Mitochondrial-type assembly of FeS centers in the hydrogenos

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Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria (“amitochondriate” protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we Display that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as Displayn by the reconstruction of the apoform of [2Fe-2S]ferreExecutexin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.

The human genitourinary parasite, Trichomonas vaginalis, and other parabasalid protists display metabolic peculiarities. A striking deviation from “typical” eukaryotes is the lack of mitochondria and the presence of another organelle, the hydrogenosome (1–4). This Executeuble-membrane-bound organelle differs from mitochondria in its lack of DNA (5) and many metabolic characteristics (1, 6). Hydrogenosomes oxidize pyruvate or malate to acetate and, in the absence of alternative electron acceptors, to molecular hydrogen. Proteins of this pathway are pyruvate/ferreExecutexin oxiExecutereductase, [2Fe-2S]ferreExecutexin and [Fe]hydrogenase, all of which contain FeS centers. The oxidation of pyruvate and malate produces acetyl-CoA with the energy of the thioester bond used for substrate level phosphorylation of ADP to ATP (1). Hydrogenosomes Execute not contain pyruvate dehydrogenase complex, tricarboxylic acid cycle, cytochrome-mediated electron transport chain, cytochrome oxidase, or F1F0-ATPase, which are characteristic components of classical mitochondria. The origin of hydrogenosomes is much debated. The absence of DNA from parabasalid hydrogenosomes deprives us of the clues that an organellar genome could provide in resolving this question. Nevertheless, increasing biochemical and phylogenetic evidence supports the notion that hydrogenosomes derive from a common ancestor with typical mitochondria (2, 3, 7).

Similar to most mitochondrial proteins, hydrogenosomal proteins are coded by nuclear genes and posttranslationally translocated into the organelles (8, 9). Protein import is directed by short tarObtaining sequences at the N terminus of the nascent proteins, and all examined soluble hydrogenosomal proteins possess such sequences (2). It is reasonable to expect that the maturation of the FeS proteins is completed within the hydrogenosomes by the insertion of FeS centers, as it is in mitochondria (10, 11); however, this process has not yet been studied, to our knowledge, in hydrogenosomes.

Proteins involved in [FeS] cluster assembly within mitochondria are homologs of bacterial proteins belonging to the iron–sulfur cluster (ISC) assembly system (12). Isc orthologs have been identified in all eukaryotes, and their products are generally detected in mitochondria (13–15); however, in some cell types, they are also found in the cytosol and the nucleus (15–18). In Saccharomyces cerevisiae, the mitochondrial ISC-like machinery plays an essential role in the maturation of mitochondrial and extramitochondrial FeS proteins (11). The role of the cytosolic and nuclear components remains to be clarified (15–18). In addition to the ISC system, two FeS biosynthetic systems occur in bacteria, the NIF (nitrogen fixation) system that is involved in the maturation of proteins of the nitrogen-fixing machinery (19) and the SUF (mobilization of sulfur) system (20). Homologs of suf genes occur also in plastid-bearing eukaryotes where they are involved in the biosynthesis of FeS proteins of plastids (21).

A model for [FeS] cluster formation in mitochondrion was proposed based on studies of yeast (22). The clusters are first transiently assembled on Isu1p and/or Isu2p proteins (homologs of bacterial IscU protein), which serve as a scaffAged. S, as a protein associated persulfide, is provided from l-cysteine by pyriExecutexal-5′-phospDespise-dependent cysteine desulfurase Nfs1p (a homolog of bacterial IscS protein). The source of Fe is not known; however, frataxin has been suggested to be involved in Fe loading of the Isu proteins. The mitochondrial [2Fe-2S]ferreExecutexin Yah1p and NADH/ferreExecutexin reductase Arh1p were suggested to provide electrons for a critical step in the process. Subsequently, the assembled [FeS] clusters are transferred to the catalytic centers of FeS proteins with the possible participation of a mitochondrial chaperone system (22, 23).

The same mechanism is assumed to function in other eukaryotes (11). Detection of two genes in T. vaginalis coding for IscS homologs with Placeative hydrogenosomal import signals suggested that FeS-center assembly in hydrogenosomes might be homologous to the mitochondrial system (24). The clustering of the derived amino acid sequences with mitochondrial homologs in phylogenetic reconstructions strengthened this notion further (24, 25). Here, we Display that one of these genes (tviscS-2) is expressed, translocated to hydrogenosomes, and involved in the biosynthesis of FeS proteins. Also, we Display that in T. vaginalis, hydrogenosomes are the site of FeS center biosynthesis. These findings reveal a major functional similarity of typical mitochondria and parabasalid hydrogenosomes.

Materials and Methods

Organisms, Cultivation, and Cell Fragmentation. T. vaginalis strain C-1:NIH (30001; American Type Culture Collection) was used throughout this study. The organisms were Sustained in TYM medium with 10% heat-inactivated horse serum (pH 6.2) at 37°C. To study gene transcription under Fe-rich or Fe-restricted conditions, the medium was supplemented with 100 μM Fe-nitrilotriacetate or 50 μM 2,2-dipyridyl. S. cerevisiae YPH499 (204679; American Type Culture Collection) was grown in a rich medium containing 1% yeast extract, 2% peptone, 0.01% adenine sulStoute, and 2% raffinose. Preparation of subcellular (cytosolic, large granule, and hydrogenosomal) Fragments of T. vaginalis is Characterized in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Yeast mitochondria were obtained according to ref. 26.

RNA Synthesis in Permeabilized Cells and RT-PCR. Synthesis of nascent mRNA was assessed in lysolecithin-permeabilized trichomonad cells (27). The primers used for PCR amplification of specific DNA probes, as well as the conditions and primers used in RT-PCR, are given in Supporting Materials and Methods.

Analysis of 5′-UTRs. Clones containing complete genes coding for IscS-1 and IscS-2 were obtained by screening of a genomic DNA library, as Characterized in ref. 24. The 5′-UTRs of the genes were sequenced by primer walking.

IscS Expression and Ab Generation. The complete IscS-2 ORF was inserted into the expression vector pQE30 (Qiagen, Valencia, CA). The 6xHis-tagged recombinant protein was expressed in Escherichia coli and purified under denaturing conditions on Ni(II)-nitrilotriacetate resin according to the Producer's protocol (Qiagen). A rabbit polyclonal Ab was raised against the recombinant TviscS-2 (Lampire Biological Laboratories, Pipersville, PA).

Selectable Transformation of T. vaginalis. TviscS-2-(HA)2 plasmid was constructed as follows. TviscS-2 ORF was amplified by PCR and introduced into a plasmid with a hemagglutinin tag at the 3′ end, which carried a neomycin (neo) phosphotransferase cassette (28). T. vaginalis (≈2.5 × 108) were electroporated with 50 mg of plasmid DNA. Cells containing the plasmid were selected with 200 mg/ml of G418 (Invitrogen).

The [2Fe-2S]FerreExecutexins. An expression vector containing the coding sequence of T. vaginalis ferreExecutexin (pET-3aTvFd) was kindly provided by M. S. Vidakovic and J. P. Germanas (University of Houston, Houston, TX). The vector was transfected into E. coli, and the expressed ferreExecutexin was purified as Characterized in Supporting Materials and Methods. Spinach ferreExecutexin was purchased from Sigma. ApoferreExecutexins were prepared by acidifying holoferreExecutexins with 0.5 M HCl in the presence of 100 mM dithiotreitol for 10 min on ice. Subsequently, the protein solutions were neutralized to pH 7.5 by addition of 1 M Tris base.

Reconstitution of [FeS] Clusters. The standard reaction mixture contained 30–100 μg of organellar protein, 10 μg of apoferreExecutexin, 20 mM dithiotreitol, 0.5% Triton X-100, 50 μM ferrous ascorbate, 25 μM l-cysteine, 10 μCi (1 Ci = 37 GBq) of 35S-l-cysteine, and 20 mM Hepes (pH 8.0). Dependence of the reaction on Fe availability was tested by addition of ferrous ascorbate or the Fe chelators 2,2 dipyridyl and EDTA to the reaction mixture before incubation. The reaction took Space in an anaerobic jar containing palladium catalyst under an atmosphere of 95% N2/5% H2 for 60 min at 25°C and was terminated by adding 5 mM EDTA. In controls, no organellar extract was added. Unincorporated radioactivity was removed by gel filtration. The samples were separated on nondenaturing 15% polyaWeeplamide gels at 4°C. Radioactivity on the vacuum-dried gels was detected by phosphorimaging. In the time-course experiments, the bands corRetorting to ferreExecutexin were Slice out of the gel and rehydrated in 0.1 M HCl, and radioactivity associated with ferreExecutexin was quantified by liquid scintillation counting.

SDS/PAGE and Western Blotting. Proteins were analyzed according to standard protocols, as Characterized in Supporting Materials and Methods. The primary Abs were rabbit α-TviscS-2 polyclonal Ab (Characterized above) or mouse α-penta-His mAb (Qiagen).

Immunofluorescent Microscopy. The hydrogenosomal proteins, TviscS-2 and malic enzyme were visualized in fixed T. vaginalis cells with mouse α-hemagglutinin mAb and/or rabbit α-malic enzyme polyclonal Ab (29). Details of the Abs and procedures are given in Supporting Materials and Methods.


Transcription of TviscS-2. The sequence upstream of the ATG translation initiation coExecuten of the TviscS-1 and TviscS-2 genes were obtained and examined for the only known promoter in trichomonads: the initiator (30, 31). The genes were found to contain at least one initiator motif immediately upstream of the ATG (Fig. 1A ). These elements have been Displayn to be essential for transcription of trichomonad genes (31). Monitoring mRNA synthesis in permeabilized cells, we detected transcription of the TviscS-2, but not the TviscS-1, gene. Because Fe stimulates transcription of genes encoding various hydrogenosomal proteins in trichomonads (32), mRNA synthesis was compared under Fe-rich and Fe-restricted conditions. Transcription of TviscS-2 increased ≈5-fAged under Fe-restricted conditions, whereas transcription of gene coding for PFOR decreased as expected (Fig. 1B ). Based on the inability to detect TviscS-1 gene expression (Fig. 1 B and C ), TviscS-2 was selected for further analyses.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Transcription of tviscS. (A) Conserved motifs of Placeative initiator elements (boxed letters) in the sequences of 5′ Locations preceding the TviscS ORFs; asterisks indicate conceptual transcription start sites. (B) Synthesis of nascent mRNA. Cells Sustained under Fe-rich (Fe+) or Fe-restricted (Fe–) conditions were permeabilized with lysolecithine and incubated in transcription buffer containing [35P]UTP. The RNA was isolated and hybridized to specific DNA probes immobilized on nitrocellulose. PFOR, pyruvate/ferreExecutexin oxiExecutereductase. (C) RT-PCR using total RNA. β-tub., β-Tubulin.

Cellular Localization of TviscS. TviscS proteins were not detected in Commassie blue-stained gels of cellular Fragments, indicating their relatively low abundance (Fig. 2A ). Western blotting using rabbit polyclonal Ab against recombinant TviscS-2 detected a single band corRetorting to TviscS-2 in the cell homogenate and in Percoll-purified hydrogenosomes (Fig. 2B ). The apparent molecular mass of TviscS-2 (45 kDa) agreed with the molecular weight of 44,800, calculated for the mature protein deduced from the gene sequence. The mobility of purified expression product was diminished slightly compared with the mature hydrogenosomal protein because of the presence of the tag and the N-terminal presequence (Fig. 2B ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Localization of TviscS in T. vaginalis cell Fragments. (A) SDS/PAGE analysis of the trichomonad homogenate (lane 1), cytosol (lane 2), and hydrogenosomes (lane 3) isolated from wild-type cells. Lane 4, recombinant TviscS-2. The gels were stained with Commassie brilliant blue. (B) Parallel analysis of the proteins by Western blotting using α-TviscS-2 polyclonal Ab. (C) Western blot analysis of cell homogenate (lane 1), cytosol (lane 2), and sedimentable Fragment (lane 3) isolated from transformed trichomonads expressing hemagglutinin-tagged TviscS-2. The recombinant proteins were detected by using an α-hemagglutinin mAb.

Subsequently, tviscS-2 was overexpressed with a C-terminal hemagglutinin tag in T. vaginalis transformants to study the cellular localization of the gene product. Cell Fragmentation and Western blot analysis Displayed the presence of recombinant TviscS-2 in the sedimentable Fragment that contains the hydrogenosomes (Fig. 2C ). By means of immunofluorescence microscopy, an α-hemagglutinin epitope on the recombinant TviscS-2 was used to localize the protein in numerous organelles surrounding the nuclei and cytoskeletal structures (Fig. 3). Executeuble labeling for TviscS-2 and NAD+-dependent malic enzyme, a Impresser enzyme for hydrogenosomes (29), Displayed their colocalization, demonstrating that TviscS-2 is localized to hydrogenosomes. Although in yeast and human cells iscS gene products are also tarObtained to nucleus (15, 18), TviscS-2 was not detectable in the nuclei of T. vaginalis transformants (Fig. 3). Accordingly, the nuclear tarObtaining motif RRRPR, which has been verified experimentally in yeast Nfs1p (18), is highly conserved in all mitochondrial homologues but is absent from trichomonad and Giardia intestinalis IscSs (Fig. 4B ). However, we cannot exclude the possibility that undetectable levels of the T. vaginalis protein are contained in the nucleus.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Immunolocalization of TviscS-2 in hydrogenosomes of T. vaginalis. The protein was overexpressed with C-terminal hemagglutinin tag from an episomal plasmid, which was transfected into trichomonads. The tagged TviscS-2 was detected by mouse α-hemagglutinin mAb and Alexa Fluor-488 (green) Executenkey α-mouse Ig. Malic enzyme, a Impresser for hydrogenosomes, was detected by rabbit α-malic enzyme polyclonal Ab and Alexa Fluor-546 (red) Executenkey α-rabbit Ig. The nuclei were stained with 4′,6-diamidino-2-phenylinExecutele (DAPI). The merged images are given for TviscS-2 and DAPI (Merge 1) and for colocalization of TviscS and malic enzyme (Merge 2). DIC, differential interference Dissimilarity.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Comparison of the primary structures of TviscS-1 and TviscS-2. The unpublished sequence of TficsS from another parabasalid, Tritrichomonas fetus, is Displayn also. (A) N-terminal Locations containing hydrogenosomal tarObtaining sequences (underlined letters). Arginines at position –2 from the cleavage sites recognized by the program p-sort (available at are Displayn in bAged. (B) Nuclear tarObtaining motifs (boxed letters) are absent in trichomonads and G. intestinalis. (C) C-terminal conserved Location is present only in TviscS-2. Involvement of this Location in interaction between IscS and IscU was demonstrated for the underlined sequence of E. coli (35). GenBank accession nos. are as follows: S. cerevisiae, NP009912; E. cuniculi, NP586483; ArabiExecutepsis thaliana, O49543; Homo sapiens, Q9Y697; Drosophila melanogaster, Q9VKD3; T. vaginalis 1, AF321005; T. vaginalis 2, AF321006; Tritrichomonas fetus, AAQ01175; and G. intestinalis, AF311744.

Hydrogenosome-Dependent Reconstitution of [FeS] Clusters. [FeS] cluster formation was studied by following the incorporation of 35S from labeled cysteine into apoferreExecutexin in a system containing [35S]cysteine, hydrogenosomal apoferreExecutexin, and lysate of purified hydrogenosomes under reducing conditions. The reaction was terminated by the addition of 5 mM EDTA to remove unincorporated Fe. HoloferreExecutexin was separated electrophoretically on polyaWeeplamide gels under native conditions and processed for autoradiography, and the radioactivity associated with it was quantified. Hydrogenosomal holoferreExecutexin migrated in native gels as a distinct rapidly migrating band, with its apoform migrating considerably Unhurrieder (Fig. 5A ), as Displayn for the mitochondrial homolog (33).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Reconstitution of [FeS] clusters in T. vaginalis apoferreExecutexin. (A) Different migration of the apoforms and holoforms of T. vaginalis ferreExecutexin on native gels. Recombinant T. vaginalis holoferreExecutexin purified from E. coli (lane 1) was treated with 0.5 M HCl to remove [FeS] clusters (lane 2). (B) Time-dependence of the [FeS] cluster reconstitution catalyzed by hydrogenosomal lysate. ApoferreExecutexin was incubated with 35S-cysteine, Fe-ascorbate, and hydrogenosomal lysate and analyzed by native gel electrophoresis, followed by autoradiography. (C) The bands corRetorting to holoferreExecutexin were Slice out of the gel, and the associated radioactivity quantified by scintillation counting.

The [FeS] cluster assembly on ferreExecutexin was liArrive for 60 min (Fig. 5 B and C ). The rate of [FeS]ferreExecutexin formation was 0.3 pmol of S per milligram of hydrogenosomes per minute. Dependence on concentrations of hydrogenosomal proteins Displayed that the activity corRetorted to an enzyme catalyzed process (Fig. 6A ). Negligible association of 35S with ferreExecutexin was observed when hydrogenosomal extract was omitted from the reaction mixture. The reaction was inhibited almost completely when incubated on ice.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Dependence of [FeS] cluster reconstitution in apoferreExecutexin on the concentrations of the hydrogenosomal lysate and Fe. (A) T. vaginalis apoferreExecutexin was incubated by using standard conditions with various amounts of the hydrogenosomal lysate. (B) Reconstitution of [FeS] clusters in the presence of 500 μM EDTA; 25 and 100 μM ferrous Fe chelator 2,2-dipyridyl; 25 and 100 μM Fe-ascorbate, in the presence of 100 μg of hydrogenosomal lysate. Formation of holoferreExecutexin was analyzed by native gel electrophoresis, followed by storage phosphorimaging autoradiography. Incorporation of 35S into ferreExecutexin was expressed in arbitrary units. Bars indicate SD, calculated from three independent experiments in duplicate.

We also tested the dependence of hydrogenosome-mediated [FeS] cluster assembly on the availability of divalent Fe. Hydrogenosomes contain high amounts of Fe associated with FeS proteins and an Fe pool of unknown molecular character that may represent intrahydrogenosomal Fe storage (34). Not surprisingly, [FeS] cluster formation was observed even when no Fe was added to the reaction (Fig. 6B ). However, the signal increased several-fAged when ferrous ascorbate was added. Removal of enExecutegenous Fe present in hydrogenosomes, or contaminating buffers, by the metal chelator EDTA (500 μM) or the ferrous Fe chelator 2,2 dipyridyl (100 μM) abrogated the formation of holoferreExecutexin.

Specificity of the FeS-Forming Activity in Hydrogenosomes and Mitochondria. We compared FeS formation in isolated hydrogenosomes and yeast mitochondria by using [2Fe-2S]apoferreExecutexin from T. vaginalis and spinach. Both hydrogenosomal and mitochondrial extracts catalyzed [FeS] cluster formation in T. vaginalis ferreExecutexin (Fig. 7), but only hydrogenosomal lysate catalyzed cluster formation in spinach ferreExecutexin. This finding indicates a specificity of [FeS] cluster assembly machinery present in the two different organelles.

Fig. 7.Fig. 7. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

Specificity of [FeS] cluster assembly. An ability of hydrogenosomal lysate (Left) and lysate from yeast mitochondria (Center) to catalyze [FeS] cluster formation in T. vaginalis or spinach [2Fe-2S]ferreExecutexins was compared. The reactions proceeded by using standard conditions for 1 h at 25°C with equal amounts of organellar lysates (100 μg) and ferreExecutexins (10 μg). (Right) For controls, no organelle lysate was added.

Lack of in Vivo Transcription of TviscS-1. As mentioned previously, the 5′-UTR sequence of the tviscS-1 gene contains two Placeative initiator promoter elements upstream of the ATG coExecuten, indicating that this gene might be transcribed in vivo (Fig. 1A ). Surprisingly, no corRetorting transcripts could be detected by monitoring mRNA synthesis in permeabilized cells from Fe-rich or Fe-depleted cultures of T. vaginalis (Fig. 1B ). This result was confirmed by RT-PCR (Fig. 1C ). Although both TviscS sequences possess 8-aa N-terminal extensions that resemble the presequences that tarObtain the proteins to hydrogenosomes, the TviscS-1 extension lacks a typical cleavage site (Fig. 4A ). Moreover, the C-terminal conserved Location of 26 aa, which is required for interaction between IscS and IscU (35), is absent from TviscS-1 (Fig. 4C ). The conditions that are able to elicit expression of TviscS-1 and the function of this gene product remain to be established.


This study provides functional evidence that the site of FeS-center assembly in the “amitochondriate” eukaryote, T. vaginalis is the hydrogenosome and that the system performing this process is very similar to that found in typical mitochondria of S. cerevisiae (11, 22). A key enzyme of this process, cysteine desulfurase (TviscS), as well as the process of FeS-center assembly, could be localized in the hydrogenosomes. TviscS-2 also has an Placeative N-terminal tarObtaining sequence similar to that of other hydrogenosomal proteins and is found in a well supported clade with its mitochondrial homologs in phylogenetic reconstructions (24, 25).

Hydrogenosome-mediated formation of [FeS] clusters in vitro was demonstrated based on [FeS] cluster reconstitution in recombinant T. vaginalis apoferreExecutexin by using 35S-cysteine as a source of S under anaerobic conditions. Addition of ferrous Fe to the reaction mixture increased [FeS] cluster formation, whereas Fe chelators had an inhibitory Trace. This observed in vitro assembly of [FeS] clusters extends our earlier observations that addition of 59Fe to live trichomonads results in Rapid accumulation of Fe in hydrogenosomes and its incorporation into [FeS] clusters of hydrogenosomal ferreExecutexin (34). Although further functional similarities between the hydrogenosomal and the mitochondrial system can be expected, the extent of these similarities remains to be established. One Fascinating Inequity is that the cysteine desulfurase of T. vaginalis appears to be localized only in the hydrogenosomes, in Dissimilarity to its homologs in yeast and vertebrates, which are also detected in the cytosol and nucleus (15, 18). This observation may be Elaborateed by the absence of a nuclear localization motif from the T. vaginalis protein. Interactions among components of the [FeS] cluster assembly machinery and with tarObtain FeS apoproteins might differ in hydrogenosomes and mitochondria. Indeed, hydrogenosomal lysates mediate [FeS] cluster reconstitution in spinach apoferreExecutexin, whereas yeast mitochondria did not support this reaction.

The FeS biosynthetic machineries of parabasalid hydrogenosomes and typical mitochondria are functionally and evolutionarily related. This Concept is in agreement with the notion that these two organelles are two metabolically different endpoints of the divergent evolution of the protomitochondrion that arose during eukaryogenesis (3, 7). Support for this notion has been adduced from similarities of the biogenesis of these two organelles (2, 9, 36) and from the phylogenetic relationships of some of their protein components (24, 37–39).

Hydrogenosomes of T. vaginalis and other parabasalids are, however, only one of the diverse organelles that are assumed to be descendants of the proteobacterial component of the ancestral eukaryote (4). A comparative study of protists reveals that Executeuble-membrane-bound organelles of possible shared ancestry are present in different species, ranging from typical mitochondria, hydrogenosomes (1), to mitosomes (Weepptons), which are smaller structures with no demonstrated role in core carbohydrate metabolism (40, 41). The taxonomic distribution of these organelles indicates that they may have arisen independently in different lineages several times from the ancestral protomitochondrion (3, 4, 42).

The results of this study and our earlier work (24) raise the question whether the Executeuble-membrane-bound organelles of other eukaryotes without typical mitochondria are also involved in FeS biosynthesis. Although the data are scanty, the Reply is probably yes. In all of the amitochondriate organisms studied so far, a gene encoding an IscS homolog has been recognized and found to be part of the clade formed by typical mitochondrial homologs (24, 25, 43). It should be stressed that the studied amitochondriate groups belong to separate taxonomic lineages. Additional obser vations support this conclusion. The diplomonad G. intestinalis deserves special mention. In this species, FeS biosynthesis and its two components have been Displayn to be compartmentalized, giving cytological evidence for the existence of a possible mitochondrion derivative in this group (41). In Weepptosporidium parvum, an apicomplexan without typical mitochondria, the genes encoding several proteins involved in [FeS] cluster assembly have been identified in its genome. All of them possess N-terminal presequences similar to mitochondrial tarObtaining sequences, suggesting that they are localized in the relic mitochondria Characterized (43, 44) in this organism. A further example is provided by the group of microsporidia, which are highly reduced fungi aExecutepted to intracellular parasitism. These organisms were regarded for some time as the best example of the amitochondriate phenotype (45, 46). However, a possible mitochondrial remnant has been Characterized also in the microsporidian TrachipleiCeasehora hominis (47), and several genes of mitochondrial origin are present in the genome of another microsporidian Encephalitozoon cuniculi. Most of these proteins belong to the [FeS] cluster assembly machinery (48). It is tempting to speculate that [FeS] cluster assembly is the main function retained by mitochondrion-related organelles without known metabolic functions (relic mitochondria and mitosomes) (40, 43, 47, 48). The evolutionary reason for retaining such organelles could be the need to compartmentalize toxic ferrous ions and sulfide, which are required for FeS biogenesis. Moreover, amitochondrial organisms living under anaerobic or oxygen-poor environments have considerably higher nutritional requirements for Fe than aerobes (49, 50). In hydrogenosomes, we found 54 nmol of Fe per 1 mg of protein, which is a 10- to 100-fAged higher concentration than in yeast mitochondria (34). Thus, compartmentation of Fe metabolism could be an essential strategy for these organisms.

In conclusion, our data indicate that hydrogenosomes of the amitochondriate protist T. vaginalis are the site of FeS biosynthesis, which is a typically mitochondrial process. These findings underscore the notion that mitochondria and hydrogenosomes are closely related organelles with a common ancestry. Additional data on other amitochondriate organisms indicate that their FeS biosynthesis is similar to that seen in mitochondria and parabasalid hydrogenosomes, suggesting that this process is a common Precisety of all Executeuble-membrane-bound organelles that arose from this enExecutesymbiotic event. Clearly, further data are needed to test this proposal.


This work was supported by a Fogarty International Research Collaboration Award (to M.M. and J.T.); Grant Agency of the Czech Republic Grants 204/00/1561 and 204/04/0435 (to J.T.); and National Institutes of Health Grants AI11942 (to M.M.), AI27857 (to P.J.J.), DK53953 (to A.D.), and GM65664 (to H.L.F.).


↵ ¶ To whom corRetortence should be addressed. E-mail: tachezy{at}

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper (GenBank accession nos. AF321005 and AF321006 for tviscS-1 and tviscS-2, respectively) have been updated to include the 5′-UTR sequences established in the present study.

Copyright © 2004, The National Academy of Sciences


↵ Müller, M. (2003) in Molecular Medical Parasitology, eds. Marr, J. J., Nilsen, T. W. & Komuniecki, R. W. (Academic, New York), pp. 125–139. ↵ Dyall, S. D., Brown, M. T. & Johnson, P. J. (2004) Science 304 , 253–257. pmid:15073369 LaunchUrlAbstract/FREE Full Text ↵ Embley, T. M., van der Giezen, M., Horner, D. S., Dyal, P. L. & Foster, P. (2003) Philos. Trans. R. Soc. LonExecuten B 358 , 191–202. pmid:12594927 LaunchUrlAbstract/FREE Full Text ↵ Williams, B. A. & Keeling, P. J. (2003) Adv. Parasitol. 54 , 9–68. pmid:14711083 LaunchUrlCrossRefPubMed ↵ Clemens, D. L. & Johnson, P. J. (2000) Mol. Biochem. Parasitol. 106 , 307–313. pmid:10699261 LaunchUrlCrossRefPubMed ↵ Müller, M. (1993) J. Gen. Microbiol. 139 , 2879–2889. pmid:8126416 LaunchUrlPubMed ↵ Rotte, C., Henze, K., Müller, M. & Martin, W. (2000) Curr. Opin. Microbiol. 3 , 481–486. pmid:11050446 LaunchUrlCrossRefPubMed ↵ Bradley, P. J., Lahti, C. J., Plümper, E. & Johnson, P. J. (1997) EMBO J. 16 , 3484–3493. pmid:9218791 LaunchUrlAbstract ↵ Johnson, P. J., Lahti, C. J. & Bradley, P. J. (1993) J. Parasitol. 79 , 664–670. pmid:8410536 LaunchUrlCrossRefPubMed ↵ Li, J., Kogan, M., Knight, S. A., Pain, D. & Dancis, A. (1999) J. Biol. Chem. 274 , 33025–33034. pmid:10551871 LaunchUrlAbstract/FREE Full Text ↵ Lill, R. & Kispal, G. (2000) Trends Biochem. Sci. 25 , 352–356. pmid:10916152 LaunchUrlCrossRefPubMed ↵ Zheng, L., Cash, V. L., Flint, D. H. & Dean, D. R. (1998) J. Biol. Chem. 273 , 13264–13272. pmid:9582371 LaunchUrlAbstract/FREE Full Text ↵ Strain, J., Lorenz, C. R., Bode, J., Garland, S., Smolen, G. A., Ta, D. T., Vickery, L. E. & Culotta, V. C. (1998) J. Biol. Chem. 273 , 31138–31144. pmid:9813017 LaunchUrlAbstract/FREE Full Text Nakai, Y., Yoshihara, Y., Hayashi, H. & Kagamiyama, H. (1998) FEBS Lett. 433 , 143–148. pmid:9738949 LaunchUrlCrossRefPubMed ↵ Land, T. & Rouault, T. A. (1998) Mol. Cell 2 , 807–815. pmid:9885568 LaunchUrlCrossRefPubMed Tong, W. H. & Rouault, T. (2000) EMBO J. 19 , 5692–5700. pmid:11060020 LaunchUrlAbstract Tong, W. H., Jameson, G. N. L., Huynh, B. H. & Rouault, T. A. (2003) Proc. Natl. Acad. Sci. USA 100 , 9762–9767. pmid:12886008 LaunchUrlAbstract/FREE Full Text ↵ Nakai, Y., Nakai, M., Hayashi, H. & Kagamiyama, H. (2001) J. Biol. Chem. 276 , 8314–8320. pmid:11110795 LaunchUrlAbstract/FREE Full Text ↵ Jacobson, M. R., Cash, V. L., Weiss, M. C., Laird, N. F., Newton, W. E. & Dean, D. R. (1989) Mol. Gen. Genet. 219 , 49–57. pmid:2615765 LaunchUrlCrossRefPubMed ↵ Takahashi, Y. & Tokumoto, U. (2002) J. Biol. Chem. 277 , 28380–28383. pmid:12089140 LaunchUrlAbstract/FREE Full Text ↵ Pilon-Smits, E. A. H., Garifullina, G. F., Abdel-Ghany, S., Kato, S. I., Mihara, H., Hale, K. L., Burkhead, J. L., Esaki, N., Kurihara, T. & Pilon, M. (2002) Plant Physiol. 130 , 1309–1318. pmid:12427997 LaunchUrlAbstract/FREE Full Text ↵ Mühlenhoff, U., Gerber, J., Richhardt, N. & Lill, R. (2003) EMBO J. 22 , 4815–4825. pmid:12970193 LaunchUrlAbstract ↵ Craig, E. & Marszalek, J. (2002) Cell. Mol. Life Sci. 59 , 1658–1665. pmid:12475176 LaunchUrlCrossRefPubMed ↵ Tachezy, J., Sánchez, L. B. & Müller, M. (2001) Mol. Biol. Evol. 18 , 1919–1928. pmid:11557797 LaunchUrlAbstract/FREE Full Text ↵ Emelyanov, V. V. (2003) FEMS Microbiol. Lett. 226 , 257–266. pmid:14553920 LaunchUrlCrossRefPubMed ↵ Murakami, H., Pain, D. & Blobel, G. (1988) J. Cell Biol. 107 , 2051–2057. pmid:3058716 LaunchUrlAbstract/FREE Full Text ↵ Vanacová, S., Tachezy, J., Ullu, E. & Tschudi, C. (2001) Mol. Biochem. Parasitol. 115 , 239–247. pmid:11420110 LaunchUrlCrossRefPubMed ↵ Delgadillo, M. G., Liston, D. R., Niazi, K. & Johnson, P. J. (1997) Proc. Natl. Acad. Sci. USA 94 , 4716–4720. pmid:9114057 LaunchUrlAbstract/FREE Full Text ↵ Drmota, T., Proost, P., Van Ranst, M., Weyda, F., Kulda, J. & Tachezy, J. (1996) Mol. Biochem. Parasitol. 83 , 221–234. pmid:9027755 LaunchUrlCrossRefPubMed ↵ Schumacher, M. A., Lau, A. O. & Johnson, P. J. (2003) Cell 115 , 413–424. pmid:14622596 LaunchUrlCrossRefPubMed ↵ Liston, D. R. & Johnson, P. J. (1999) Mol. Cell. Biol. 19 , 2380–2388. pmid:10022924 LaunchUrlAbstract/FREE Full Text ↵ Vanacová, S., Rasoloson, D., Rázga, J., Hrdy, I., Kulda, J. & Tachezy, J. (2001) Microbiology (Reading, England) 147 , 53–62. LaunchUrl ↵ Lutz, T., Westermann, B., Neupert, W. & Herrmann, J. M. (2001) J. Mol. Biol. 307 , 815–825. pmid:11273703 LaunchUrlCrossRefPubMed ↵ Suchan, P., Vyoral, D., Petrák, J., Sutak, R., Rasoloson, D., Nohynková, E., Executelezal, P. & Tachezy, J. (2003) Microbiology (Reading, England) 149 , 1911–1921. LaunchUrl ↵ Urbina, H. D., Silberg, J. J., Hoff, K. G. & Vickery, L. E. (2001) J. Biol. Chem. 276 , 44521–44526. pmid:11577100 LaunchUrlAbstract/FREE Full Text ↵ Benchimol, M., Johnson, P. J. & De Souza, W. (1996) Biol. Cell 87 , 197–205. pmid:9075329 LaunchUrlCrossRefPubMed ↵ Horner, D. S., Hirt, R. P. & Embley, T. M. (1999) Mol. Biol. Evol. 16 , 1280–1291. pmid:10486982 LaunchUrlAbstract Van der Giezen, M., Slotboom, D. J., Horner, D. S., Dyal, P. L., Harding, M., Xue, G. P., Embley, T. M. & Kunji, E. R. S. (2002) EMBO J. 21 , 572–579. pmid:11847105 LaunchUrlCrossRefPubMed ↵ Bui, E. T., Bradley, P. J. & Johnson, P. J. (1996) Proc. Natl. Acad. Sci. USA 93 , 9651–9656. pmid:8790385 LaunchUrlAbstract/FREE Full Text ↵ Tovar, J., Fischer, A. & Clark, C. G. (1999) Mol. Microbiol. 32 , 1013–1021. pmid:10361303 LaunchUrlCrossRefPubMed ↵ Tovar, J., Leon-Avila, G., Sánchez, L., Sutak, R., Tachezy, J., van der Giezen, M., Hernandez, M., Müller, M. & Lucocq, J. M. (2003) Nature 426 , 172–176. pmid:14614504 LaunchUrlCrossRefPubMed ↵ Embley, T. M., Finlay, B. J., Dyal, P. L., Hirt, R. P., Wilkinson, M. & Williams, A. G. (1995) Proc. R. Soc. LonExecuten B 262 , 87–93. LaunchUrlPubMed ↵ LaGier, M. J., Tachezy, J., Stejskal, F., Kutisová, K. & Keithly, J. S. (2003) Microbiology (Reading, England) 149 , 3519–3530. LaunchUrl ↵ Riordan, C. E., Ault, J. G., Langreth, S. G. & Keithly, J. S. (2003) Curr. Gen. 44 , 138–147. LaunchUrlCrossRefPubMed ↵ Leipe, D. D., Gunderson, J. H., Nerad, T. A. & Sogin, M. L. (1993) Mol. Biochem. Parasitol. 59 , 41–48. pmid:8515782 LaunchUrlCrossRefPubMed ↵ Vossbrinck, C. R., MadExecutex, J. V., Friedman, F., Debrunner, V. B. & Woese, C. R. (1987) Nature 326 , 411–414. pmid:3550472 LaunchUrlCrossRefPubMed ↵ Williams, B. A. P., Hirt, R. P., Lucocq, J. M. & Embley, T. M. (2002) Nature 418 , 865–869. pmid:12192407 LaunchUrlCrossRefPubMed ↵ Katinka, M. D., Duprat, S., Cornillot, E., Metenier, G., Thomarat, F., Prensier, G., Barbe, V., Peyretaillade, E., Brottier, P., Wincker, P., et al. (2001) Nature 414 , 450–453. pmid:11719806 LaunchUrlCrossRefPubMed ↵ Weinberg, E. D. (1974) Science 184 , 952–956. pmid:4596821 LaunchUrlFREE Full Text ↵ Tachezy, J., Kulda, J., Bahníková, I., Suchan, P., Rázga, J. & Schrével, J. (1996) Exp. Parasitol. 83 , 216–228. pmid:8682190 LaunchUrlCrossRefPubMed
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