cytosine to uridine editing of mitochondrial tRNATrp, inevitably leading to the disruption of mitochondrial translation [21]. It thus appears that it is primarily the lack of thiolation, which causes the growth phenotype of procyclic T. brucei interfered against Nfs, since a similar drop of cysteine desulfurase activity in the SCL RNAi cells is insufficient to markedly slow their growth. Consequently, it appears that in the absence of one Nfs type enzyme in a given cellular compartment, the other Nfs type protein or another as yet unknown protein with an overlapping activity upholds the cysteine desulfurase and SCL activities at a level sufficient for survival, although at levels significantly lower than those in the wild type cells. This is not particularly surprising in the case of mitochondrial and cytosolic SCL activities, which remain relatively high in both knock-downs. However, it is quite unexpected in the case of the mitochondrial cysteine desulfurase activity, which drops in the SCL knock-downs to only about 15% of the wild type level, yet the cells are still able to retain unabated growth.

By mass spectrometry analysis we have shown that, like in other eukaryotes, T. brucei Nfs co-purifies with its highly conserved binding partner Isd11 (Paris Z., Changmai P. J.L., unpubl. results), while SCL does not seem to stably interact with any other protein (this work). Yet SCL is still capable of strong cysteine desulfurase activity in vitro, although the same activity of the Nfs protein in microsporidia was shown to be strongly potentiated by bound Isd11 [4], the knock-down of which is lethal in yeast [2, 3] as well as in trypanosomes (Paris Z., Changmai P., and J.L., unpubl. results). In E. coli, the deletion of one Nfs-like protein is not lethal, which was attributed to complementation by another Nfs-like protein, SufS. We propose that in a similar fashion, Nfs can fully complement SCL, however, SCL can only partially fulfill the functions of Nfs, perhaps because it is incapable of binding Isd11.

Experimental procedures

Phylogenetic analysis

Available homologues for genes encoding Nfs/IscS, NifS and selenocysteine lyases (SCL) from prokaryotes and eukaryotes were downloaded from GeneBank. Special attention was placed on using genes in question with the function confirmed experimentally. The amino acid sequences of the genes were aligned using Kalign [27]; ambiguously aligned regions and gaps were excluded form further analysis. Phylogenetic trees were computed using maximum likelihood (ML) (PhyML; [28]), maximum parsimony (MP) (PAUP* b4.10; [29]) and neighbor joining (NJ) (AsaturA; [30]; the particular method is designed to deal with saturation of amino acid positions). The model for amino acid substitutions (WAG+I+Γ) was inferred from the dataset using ProtTest [31]. Analogously, all parameters for ML analysis (likelihood of the tree is ln=-53614.118606; gamma shape parameter=1.249; proportion of invariants=0.012) were derived from the particular dataset. The robustness of constructed trees was tested by bootstrap analyses (ML in 300 replicates; MP and NJ with 1000 replicates) and is indicated in Fig. 1. Both T. brucei genes are highlighted.

Plasmid constructs, transfection, RNAi induction and growth curves

The T. brucei procyclic cell lines with inducible ablation of either Nfs or Nfs-like protein were described previously [20, 21]. Synthesis of double stranded RNA was induced by the addition of 1 μg/ml tetracycline. Two clonal cell lines (A and D), in which the Nfs-like mRNA was targeted, were obtained by limiting dilution in plates at 27°C in the presence of 5% CO2. An HA3-tagged Nfs-like fusion protein expressed from the pJH54 vector was electroporated into the 29-13 procyclics as described elsewhere [21]. Next, mitochondrial and cytosolic fractions were obtained from cells resistant to 1 μg/ml tetracycline, and used for immunodetection of the HA3-tagged protein.

Northern and Western blots

Detection of Nfs-like mRNA isolated from the non-induced cells and cells 2 and 4 days of RNAi induction was carried by Northern blot analysis using a random primed labeled probe and formaldehyde gel electrophoresis of total RNA following standard protocols [32]. All antibodies used for Western blots were generated against T. brucei proteins over-expressed in E. coli. Cell lysates corresponding to 5 × 106 procyclic cells/lane were separated on a 12% SDS-PAGE gel and blotted. The polyclonal rabbit antibodies against IscU, MRP2 (mitochondrial RNA binding protein 2), GAP1 (guide RNA-binding protein 1), PHB1 (prohibitin) and enolase were used at 1:1,000, 1:1,000, 1:1,000, 1:1,000 and 1: 150,000, respectively [33,34,47]. The polyclonal chicken antibodies against Nfs were used at 1:500. Secondary anti-rabbit immunoglobulin G antibodies (1:1,000) (Sevapharma) coupled to horseradish peroxidase were visualized using the ECL kit (Amersham Biosciences). For the detection of the Nfs-like protein, lysates from cells stably expressing the SCL protein HA3-tagged at its C-terminus were separated and blotted as described above, and the membranes were treated with anti-HA3-tag mouse monoclonal antibody, followed by chicken anti-mouse antibody coupled to horse-radish peroxidase. Western blot bands were quantified with software Bio-Rad quantity one.

TAP-tag analysis

The whole Nfs-like gene was PCR amplified and cloned into pLew79-MHT vector which contains c-myc, His, calmodulin binding peptide and protein A tags in that order. The last two tags are separated by a TEV protease cleavage site [35]. Upon linearization by NotI, the resulting construct was transfected into the T. brucei 29-13 procyclic strain. The Nfs-like-TAP cells, checked for inducible and tightly regulated expression, were induced for 48 hrs by the addition of 1 μg/ml of tetracycline to the medium. The TAP purification was performed as described elsewhere [33].

Digitonin fractionation and subcellular localization

Purification of mitochondrial vesicles isolated by hypotonic lysis from 5 × 108 cells was performed by digitonin fractionation as described elsewhere [21]. Pelleted mitochondrial vesicles were stored at −80 °C until further use. Subcellular localization of the expressed tagged protein within the cell was determined by immunofluorescence assay using anti-Myc polyclonal antibody (Invitrogen). Briefly the cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% fetal bovine serum, and incubated with anti-Myc antibody at 1:100 dilution. After washing, the cells were incubated with anti-rabbit FITC-conjugated antibody (1:250 dilution) (Sigma), washed, and treated with 4′,6-diamidino-2-phenylindole (DAPI) stain to visualize DNA. Co-localization analysis was performed using mAb56 against the mitochondrial MRP1/MRP2 complex [36] coupled with TexasR Red-X conjugated secondary antibody (Invitrogen). Phase-contrast images of the cells and their fluorescence were captured with a Nikon fluorescence microscope equipped with a camera and appropriate filters.

Enzyme essays

Cysteine desulfurase activity was assayed at 25 °C essentially as described [37]. Briefly, protein extract was added to a reaction mixture containing 25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 μM pyridoxal 5′-phosphate, 1 mM dithiothreitol and 500 μM cysteine. The reaction was stopped by the addition of 20 μl of 20 mM N,N-dimethyl-p-phenylenediamine in 7.2M HCl. Methylene blue was formed by the addition of 20 μlof 30 mM FeCl3 in 1.2 M HCl and was assayed by measuring theabsorbance at 670 nm. The selenocysteine lyase activity was measuredas described elsewhere [38]. In short, a 100 μl reaction mixture of 0.12M tricine, 10mM selenocysteine, 50mM DTT, and 0.2mM pyridoxal phosphate was allowed to incubate for 30 min, before being stopped with lead acetate. The formation of lead-selenide was quantified at 400 nm.

Acknowledgments

We thank Ondřej Šmíd (Charles University, Prague) and Milan Jirk (Biology Centre, České Budějovice) for their valuable contributions at an early stage of this project. We also thank Aswini Panigrahi (Seattle Biomedical Research Institute, Seattle) for help with the TAP-tag study. This work was supported by the Grant Agency of the Czech Republic 204/09/1667, the Ministry of Education of the Czech Republic (LC07032 and 2B06129 and 6007665801) and the Praemium Academiae award to J.L. and by National Institutes of Health (AI065935) to K.D.S.

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