1 Wadsworth Center, New York State Department of Health, PO Box 22002, Albany, NY 12201-2002, USA
2 First Faculty of Medicine, Charles University, Prague, Czech Republic
3 Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
Correspondence
Michael J. LaGier
michael.lagier{at}noaa.gov
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ABSTRACT |
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Present address: University of Miami, CIMAS, 4600 Rickenbacker Causeway, Miami, FL 33149, USA.
The GenBank accession numbers for the sequences reported in this paper are AY029212 (CpIscS) and AY078500 (CpIscU).
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INTRODUCTION |
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Ironsulfur clusters [FeS] are ubiquitous, and [FeS]-containing proteins play critical metabolic, regulatory and signalling roles in essentially all forms of life, from archaea to man (Johnson et al., 1998; Lill & Kispal, 2000
; Gerber & Lill, 2002
). Furthermore, because [FeS] have a broad range of redox potentials, many [FeS]-containing proteins are key components in electron-transfer reactions. Although [FeS] have been proposed to be among the earliest catalytic centers in biochemical evolution (Cammack, 1971
, 1983
), little is known about their biosynthesis in eukaryotes (Seeber, 2002
). Nonetheless, the ancient character of [FeS] is supported by recent observations that proteins involved in [FeS] biosynthesis are highly conserved in prokaryotes and eukaryotes (Mühlenhoff & Lill, 2000
; Tachezy et al., 2001
; Tovar et al., 2003
), including members of the Apicomplexa (Seeber, 2002
).
Among the proteins involved in [FeS] assembly, the two best characterized and most highly conserved are a pyridoxal-5'-phosphate (PLP)-dependent cysteine desulfurase which catalyses the formation of L-alanine and elementary sulfur using L-cysteine as a substrate (IscS), and the protein IscU, which serves as a scaffolding for the assembly of [FeS] prior to their incorporation into apoproteins (Mühlenhoff & Lill, 2000).
In most eukaryotes [FeS] assembly and maturation is primarily confined to mitochondria and chloroplasts (reviewed by Lill & Kispal, 2000). Until very recently, the cellular localization of [FeS] assembly in amitochondriate protists was completely unknown. Now, however, in addition to the hydrogenosomes of the parabasalid Trichomonas vaginalis (Tachezy et al., 2001
), mitochondrion-like organelles discovered in the diplomonad Giardia intestinalis have been shown to participate in [FeS] biosynthesis (Tovar et al., 2003
). All of these organelles are thought to be descendents of a common endosymbiont which gave rise to both hydrogenosomes and mitochondria (Martin & Müller, 1998
; Dyall & Johnson, 2000
; Rotte et al., 2000
; Martin et al., 2001
). Putative mitochondrial relicts have also been identified in the parasitic amoeba Entamoeba histolytica (mitosome, Tovar et al., 1999
; crypton, Mai et al., 1999
) and the microsporidian Trachipleistophora hominis (Williams et al., 2002
). Although similar structures have not yet been observed in the microsporidian Encephalitozoon cuniculi, even this species contains genes of probable mitochondrial origin (Katinka et al., 2001
).
Recent data indicate that the ribosome-studded organelle of C. parvum sporozoites is a relict mitochondrion (Riordan et al., 2003). Significantly, the C. parvum mitochondrion is limited by a double membrane, contains internal membranous cristae-like compartments, and both a mitochondrial-type, nucleus-encoded chaperone protein (Cpn60; GenBank AAC32614) and heat-shock protein 70 (Cp-mtHSP70; GenBank AY235430) are localized to it (Riordan et al., 2003
; J. R. Slapeta & J. S. Keithly, unpublished observations). Although the physiological functions of the C. parvum mitochondrion, as well as other mitochondrial remnants, are unknown (Katinka et al., 2001
; Williams et al., 2002
), mitochondria-type [FeS] assembly machinery has been demonstrated for the amitochondriate protist G. intestinalis (Tovar et al., 2003
), and suggested for T. vaginalis (Tachezy et al., 2001
), E. histolytica (GenBank accession AY040613) and E. cuniculi (Katinka et al., 2001
). Here we propose that IscS and IscU in the apicomplexan C. parvum may also contribute to [FeS] assembly within its relict mitochondrion.
As an initial step in addressing this hypothesis, nucleus-encoded IscS and IscU genes have been isolated from C. parvum, transcription in sporozoites documented, and the N-terminal signal sequences of these two [FeS] homologues reconstructed in a vector to deliver green fluorescent protein (GFP) to the yeast mitochondrial network.
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METHODS |
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Molecular cloning.
Partial sequences of C. parvum genes encoding homologues of proteins involved in [FeS] assembly were identified by scanning genomic DNA (gDNA) sequences deposited into the GenBank database as part of an ongoing C. parvum genomic sequence survey (GSS; Strong & Nelson, 2000). These initial C. parvum database searches were performed using BLAST (Altschul et al., 1997
) 2.0 (Expected value set to 10) at the National Center for Biotechnology Information (NCBI) homepage (http://www3.ncbi.nlm.nih.gov/BLAST) using IscS (GenBank M98808) and IscU (GenBank NP014869) homologues from the yeast Saccharomyces cerevisiae (Mühlenhoff & Lill, 2000
) as query sequences. GSS clones containing gDNA fragments of putative C. parvum IscS (GenBank AQ449762) and IscU (GSS contig #1309) homologues were identified from this database.
Next, PCR was used to amplify portions of both clones directly from isolated sporozoite gDNA. The resultant amplicons were extracted from agarose gels and utilized as probes to screen plasmid-based C. parvum gDNA libraries transformed into the bacterium Escherichia coli (Zhu et al., 2000). Two gDNA libraries of KSU-1 sporozoites constructed within pBluescript SK+ (Stratagene) at the EcoRI or HindIII sites were utilized for cloning. The final ORFs of both CpIscS and CpIscU were obtained by screening both gDNA libraries using gene-walking (LaGier et al., 2001
; Zhu et al., 2000
). All clones represent at least two identical overlapping clones obtained from each library. Each clone was sequenced twice on both strands by automated sequencing. Oligonucleotides for sequencing and PCR were synthesized, and automated sequencing was performed by the Molecular Genetics Core Facility at the Wadsworth Center (USA) or by Generi Biotech (Czech Republic). Sequence analyses, including clone assembly, protein translation and primary sequence analysis used the GCG Wisconsin Package UNIX version 9.1 (Genetics Computer Group), and Lasergene system (DNASTAR). The C. parvum IscS and IscU sequences have been submitted to GenBank under accession numbers AY029212 and AY078500, respectively.
Sequence alignments and phylogenetic analysis.
Nucleotide and protein databases at NCBI were queried by BLAST 2.0 (Expected value set to 10) using the complete ORF of CpIscS or CpIscU. Sequences of interest were extracted from the various databases using the BlastAli program (http://www.joern-lewin.de/). The IscS and IscU sequences of C. parvum were aligned to sequences from the eubacterium Azotobacter vinelandii, mitosome-bearing diplomonad G. intestinalis, hydrogenosome-bearing parabasalid Trichomonas vaginalis (IscS only), mitochondrion-bearing apicomplexan Plasmodium falciparum (IscS), yeast S. cerevisiae, and microsporidian Encephalitozoon cuniculi using the default parameters of CLUSTAL_X (Thompson et al., 2000). For phylogenetic analysis, CpIscS was aligned to sequences from 24 selected taxa using CLUSTAL_X (Thompson et al., 2000
) and was further edited visually using the ED program of MUST (Philippe, 1993
). The alignment of the 24 IscS taxa resulted in 361 shared amino acid positions.
Phylogenetic relationships were analysed by the neighbour-joining (NJ) and maximum-parsimony (MP) methods using PHYLIP, version 3.6 (Felsenstein, 1989), and by the maximum-likelihood (ML) method using the PROTML program in MOLPHY, version 2.3 (Adachi & Hasegawa, 1996
). The ML tree was constructed by local rearrangement of an NJ tree using the JonesTaylorThornton model of amino acid substitutions with the F-option to account for amino acid frequencies in the dataset. User-defined trees were analysed to compare alternative topologies (Kishino & Hasegawa, 1989
). The local bootstrap proportion value was calculated for each internal branch of the ML tree using a local rearrangement option of the PROTML program. Bootstrap support (bootstrap proportion, BP) for distance and parsimony analyses was based on 100 re-sampled datasets using SEQBOOT, PHYLIP, version 3.6.
Prediction of mitochondrial targeting sequences.
Two in silico methods were used for predicting N-terminal mitochondrial targeting peptides, TargetP (Emanuelsson et al., 2000) and MitoProt (Claros & Vincens, 1996
; Emanuelsson & von Heijne, 2001
). Specifically, potential N-terminal peptides were identified for both CpIscS and CpIscU using TargetP (http://www.cbs.dtu.dk/services/TargetP/) and MitoProt (http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter). In the analyses of CpIscS and CpIscU, TargetP was used in the winner-takes-all mode without setting a specificity cut-off for targeting, and MitoProt was implemented using default parameters. The entire ORFs of CpIscS or CpIscU were used as query templates during analysis with both MitoProt and Target P. MitoProt was also used for the prediction of putative signal peptide cleavage sites (default settings). Prediction of
-helical structures was performed using the secondary structure program Predator at the Predict Protein server (http://www.embl-heidelberg.de/predictprotein/predictprotein.html). The reliability classes of TargetP decrease from a score of 1 (97 % specificity) to 5 (53 % specificity). The reliability feature of TargetP is an indication of the level of certainty in a prediction for a given sequence (Emanuelsson et al., 2000
).
Southern blotting.
For Southern blot analysis, 2·5 µg of sporozoite gDNA per lane was digested with restriction endonucleases, separated by electrophoresis in 0·75 % agarose gels and transferred to Zeta-probe Nylon membranes in a 0·4 M NaOH solution (Bio-Rad). Fragments of the CpIscS and CpIscU ORFs, 758 and 447 bp respectively, were PCR-amplified from isolated sporozoite gDNA using gene-specific primers (Table 1) and the resultant amplicons were extracted from agarose gels (Maniatis et al., 1989
). Next, the isolated gDNA fragments of CpIscS and CpIscU were random-primer labelled with [
-32P]dATP (Boehringer Mannheim), and used as probes for Southern blot analyses under conditions of high stringency (Maniatis et al., 1989
).
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Prior to cDNA synthesis, DNase-treated RNA was tested for the presence of gDNA contamination using standard PCR (Maniatis et al., 1989) and oligonucleotide primers specific for the C. parvum 18S rRNA gene (Abrahamsen & Schroeder, 1999
). More specifically, following extraction from sporozoites, 250 ng total RNA was PCR amplified for 35 cycles, and agarose gel electrophoresis was used to confirm the absence of gDNA in RNA preparations as indicated by a lack of detectable 18s rRNA amplicons.
The amplification of cDNA via standard PCR (Maniatis et al., 1989) for 35 cycles (94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min) was performed using primers specific for CpIscS or CpIscU (Table 1
). Each reaction contained 0·2 mM gene-specific primers, 2 µl gDNA-free cDNA, 0·8 mM dNTPs, 2 mM MgCl2, 10x PCR reaction buffer (Promega), and 5 U Taq polymerase (Promega). Following agarose gel electrophoresis, amplicons of the expected sizes were sequenced to confirm identity.
Targeting of predicted C. parvum signal peptides in S. cerevisiae.
The predicted mitochondrial signal peptides of CpIscS and CpIscU were amplified via PCR from IOWA gDNA using oligonucleotide primers with 5' restriction cut-sites, EcoRI and BamHI (Table 1). The mitochondrion-targeted plasmid pYX223-mtGFP (Westermann & Neupert, 2000
) was a gift from B. Westermann (Ludwig-Maxilians-Universität, Müchen, Germany). This plasmid contains a known mitochondrial pre-sequence from protein 9 (proteolipid subunit) of the F0 part of the F1F0 ATPase of Neurospora crassa ligated to GFP, and has an inducible galactose promoter. The first 168 bp (56 aa) of the CpIscS gene and the first 111 bp (37 aa) of the CpIscU gene, respectively, were generated by PCR from gDNA by adding EcoRI and BamHI linker sequences to the 5' and 3' end of these genes, respectively. The N. crassa mitochondrial pre-sequence was digested from the plasmid using EcoRI and BamHI, and then was replaced with the putative C. parvum pre-sequences. The new recombinant plasmids pCpIscS-56-GFP and pCpIscU-37-GFP were amplified in Ultracompetent XL-10 gold cells (Stratagene), purified, and sequenced to confirm identities. These were then transfected using the standard lithium acetate method (Burke et al., 2000
) into S. cerevisiae strain W303 (Gaxiola et al., 1998
), a gift of D. Kornitzer (Israel Institute of Technology, Haifa, Israel), and plated onto 2 % galactose-containing histidine-free synthetic complete agar to induce expression of the GFP. Only cells transformed with the recombinant plasmids grew on selective plates, and samples from these colonies were prepared for examination by growing for 3 days at 30 °C in histidine-free synthetic complete liquid broth and immobilizing with 0·5 % low-melt agarose on slides for fluorescence microscopy. Yeast cells were observed with an Olympus BX 51 microscope equipped for differential interference contrast and fluorescent microscopy. All photomicrographs were processed identically following capture with a colour digital camera (Alpha Innotech) and documented using Adobe Photoshop 6.1 (Adobe Systems). For excitation of GFP, a 450490 nm band pass filter was used, and emitted light observed with a 520 nm pass filter.
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RESULTS |
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While a number of potential initiation codons were noted at the 5' end of CpIscS and CpIscU, the first in-frame ATG triplets (16ATG and 10ATG, respectively) have been designated the putative translation start sites. This interpretation is supported by the presence of purines at positions -3 and +4, matching consensus nucleotides for start codon sequences (Kozak, 1989) in eukaryotic mRNAs (data not shown).
CpIscS and CpIscU are single-copy genes
Genomic DNA isolated from C. parvum sporozoites was digested with EcoRI or HindIII, and then used for Southern blot analysis. Using 758 bp of the CpIscS ORF as a probe, hybridizing fragments of 1·2 and 8·0 kb were observed (Fig. 1a). Identically prepared Southern blots probed with a 447 bp CpIscU probe hybridized with fragments of 2·0 and 2·6 kb (Fig. 1b
). Overall, these data suggest that CpIscS and CpIscU are single-copy genes.
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CpIscS phylogeny
The phylogenetic relationship among major IscS clusters was examined using 361 aa within conserved domain contigs from a dataset of 25 sequences that included IscS from C. parvum and four other protists (two T. vaginalis isoforms), six from proteobacteria, and 14 from other eukaryotic taxa (Fig. 3). The topology of this ML tree essentially agrees with that for the C. parvum mitochondrial genes AK2 (adenylate kinase 2; Riordan et al., 1999
) and Cpn60 (Riordan et al., 2003
), as well as to a previous tree of eukaryotic-proteobacterial IscS constructed using the ML method (Tachezy et al., 2001
). The NJ and ML methods confirmed the close relationship between eukaryotic IscS with high bootstrap support (Fig. 3
, BP=100 and 94 %, respectively). Six branches were defined on the tree: (1) Protista, (2) Fungi, (3) Arabidopsis and Dictyostelium, (4) Metazoa, (5) Rickettsia, and (6) Proteobacteria. Consistent with previous analyses, the Proteobacteria were recovered as a sister group to the mitochondrion-bearing eukaryote clades (Fig. 3
) with robust bootstrap support (BP=99/100/100 %), suggesting a common ancestral origin for IscS in [FeS] assembly (Dyall & Johnson, 2000
; Mullenhoff & Lill, 2000
; Martin et al., 2001
; Tachezy et al., 2001
). Although CpIscS clustered with significant bootstrap support (BP=75 %) together with IscS homologues of the protists G. intestinalis, T. vaginalis and P. falciparum within the mitochondrion-bearing eukaryotes (Fig. 3
), the placements between and among protist taxa are not clearly resolved. The two most likely reasons for poor resolution among protists in this study are: (i) lack of species diversity in the IscS database, and (ii) presence of lineages which are often fast-evolving and which can be misplaced due to long-branch attraction artefact (Philippe & Laurent, 1998
; Tachezy et al., 2001
). Long-branch attraction artefact is probably responsible for relationships here among trichomonads, diplomonads and apicomplexans (Fig. 3
).
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Primary sequence analysis of CpIscU
The primary sequence of CpIscU was compared to IscU homologues from four diverse taxa, including the eubacteria A. vinelandii and E. coli, the yeast S. cerevisiae (two isoforms), and the diplomonad G. intestinalis. Most importantly, CpIscU contains the three cysteine residues (positions 60, 85 and 128) previously shown to be essential for [FeS] scaffolding of S. cerevisiae IscU (Garland et al., 1999), and interaction with a mitochondrial chaperone (Hsp70; Tokumoto et al., 2002
). CpIscU also possesses the conserved Asp62 residue thought to play a role in the release of transient [FeS] for eventual delivery to apoproteins (Mühlenhoff & Lill, 2000
).
Similar to CpIscS, phylogenetic analysis supported CpIscU clustering with mitochondria-type homologues (data not shown). Like G. intestinalis IscU (Tovar et al., 2003), the N-terminal sequence of CpIscU is rich in hydroxylated and basic amino acids, and MitoProt predicted the mitochondrial localization of CpIscU with high confidence (P>0·96). Furthermore, the CpIscU predicted N-terminal cleavage site at Ser27 is downstream just one residue from Tyr35, the cleavage site of the human mitochondria-type IscU sequence (Tong & Rouault, 2000
). According to Predator software (section 2.5), these 27 aa have the potential to form the amphiphilic
-helical structures 3QLRQLIDKRIL13 and 21CQRLFYSDTVHDHF34 necessary for targeting mitochondria (Fig. 4
, Table 2
). Therefore, although neither in silico method predicted a typical cleavage site, these findings suggest that the N-terminal pre-sequence might target CpIscU to the relict mitochondrion of C. parvum. In addition, not all targeting sequences follow the rules' for cleavage (Taylor et al., 2001
), and most of the prediction programs, including MitoProtI and TargetP, are based upon targeting sequences from mammals, yeast, and other model organisms. Therefore, it is not too surprising that these in silico methods vary in their ability to exactly predict cleavage sites in protists. For example, neither C. parvum Cpn60 nor mt-HSP70 perfectly fit the cleavage rules, yet both have been shown by immunogold labelling to properly target the relict mitochondrion of C. parvum (Riordan et al., 2003
; J. R. Slapeta & J. S. Keithly, unpublished results). Furthermore, unlike CpIscS and GiIscU, both Giardia IscS and the microsporidian Trachipleistophora mtHsp70 lack typical N-terminal signals, but both are efficiently targeted into remnant mitochondrion-derived organelles (Tovar et al., 2003
; Williams et al., 2002
).
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Targeting of GFP to yeast mitochondria using C. parvum signal peptides
As mentioned previously, plasmid vectors were constructed to test for the ability of the N-terminal signal peptides of CpIscS and CpIscU to deliver proteins to the mitochondrial network of yeast. Both pCpIscS-56-GFP and pCpIscU-37-GFP (see Methods) can correctly target GFP to the mitochondrial network of transformed yeast, as indicated by a sinuous intracellular staining pattern (Fig. 6). This pattern is consistent with previous studies describing the mitochondria of S. cerevisiae as branched, tubular networks located below the cell cortex (Hoffman & Avers, 1973
). As expected, yeast transfected with pYX223-mtGFP lacking the N. crassa mitochondrial targeting sequence showed a cytoplasmic localization of GFP, confirming the dependence of an N-terminal targeting sequence for trafficking of GFP to yeast mitochondria (data not shown). Intact pYX223-mtGFP was used to transfect yeast as a positive control. As shown in Fig. 6(f)
, yeast containing pYX223-mtGFP localize GFP to the mitochondrial network of S. cerevisiae.
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DISCUSSION |
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Here we show by genetic analysis that the apicomplexan C. parvum, like other protists, encodes two key components of [FeS] assembling machinery the PLP-dependent cysteine desulfurase (IscS), and IscU, a protein providing scaffolding for transient [FeS] formation (Lill & Kispal, 2000; Gerber & Lill, 2002
). Both sequence and phylogenetic analyses suggest that CpIscS and CpIscU share a common ancestry from a proteobacterium. Our data are also consistent with the observation that most protist IscS homologues belong to group I, which includes those of bacteria and eukaryotic mitochondria (Tachezy et al., 2001
), rather than group II IscS homologues, which include a diverse group of bacteria and plastid-containing eukaryotes.
Both pCpIscS-56-GFP and pCpIscU-37-GFP (see Methods) delivered GFP into the S. cerevisiae mitochondrial network, indicating that CpIscS and CpIscU contain N-terminal sequence elements that are interpreted by yeast as signals for import into the organelle. Interestingly, the CpIscU predicted N-terminal cleavage site at Ser27 is downstream just one residue from Tyr35, the cleavage site of the human IscU mitochondria-type isoform (Tong & Rouault, 2000), whereas the G. intestinalis IscU mitochondrial cleavage site at Glu27 is two residues upstream of the human site (Tovar et al., 2003
). Therefore, it appears that although some divergence has occurred among protist [FeS] protein mitochondrial targeting signals, sufficient sequence information is retained to properly deliver nucleus-encoded IscU into the mitochondrial compartment.
[FeS] biogenesis also requires reduced iron (Scheffler, 1999), which is highly toxic for cells (Scheffler, 1999
; Gerber & Lill, 2002
), and therefore a reason for retaining mitochondrial compartments even if they have become altered (Roger et al., 1998
; Katinka et al., 2001
; Williams et al., 2002
). Recent evidence has shown, for example, that 59Fe can be incorporated into the hydrogenosome of the cattle parasite Tritrichomonas foetus, and that this requires ferredoxin and other peptides within the [FeS] machinery (Suchan et al., 2003
). Whether C. parvum might also incorporate iron into the relict mitochondrion is not yet known, but certainly the fact that this apicomplexan possesses IscS, IscU, ferredoxin and mtHsp70 homologues with mitochondrial targeting signals suggests that [FeS] biogenesis may be one of the essential biochemical pathways of the endosymbiont retained by parasitic protists after reductive evolution of the mitochondrial relict.
The localization of human IscS and IscU homologues is regulated through translation initiation at alternative in-frame AUG sites (Land & Rouault, 1998), and alternative splicing of a common pre-mRNA, respectively (Tong & Rouault, 2000
). That is, human IscS and IscU can be targeted to mitochondria, the cytosol or nucleus (IscS only) as a consequence of these post-transcriptional events. Although alternative splicing is unlikely to regulate CpIscU targeting since the gene consists of a single exon, only the intracellular localization of CpIscS and CpIscU in sporozoites can directly ascertain whether [FeS] assembly occurs within the relict mitochondrion of C. parvum.
In summary, sequence and phylogenetic analyses indicate that C. parvum IscS and IscU are mitochondrial isotypes, and that like the [FeS] assembly proteins found in the genomes of the amitochondriate protists Entamoeba histolytica (http://www.sanger.ac.uk/Projects/E_histolytica/; http://www.tigr.org/tdb/e2k1/eha1/), Giardia intestinalis (McArthur et al., 2000), Trichomonas vaginalis (Tachezy et al., 2001
), and the microsporidian Encephalitozoon cuniculi (Katinka et al., 2001
), arose by a symbiogenic event from a common proteobacterial ancestor. Although these data lend support to the hypothesis that one of the functions retained by the relict mitochondrion of C. parvum is the assembly and maturation of [FeS], this hypothesis awaits both definitive localization of CpIscS and CpIscU to the organelle and measurement of enzyme activities for both enzymes.
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ACKNOWLEDGEMENTS |
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Received 25 March 2003;
revised 28 August 2003;
accepted 9 September 2003.