Mitochondrial-Type Assembly of Fes Centers in the Hydrogenosomes of the Amitochondriate Eukaryote Trichomonas Vaginalis

Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis

Robert Sutak*, Pavel Dolezal*, Heather L. Fiumera†, Ivan Hrdy*, Andrew Dancis†, Maria Delgadillo-Correa‡, Patricia J. Johnson‡, Miklo´ sMu¨ ller§, and Jan Tachezy*¶

*Department of Parasitology, Faculty of Science, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic; †Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104; ‡Department of Microbiology, Immunology, and Molecular Genetics, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095; and §The Rockefeller University, 1230 York Avenue, New York, NY 10021

Edited by William Trager, The Rockefeller University, New York, NY, and approved May 28, 2004 (received for review February 25, 2004) Mitochondria are the site of assembly of FeS centers of mitochondria (10, 11); however, this process has not yet been studied, to drial and cytosolic FeS proteins. Various microaerophilic or anaer- our knowledge, in hydrogenosomes. obic unicellular eukaryotes lack typical mitochondria (‘‘amito- Proteins involved in [FeS] cluster assembly within mitochondria chondriate’’ protists). In some of these organisms, a metabolically are homologs of bacterial proteins belonging to the iron–sulfur different organelle, the hydrogenosome, is found, which is cluster (ISC) assembly system (12). Isc orthologs have been iden- thought to derive from the same proteobacterial ancestor as tified in all eukaryotes, and their products are generally detected in mitochondria. Here, we show that hydrogenosomes of Trichomo- mitochondria (13–15); however, in some cell types, they are also nas vaginalis, a human genitourinary parasite, contain a key found in the cytosol and the nucleus (15–18). In Saccharomyces enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), cerevisiae, the mitochondrial ISC-like machinery plays an essential which is phylogenetically related to its mitochondrial homologs. role in the maturation of mitochondrial and extramitochondrial FeS Hydrogenosomes catalyze the enzymatic assembly and insertion of proteins (11). The role of the cytosolic and nuclear components FeS centers into apoproteins, as shown by the reconstruction of the remains to be clarified (15–18). In addition to the ISC system, two apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from FeS biosynthetic systems occur in bacteria, the NIF (nitrogen labeled cysteine. Our results indicate that the biosynthesis of FeS fixation) system that is involved in the maturation of proteins of the proteins is performed by a homologous system in mitochondriate nitrogen-fixing machinery (19) and the SUF (mobilization of and amitochondriate eukaryotes and that this process is inherited sulfur) system (20). Homologs of suf genes occur also in plastid- from the proteobacterial ancestor of mitochondria. bearing eukaryotes where they are involved in the biosynthesis of FeS proteins of plastids (21). he human genitourinary parasite, Trichomonas vaginalis, and A model for [FeS] cluster formation in mitochondrion was proposed based on studies of yeast (22). The clusters are first Tother parabasalid protists display metabolic peculiarities. A ͞ striking deviation from ‘‘typical’’ eukaryotes is the lack of transiently assembled on Isu1p and or Isu2p proteins (homologs mitochondria and the presence of another organelle, the hydro- of bacterial IscU protein), which serve as a scaffold. S, as a protein associated persulfide, is provided from L-cysteine by genosome (1–4). This double-membrane-bound organelle dif- Ј fers from mitochondria in its lack of DNA (5) and many pyridoxal-5 -phosphate-dependent cysteine desulfurase Nfs1p (a metabolic characteristics (1, 6). Hydrogenosomes oxidize pyru- homolog of bacterial IscS protein). The source of Fe is not vate or malate to acetate and, in the absence of alternative known; however, frataxin has been suggested to be involved in Fe loading of the Isu proteins. The mitochondrial [2Fe- electron acceptors, to molecular hydrogen. Proteins of this ͞ pathway are pyruvate͞ferredoxin oxidoreductase, [2Fe- 2S]ferredoxin Yah1p and NADH ferredoxin reductase Arh1p 2S]ferredoxin and [Fe]hydrogenase, all of which contain FeS were suggested to provide electrons for a critical step in the centers. The oxidation of pyruvate and malate produces acetyl- process. Subsequently, the assembled [FeS] clusters are trans- CoA with the energy of the thioester bond used for substrate ferred to the catalytic centers of FeS proteins with the possible level phosphorylation of ADP to ATP (1). Hydrogenosomes do participation of a mitochondrial chaperone system (22, 23). not contain pyruvate dehydrogenase complex, tricarboxylic acid The same mechanism is assumed to function in other eu- cycle, cytochrome-mediated electron transport chain, cyto- karyotes (11). Detection of two genes in T. vaginalis coding for IscS homologs with putative hydrogenosomal import signals chrome oxidase, or F1F0-ATPase, which are characteristic components of classical mitochondria. The origin of hydrogenosomes suggested that FeS-center assembly in hydrogenosomes might be is much debated. The absence of DNA from parabasalid hydro- homologous to the mitochondrial system (24). The clustering of genosomes deprives us of the clues that an organellar genome the derived amino acid sequences with mitochondrial homologs could provide in resolving this question. Nevertheless, increasing in phylogenetic reconstructions strengthened this notion further tviscS-2 biochemical and phylogenetic evidence supports the notion that (24, 25). Here, we show that one of these genes ( )is expressed, translocated to hydrogenosomes, and involved in the hydrogenosomes derive from a common ancestor with typical biosynthesis of FeS proteins. Also, we show that in T. vaginalis, mitochondria (2, 3, 7). hydrogenosomes are the site of FeS center biosynthesis. These 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 This paper was submitted directly (Track II) to the PNAS ofﬁce. short targeting sequences at the N terminus of the nascent Data deposition: The sequences reported in this paper (GenBank accession nos. AF321005 proteins, and all examined soluble hydrogenosomal proteins and AF321006 for tviscS-1 and tviscS-2, respectively) have been updated to include the possess such sequences (2). It is reasonable to expect that the 5Ј-UTR sequences established in the present study. maturation of the FeS proteins is completed within the hydro- ¶To whom correspondence should be addressed. E-mail: [email protected]. genosomes by the insertion of FeS centers, as it is in mitochon- © 2004 by The National Academy of Sciences of the USA

10368–10373 ͉ PNAS ͉ July 13, 2004 ͉ vol. 101 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401319101 Downloaded by guest on September 27, 2021 findings reveal a major functional similarity of typical mitochondria and parabasalid hydrogenosomes. Materials and Methods Organisms, Cultivation, and Cell Fractionation. T. vaginalis strain C-1:NIH (30001; American Type Culture Collection) was used throughout this study. The organisms were maintained 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 ␮M Fe- nitrilotriacetate or 50 ␮M 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% ade- nine sulfate, and 2% raffinose. Preparation of subcellular (cytosolic, large granule, and hydrogenosomal) fractions of T. vaginalis is described in Supporting Materials and Methods, which Fig. 1. Transcription of tviscS.(A) Conserved motifs of putative initiator is published as supporting information on the PNAS web site. elements (boxed letters) in the sequences of 5Ј regions preceding the TviscS Yeast mitochondria were obtained according to ref. 26. ORFs; asterisks indicate conceptual transcription start sites. (B) Synthesis of nascent mRNA. Cells maintained under Fe-rich (Feϩ) or Fe-restricted (FeϪ) RNA Synthesis in Permeabilized Cells and RT-PCR. Synthesis of conditions were permeabilized with lysolecithine and incubated in transcrip- 35 nascent mRNA was assessed in lysolecithin-permeabilized tion buffer containing [ P]UTP. The RNA was isolated and hybridized to ͞ trichomonad cells (27). The primers used for PCR amplification speciﬁc DNA probes immobilized on nitrocellulose. PFOR, pyruvate ferredoxin oxidoreductase. (C) RT-PCR using total RNA. ␤-tub., ␤-Tubulin. of specific DNA probes, as well as the conditions and primers used in RT-PCR, are given in Supporting Materials and Methods.

؅ by adding 5 mM EDTA. In controls, no organellar extract was Analysis of 5 -UTRs. Clones containing complete genes coding for added. Unincorporated radioactivity was removed by gel filtra- IscS-1 and IscS-2 were obtained by screening of a genomic DNA Ј tion. The samples were separated on nondenaturing 15% poly- EVOLUTION library, as described in ref. 24. The 5 -UTRs of the genes were acrylamide gels at 4°C. Radioactivity on the vacuum-dried gels sequenced by primer walking. was detected by phosphorimaging. In the time-course experiments, the bands corresponding to ferredoxin were cut out of the IscS Expression and Ab Generation. The complete IscS-2 ORF was gel and rehydrated in 0.1 M HCl, and radioactivity associated inserted into the expression vector pQE30 (Qiagen, Valencia, with ferredoxin was quantified by liquid scintillation counting. CA). The 6xHis-tagged recombinant protein was expressed in Escherichia coli and purified under denaturing conditions on SDS͞PAGE and Western Blotting. Proteins were analyzed according Ni(II)-nitrilotriacetate resin according to the manufacturer’s to standard protocols, as described in Supporting Materials and protocol (Qiagen). A rabbit polyclonal Ab was raised against the Methods. The primary Abs were rabbit ␣-TviscS-2 polyclonal Ab recombinant TviscS-2 (Lampire Biological Laboratories, Pip- (described above) or mouse ␣-penta-His mAb (Qiagen). ersville, PA). Immunofluorescent Microscopy. The hydrogenosomal proteins, Selectable Transformation of T. vaginalis. TviscS-2-(HA) plasmid 2 TviscS-2 and malic enzyme were visualized in fixed T. vaginalis was constructed as follows. TviscS-2 ORF was amplified by PCR ␣ ͞ ␣ and introduced into a plasmid with a hemagglutinin tag at the 3Ј cells with mouse -hemagglutinin mAb and or rabbit -malic end, which carried a neomycin (neo) phosphotransferase cas- enzyme polyclonal Ab (29). Details of the Abs and procedures sette (28). T. vaginalis (Ϸ2.5 ϫ 108) were electroporated with 50 are given in Supporting Materials and Methods. mg of plasmid DNA. Cells containing the plasmid were selected Results with 200 mg͞ml of G418 (Invitrogen). Transcription of TviscS-2. The sequence upstream of the ATG The [2Fe-2S]Ferredoxins. An expression vector containing the coding translation initiation codon of the TviscS-1 and TviscS-2 genes were sequence of T. vaginalis ferredoxin (pET-3aTvFd) was kindly obtained and examined for the only known promoter in provided by M. S. Vidakovic and J. P. Germanas (University of trichomonads: the initiator (30, 31). The genes were found to Houston, Houston, TX). The vector was transfected into E. coli, and contain at least one initiator motif immediately upstream of the the expressed ferredoxin was purified as described in Supporting ATG (Fig. 1A). These elements have been shown to be essential for Materials and Methods. Spinach ferredoxin was purchased from transcription of trichomonad genes (31). Monitoring mRNA syn- Sigma. Apoferredoxins were prepared by acidifying holoferredox- thesis in permeabilized cells, we detected transcription of the ins with 0.5 M HCl in the presence of 100 mM dithiotreitol for 10 TviscS-2, but not the TviscS-1, gene. Because Fe stimulates tran- min on ice. Subsequently, the protein solutions were neutralized to scription of genes encoding various hydrogenosomal proteins in pH 7.5 by addition of1MTrisbase. trichomonads (32), mRNA synthesis was compared under Fe-rich and Fe-restricted conditions. Transcription of TviscS-2 increased Reconstitution of [FeS] Clusters. The standard reaction mixture Ϸ5-fold under Fe-restricted conditions, whereas transcription of contained 30–100 ␮g of organellar protein, 10 ␮g of apoferre- gene coding for PFOR decreased as expected (Fig. 1B). Based on doxin, 20 mM dithiotreitol, 0.5% Triton X-100, 50 ␮M ferrous the inability to detect TviscS-1 gene expression (Fig. 1 B and C), ascorbate, 25 ␮M L-cysteine, 10 ␮Ci (1 Ci ϭ 37 GBq) of TviscS-2 was selected for further analyses. 35S-L-cysteine, and 20 mM Hepes (pH 8.0). Dependence of the reaction on Fe availability was tested by addition of ferrous Cellular Localization of TviscS. TviscS proteins were not detected in ascorbate or the Fe chelators 2,2 dipyridyl and EDTA to the Commassie blue-stained gels of cellular fractions, indicating reaction mixture before incubation. The reaction took place in their relatively low abundance (Fig. 2A). Western blotting using an anaerobic jar containing palladium catalyst under an atmo- rabbit polyclonal Ab against recombinant TviscS-2 detected a ͞ sphere of 95% N2 5% H2 for 60 min at 25°C and was terminated single band corresponding to TviscS-2 in the cell homogenate

Sutak et al. PNAS ͉ July 13, 2004 ͉ vol. 101 ͉ no. 28 ͉ 10369 Downloaded by guest on September 27, 2021 Fig. 2. Localization of TviscS in T. vaginalis cell fractions. (A) SDS͞PAGE 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 ␣-TviscS-2 polyclonal Ab. (C) Western blot analysis of cell homogenate (lane 1), cytosol (lane 2), and sedimentable fraction (lane 3) isolated from transformed trichomonads ex- Fig. 3. Immunolocalization of TviscS-2 in hydrogenosomes of T. vaginalis. pressing hemagglutinin-tagged TviscS-2. The recombinant proteins were de- The protein was overexpressed with C-terminal hemagglutinin tag from an tected by using an ␣-hemagglutinin mAb. episomal plasmid, which was transfected into trichomonads. The tagged TviscS-2 was detected by mouse ␣-hemagglutinin mAb and Alexa Fluor-488 (green) donkey ␣-mouse Ig. Malic enzyme, a marker for hydrogenosomes, was detected by rabbit ␣-malic enzyme polyclonal Ab and Alexa Fluor-546 (red) and in Percoll-purified hydrogenosomes (Fig. 2B). The apparent donkey ␣-rabbit Ig. The nuclei were stained with 4Ј,6-diamidino-2- molecular mass of TviscS-2 (45 kDa) agreed with the molecular phenylindole (DAPI). The merged images are given for TviscS-2 and DAPI weight of 44,800, calculated for the mature protein deduced from (Merge 1) and for colocalization of TviscS and malic enzyme (Merge 2). DIC, the gene sequence. The mobility of purified expression product differential interference contrast. was diminished slightly compared with the mature hydrogenosomal protein because of the presence of the tag and the Hydrogenosome-Dependent Reconstitution of [FeS] Clusters. [FeS] N-terminal presequence (Fig. 2B). cluster formation was studied by following the incorporation of Subsequently, tviscS-2 was overexpressed with a C-terminal 35S from labeled cysteine into apoferredoxin in a system con- hemagglutinin tag in T. vaginalis transformants to study the taining [35S]cysteine, hydrogenosomal apoferredoxin, and lysate cellular localization of the gene product. Cell fractionation and of purified hydrogenosomes under reducing conditions. The Western blot analysis showed the presence of recombinant reaction was terminated by the addition of 5 mM EDTA to TviscS-2 in the sedimentable fraction that contains the hydro- remove unincorporated Fe. Holoferredoxin was separated elec- genosomes (Fig. 2C). By means of immunofluorescence micros- ␣ trophoretically on polyacrylamide gels under native conditions copy, an -hemagglutinin epitope on the recombinant TviscS-2 and processed for autoradiography, and the radioactivity asso- was used to localize the protein in numerous organelles sur- ciated with it was quantified. Hydrogenosomal holoferredoxin rounding the nuclei and cytoskeletal structures (Fig. 3). Double ϩ migrated in native gels as a distinct rapidly migrating band, with labeling for TviscS-2 and NAD -dependent malic enzyme, a its apoform migrating considerably slower (Fig. 5A), as shown marker enzyme for hydrogenosomes (29), showed their colocal- for the mitochondrial homolog (33). ization, demonstrating that TviscS-2 is localized to hydrogeno- The [FeS] cluster assembly on ferredoxin was linear for 60 min somes. Although in yeast and human cells iscS gene products are (Fig. 5 B and C). The rate of [FeS]ferredoxin formation was 0.3 also targeted to nucleus (15, 18), TviscS-2 was not detectable in pmol of S per milligram of hydrogenosomes per minute. Depen- the nuclei of T. vaginalis transformants (Fig. 3). Accordingly, the dence on concentrations of hydrogenosomal proteins showed that nuclear targeting motif RRRPR, which has been verified exper- the activity corresponded to an enzyme catalyzed process (Fig. 6A). imentally in yeast Nfs1p (18), is highly conserved in all mito- Negligible association of 35S with ferredoxin was observed when chondrial homologues but is absent from trichomonad and hydrogenosomal extract was omitted from the reaction mixture. Giardia intestinalis IscSs (Fig. 4B). However, we cannot exclude The reaction was inhibited almost completely when incubated the possibility that undetectable levels of the T. vaginalis protein on ice. are contained in the nucleus. We also tested the dependence of hydrogenosome-mediated

10370 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401319101 Sutak et al. Downloaded by guest on September 27, 2021 Fig. 5. Reconstitution of [FeS] clusters in T. vaginalis apoferredoxin. (A) Different migration of the apoforms and holoforms of T. vaginalis ferredoxin on native gels. Recombinant T. vaginalis holoferredoxin 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. Apoferredoxin was incubated with 35S-cysteine, Fe-ascorbate, and hydrogenosomal lysate and analyzed by native gel electrophoresis, followed by autoradiography. (C) The bands corresponding to holoferredoxin were cut out of the gel, and the associated radioactivity quantiﬁed by scintillation counting.

that this gene might be transcribed in vivo (Fig. 1A). Surprisingly, no corresponding transcripts could be detected by monitoring

mRNA synthesis in permeabilized cells from Fe-rich or Fe-depleted EVOLUTION 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 target Fig. 4. Comparison of the primary structures of TviscS-1 and TviscS-2. The the proteins to hydrogenosomes, the TviscS-1 extension lacks a unpublished sequence of TﬁcsS from another parabasalid, Tritrichomonas typical cleavage site (Fig. 4A). Moreover, the C-terminal conserved fetus, is shown also. (A) N-terminal regions containing hydrogenosomal targeting sequences (underlined letters). Arginines at position Ϫ2 from the cleavage sites recognized by the program P-SORT (available at http:͞͞ psort.nibb.ac.jp) are shown in bold. (B) Nuclear targeting motifs (boxed letters) are absent in trichomonads and G. intestinalis. (C) C-terminal conserved region is present only in TviscS-2. Involvement of this region 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; Arabidopsis thaliana, O49543; Homo sapiens, Q9Y697; Drosophila melanogaster, Q9VKD3; T. vaginalis 1, AF321005; T. vaginalis 2, AF321006; Tritrichomonas fetus, AAQ01175; and G. intestinalis, AF311744.

[FeS] cluster assembly on the availability of divalent Fe. Hydro- genosomes 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-fold when ferrous ascorbate was added. Removal of endogenous Fe present in hydrogenosomes, or contaminating buffers, by the metal chelator EDTA (500 ␮M) or the ferrous Fe chelator 2,2 dipyridyl (100 ␮M) abrogated the formation of holoferredoxin.

Specificity of the FeS-Forming Activity in Hydrogenosomes and Mi- tochondria. We compared FeS formation in isolated hydrogenosomes and yeast mitochondria by using [2Fe-2S]apoferredoxin from T. vaginalis and spinach. Both hydrogenosomal and mitochondrial extracts catalyzed [FeS] cluster formation in T. vagi- Fig. 6. Dependence of [FeS] cluster reconstitution in apoferredoxin on the nalis ferredoxin (Fig. 7), but only hydrogenosomal lysate cata- concentrations of the hydrogenosomal lysate and Fe. (A) T. vaginalis apo- lyzed cluster formation in spinach ferredoxin. This finding ferredoxin was incubated by using standard conditions with various amounts indicates a specificity of [FeS] cluster assembly machinery of the hydrogenosomal lysate. (B) Reconstitution of [FeS] clusters in the presence of 500 ␮M EDTA; 25 and 100 ␮M ferrous Fe chelator 2,2-dipyridyl; 25 present in the two different organelles. and 100 ␮M Fe-ascorbate, in the presence of 100 ␮g of hydrogenosomal lysate. Formation of holoferredoxin was analyzed by native gel electrophoresis, Lack of in Vivo Transcription of TviscS-1. As mentioned previously, followed by storage phosphorimaging autoradiography. Incorporation of 35S the 5Ј-UTR sequence of the tviscS-1 gene contains two putative into ferredoxin was expressed in arbitrary units. Bars indicate SD, calculated initiator promoter elements upstream of the ATG codon, indicating from three independent experiments in duplicate.

Sutak et al. PNAS ͉ July 13, 2004 ͉ vol. 101 ͉ no. 28 ͉ 10371 Downloaded by guest on September 27, 2021 tral eukaryote (4). A comparative study of protists reveals that double-membrane-bound organelles of possible shared ancestry are present in different species, ranging from typical mitochondria, hydrogenosomes (1), to mitosomes (cryptons), which are smaller structures with no demonstrated role in core carbohy- drate metabolism (40, 41). The taxonomic distribution of these organelles indicates that they may have arisen independently in different lineages several times from the ancestral protomito- Fig. 7. Speciﬁcity of [FeS] cluster assembly. An ability of hydrogenosomal chondrion (3, 4, 42). lysate (Left) and lysate from yeast mitochondria (Center) to catalyze [FeS] The results of this study and our earlier work (24) raise the cluster formation in T. vaginalis or spinach [2Fe-2S]ferredoxins was compared. question whether the double-membrane-bound organelles of The reactions proceeded by using standard conditions for1hat25°C with other eukaryotes without typical mitochondria are also involved equal amounts of organellar lysates (100 ␮g) and ferredoxins (10 ␮g). (Right) For controls, no organelle lysate was added. in FeS biosynthesis. Although the data are scanty, the answer is probably yes. In all of the amitochondriate organisms studied so far, a gene encoding an IscS homolog has been recognized and region of 26 aa, which is required for interaction between IscS and found to be part of the clade formed by typical mitochondrial IscU (35), is absent from TviscS-1 (Fig. 4C). The conditions that are homologs (24, 25, 43). It should be stressed that the studied able to elicit expression of TviscS-1 and the function of this gene amitochondriate groups belong to separate taxonomic lineages. product remain to be established. Additional observations support this conclusion. The diplomonad G. intestinalis deserves special mention. In this Discussion species, FeS biosynthesis and its two components have been This study provides functional evidence that the site of FeS- shown to be compartmentalized, giving cytological evidence for center assembly in the ‘‘amitochondriate’’ eukaryote, T. vaginalis the existence of a possible mitochondrion derivative in this group is the hydrogenosome and that the system performing this (41). In Cryptosporidium parvum, an apicomplexan without process is very similar to that found in typical mitochondria of typical mitochondria, the genes encoding several proteins in- S. cerevisiae (11, 22). A key enzyme of this process, cysteine volved in [FeS] cluster assembly have been identified in its desulfurase (TviscS), as well as the process of FeS-center as- genome. All of them possess N-terminal presequences similar to sembly, could be localized in the hydrogenosomes. TviscS-2 also mitochondrial targeting sequences, suggesting that they are has an putative N-terminal targeting sequence similar to that of localized in the relic mitochondria described (43, 44) in this other hydrogenosomal proteins and is found in a well supported organism. A further example is provided by the group of clade with its mitochondrial homologs in phylogenetic recon- microsporidia, which are highly reduced fungi adopted to intra- structions (24, 25). cellular parasitism. These organisms were regarded for some Hydrogenosome-mediated formation of [FeS] clusters in vitro time as the best example of the amitochondriate phenotype (45, was demonstrated based on [FeS] cluster reconstitution in recom- 46). However, a possible mitochondrial remnant has been de- binant T. vaginalis apoferredoxin by using 35S-cysteine as a source scribed also in the microsporidian Trachipleistophora hominis of S under anaerobic conditions. Addition of ferrous Fe to the (47), and several genes of mitochondrial origin are present in the reaction mixture increased [FeS] cluster formation, whereas Fe genome of another microsporidian Encephalitozoon cuniculi. chelators had an inhibitory effect. This observed in vitro assembly Most of these proteins belong to the [FeS] cluster assembly of [FeS] clusters extends our earlier observations that addition of machinery (48). It is tempting to speculate that [FeS] cluster 59Fe to live trichomonads results in fast accumulation of Fe in assembly is the main function retained by mitochondrion-related hydrogenosomes and its incorporation into [FeS] clusters of hy- organelles without known metabolic functions (relic mitochon- drogenosomal ferredoxin (34). Although further functional simi- dria and mitosomes) (40, 43, 47, 48). The evolutionary reason for larities between the hydrogenosomal and the mitochondrial system retaining such organelles could be the need to compartmentalize can be expected, the extent of these similarities remains to be toxic ferrous ions and sulfide, which are required for FeS established. One interesting difference is that the cysteine desul- biogenesis. Moreover, amitochondrial organisms living under furase of T. vaginalis appears to be localized only in the hydrogeno- anaerobic or oxygen-poor environments have considerably somes, in contrast to its homologs in yeast and vertebrates, which higher nutritional requirements for Fe than aerobes (49, 50). In are also detected in the cytosol and nucleus (15, 18). This obser- hydrogenosomes, we found 54 nmol of Fe per 1 mg of protein, vation may be explained by the absence of a nuclear localization which is a 10- to 100-fold higher concentration than in yeast motif from the T. vaginalis protein. Interactions among components mitochondria (34). Thus, compartmentation of Fe metabolism of the [FeS] cluster assembly machinery and with target FeS could be an essential strategy for these organisms. apoproteins might differ in hydrogenosomes and mitochondria. In conclusion, our data indicate that hydrogenosomes of the amitochondriate protist T. vaginalis are the site of FeS biosyn- Indeed, hydrogenosomal lysates mediate [FeS] cluster reconstitu- thesis, which is a typically mitochondrial process. These findings tion in spinach apoferredoxin, whereas yeast mitochondria did not underscore the notion that mitochondria and hydrogenosomes support this reaction. are closely related organelles with a common ancestry. Addi- The FeS biosynthetic machineries of parabasalid hydrogeno- tional data on other amitochondriate organisms indicate that somes and typical mitochondria are functionally and evolution- their FeS biosynthesis is similar to that seen in mitochondria and arily related. This idea is in agreement with the notion that these parabasalid hydrogenosomes, suggesting that this process is a two organelles are two metabolically different endpoints of the common property of all double-membrane-bound organelles divergent evolution of the protomitochondrion that arose during that arose from this endosymbiotic event. Clearly, further data eukaryogenesis (3, 7). Support for this notion has been adduced are needed to test this proposal. from similarities of the biogenesis of these two organelles (2, 9, 36) and from the phylogenetic relationships of some of their This work was supported by a Fogarty International Research Collab- protein components (24, 37–39). oration Award (to M.M. and J.T.); Grant Agency of the Czech Republic Hydrogenosomes of T. vaginalis and other parabasalids are, Grants 204͞00͞1561 and 204͞04͞0435 (to J.T.); and National Institutes however, only one of the diverse organelles that are assumed to of Health Grants AI11942 (to M.M.), AI27857 (to P.J.J.), DK53953 be descendants of the proteobacterial component of the ances- (to A.D.), and GM65664 (to H.L.F.).

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