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í Petrák2
ut'ák1
nková3
al1
1 Department of Parasitology, Faculty of Science, Charles University, Vini
ná 7, 128 44, Prague 2, Czech Republic
2 Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 44, Prague 2, Czech Republic
3 Department of the Tropical Medicine, 1st Faculty of Medicine, Charles University, Faculty Hospital Bulovka, Studnikova 7, 128 00, Prague 2, Czech Republic
4 Johns Hopkins University, Bloomberg School of Public Health, W. Harry Feinstone Department of Molecular Microbiology and Immunology, 615 North Wolfe Street, Baltimore 21205, MD, USA
Correspondence
Jan Tachezy
tachezy{at}natur.cuni.cz
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AF545472.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To cover its nutritional requirements, Tt. foetus is able to utilize lactoferrin, transferrin or low-molecular-mass iron complexes (Tachezy et al., 1996; Tachezy et al., 1998). Lactoferrin is internalized via receptor-mediated endocytosis, while transferrin-bound iron and iron from low-molecular-mass complexes is acquired by means of carrier-mediated transport (Tachezy et al., 1996, 1998). The high nutritional requirements of Tt. foetus and other amitochondrial protists such as Giardia intestinalis and Entamoeba histolytica are ascribed to the importance of FeS protein in their energy metabolism (Payne et al., 1993; Ellis et al., 1993; Weinbach et al., 1980; Gorrell et al., 1984; Müller, 1988). They possess ferredoxin-mediated low-redox potential electron transport, which is linked to pyruvate : ferredoxin oxidoreductase (PFOR), a key FeS-containing enzyme of pyruvate metabolism (Weinbach et al., 1980; Brown et al., 1998; Ellis et al., 1993; Kulda, 1999). In trichomonads, PFOR-dependent pyruvate metabolism takes place in specific ATP-generating organelles called hydrogenosomes (Dyall & Johnson, 2000; Müller, 1993). Several authors have reported that iron-restricted nutritional conditions cause a decrease in the metabolic activity of hydrogenosomes (Tachezy et al., 1996; Va
áová et al., 2001; Gorrell, 1985; Peterson & Alderete, 1984). This phenomenon are associated with a down-regulation of the genes encoding PFOR and other hydrogenosomal proteins (Vaáová et al., 2001). Similar changes have been observed in metronidazole-resistant organisms. Metronidazole and other 5-nitroimidazoles, which are used for the treatment of trichomoniasis, are reductively activated in hydrogenosomes. The [2Fe–2S] ferredoxin is considered to be the major hydrogenosomal electron carrier responsible for drug activation. In the absence of metronidazole, ferredoxin transports electrons from PFOR to [Fe]-hydrogenase, where molecular hydrogen is formed. When metronidazole is present, the ferredoxin-transported electrons are preferentially captured by the drug, which results in a release of cytotoxic nitro-radicals (Kulda, 1999). A decreased expression or absence of ferredoxin, PFOR and other hydrogenosomal proteins has been found in metronidazole-resistant Trichomonas vaginalis (Rasoloson et al., 2001, 2002; Quon et al., 1992) and Tt. foetus (Land et al., 2001).
The mechanisms of intracellular iron transport and its delivery to organelles are poorly understood processes in eukaryotes and virtually unknown in parasitic protists. Several authors proposed that iron is transported within the cell in a complex with low-molecular-mass ligands; however, their molecular basis remains unclear (Jacobs, 1977; Weaver & Pollack, 1989; Bohnke & Matzanke, 1995). ‘Mobile’ iron is also referred to as the labile-iron pool (LIP) as it is easily removed from ligands by iron chelators. It can be visualized and quantified by means of native gradient electrophoresis followed by storage phosphorimaging (Vyoral & Petrák, 1998b; Vyoral et al., 1998). An alternative model for intracellular iron transport was proposed by Richardson et al. (1996). They suggested that iron is transported within the cell in endosomal vesicles and delivered to various cellular compartments by means of direct protein–protein contact transport without the contribution of low-molecular-mass iron ligands.
In mitochondria, iron is required for the biogenesis of FeS proteins (Lill & Kispal, 2000). The source of sulfur for FeS cluster assembly is cysteine, from which molecular sulfur is released by IscS, a PLP-dependent cysteine desulfurase (Zheng et al., 1994). It is likely that in trichomonads IscS-mediated FeS cluster formation occurs within the hydrogenosomes (Tachezy et al., 2001). Unlike sulfur, the source of iron is unknown in both mitochondria and hydrogenosomes. Here we use native gel electrophoresis to trace iron uptake and distribution in Tt. foetus. A pathway to the hydrogenosomes is defined, mediating iron accumulation in that organelle and delivery to the resident FeS protein ferredoxin.
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), metronidazole-resistant strain Lub-1 MR100 (Tachezy et al., 1998), G. intestinalis strain Portland-1 (ATCC 30888) and E. histolytica strain HM-1 : IMSS (ATCC 30459).
Trichomonads were maintained in TYM medium (Diamond, 1957) with 10 % heat-inactivated horse serum at pH 7·2. G. intestinalis cells were grown in TYI-S-33 medium supplemented with 10 % heat-inactivated bovine serum and 0·1 % bovine bile at pH 6·8 (Keister, 1983). For cultivation of E. histolytica parasites, TYI-S-33 medium with 10 % heat-inactivated bull serum and a complex vitamin mixture was used (Diamond et al., 1978). K-562 human leukaemic cells (ATCC CCL-243) were maintained in Iscove's modified Dulbecco's medium (Sigma) supplemented with 10 % fetal bovine serum.
Reagents.
Bovine and human apotransferrins and lactoferrins were purchased from Sigma, desferrioxamine (DFO) was obtained from Ciba-Geigy, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, Deferiprone) was kindly provided by Dr G. J. Kontoghiorges (Royal Free Hospital, London). Radiolabelled iron(III)-nitrilotriacetate (Fe-NTA) was prepared according to Bates & Wernicke (1971) using 59FeCl3 (NEN) and NTA-disodium salt (Sigma) in a molar ratio of 1 : 4.
Iron desaturation and saturation of lactoferrin and transferrin.
Bovine and human lactoferrins were iron-desaturated according to Mazurier & Spik (1980). Saturation of apolactoferrin and apotransferrin with iron using the 59Fe-NTA complex was performed as described previously (Tachezy et al., 1996). The proteins were about 80–95 % iron-saturated as determined by incorporated radioactivity. Non-radioactive diferric transferrin was prepared by the same method using a non-radioactive Fe-NTA complex.
Growth experiments.
To test iron-dependent stimulation of cell growth, TYM and TYI-S-33 media were depleted of iron by addition of 100 µM 2,2-dipyridyl and supplemented with serial concentrations of Fe-NTA, Fe-transferrin or Fe-lactoferrin. Tt. foetus suspensions [1x105 cells (ml depleted medium)-1] were placed on 96-well microtitre plates (250 µl aliquots per well) and the plates were incubated in anaerobic jars at 37 °C for 30 h. G. intestinalis and E. histolytica aliquots of 1 ml (1x105 cells) per well were placed on 24-well microtitre plates and incubated for 48 and 72 h, respectively. Cultivation was stopped by addition of 25 µl 1 % formaldehyde in PBS per well and, prior to counting, the plates were left for 3–12 h in a refrigerator which led to quantitative detachment of the formaldehyde-fixed cells.
Incorporation of [59Fe]iron.
The parasites were washed three times in NaCl-HEPES buffer (0·14 M NaCl, 10 mM HEPES, pH 7·4), resuspended to a density of 5x107 ml-1 and 100 µl aliquots were placed into 1·5 ml microtubes. The cells were preincubated at 37 °C for 15 min. Subsequently, NaCl-HEPES buffer and 59Fe-NTA, 59Fe-transferrin or 59Fe-lactoferrin were added to the cell suspension to give 150 ng Fe ml-1 in a final volume of 250 µl. The cells were incubated at 37 °C for 60 min, washed three times in NaCl-HEPES buffer to remove the unbound radioactivity and stored at -70 °C until further processing.
Subcellular fractionation.
Tt. foetus cells (5x108) were incubated with 59Fe-NTA as described above in 25 ml NaCl-HEPES buffer. The parasites were washed three times in sucrose-Tris (ST) buffer (0·25 M sucrose, 10 mM Tris, 0·5 mM KCl, pH 7·2) to remove the unbound radioactivity and resuspended at a density of about 5x108 cells ml-1. Cytosolic, microsomal, hydrogenosomal and lysosomal fractions were obtained by means of differential and gradient centrifugation. All steps were performed at 4 °C in ST buffer supplemented with 10 µg leupeptin ml-1 and 50 µg L-tosyl lysyl chloromethyl ketone ml-1. The cells were disrupted by 10 strokes in a Potter–Elvehiem homogenizer. The homogenate was diluted twice with ST buffer and centrifuged for 10 min at 700 g to remove the cell debris. The supernatant was spun for 10 min at 10 300 g, resulting in a large granule fraction (sediment) and a crude cytosolic fraction (supernatant). The crude cytosolic fraction was centrifuged for 30 min at 140 000 g to obtain the final cytosolic fraction (supernatant) and microsomes in the sediment. The large granule fraction was loaded on a Percoll cushion (ST buffer containing 20 % Percoll; Amersham) and centrifuged for 30 min at 20 000 g to obtain hydrogenosomal and lysosomal fractions. Both fractions were washed twice with 10 vols ST buffer to remove the Percoll. All fractions were counted for total 59Fe radioactivity and the protein content was assayed by the method of Lowry. Assays for marker enzymes [NAD-dependent malate dehydrogenase (decarboxylating), NADH oxidase and acid phosphatase)] were performed as described by Drmota et al. (1996), Rasoloson et al. (2002) and Müller (1973).
Kinetics of 59Fe uptake.
In a time-course experiment, 5x106 Tt. foetus cells were incubated with 59Fe-NTA as described above in 250 µl NaCl-HEPES buffer for different time intervals (3–180 min). The parasites were immediately washed three times in ice-cold NaCl-HEPES buffer and stored at -70 °C.
In pulse–chase experiments, the cells (5x108) were incubated with 59Fe-NTA as described above in 25 ml NaCl-HEPES buffer for 10 min. The parasites were washed three times in ice-cold NaCl-HEPES buffer to remove the unbound radioactivity and resuspended in 25 ml NaCl-HEPES buffer pre-warmed to 37 °C. The cell suspension was reincubated in the absence of radioactive iron for 0, 60 or 150 min at 37 °C, washed twice in ST buffer and the cells were immediately fractionated as described above.
Sample preparation, electrophoresis and storage phosphorimaging.
Samples were solubilized by the addition of 20 % Triton X-100 to give a final detergent concentration of 1·5 % at 4 °C for 10 min. The lysates were vortexed and centrifuged at 4 °C for 20 min at 15 000 g. Both pellets and supernatants were counted for 59Fe radioactivity. Supernatants were then mixed with sample buffer (10 % sucrose with a trace amount of bromophenol blue stain) and aliquots corresponding to 500–2500 c.p.m. were applied to the sample wells for the electrophoretic separation. Protein load ranged from 20 to 600 µg per well. In the experiments focused on iron chelatability, DFO or Deferiprone was added to the samples prior to loading on the gel to give final Fe concentrations of 25, 100 or 500 µM.
Linear 3–20 % polyacrylamide gradient gels containing 1·5 % Triton X-100 were prepared for separation of iron-binding proteins as described by Vyoral et al. (1998). Separation of hydrogenosome-bound iron was performed using a 4–27 % linear polyacrylamide gradient to obtain better resolution at the front of the electrophoretogram. Electrophoresis was performed using a Hoeffer SE 600 vertical electrophoresis system with external cooling set to 4 °C at 110 mA constant current for two gel gradients. The electrode buffer contained 0·025 M Tris, 0·192 M glycine, pH 8·3. The run was stopped when haemoglobin, which was used as a marker, migrated to the middle of the gradient gel. The gels were then vacuum-dried, exposed to Storage Phophor Screen GP (Amersham) at room temperature for 24 h, scanned at a resolution of 100 µm per pixel using PhosphorImager SI (Amersham) and analysed using ImageQuantNT analysis software (Amersham). The dried gradient gels were rehydrated and stained in Coomassie brilliant blue solution after storage phosphorimaging analysis. After 2 days the stain was replaced by a solution containing 40 % methanol and 10 % acetic acid and the gels were destained overnight.
Molecular mass estimation of the LIP by ultrafiltration.
The cytosolic fraction isolated from 59Fe-NTA-labelled Tt. foetus cells (100 µl with a radioactivity of about 4000 c.p.m.) was added to 400 µl NaCl-HEPES buffer (140 mM NaCl, 10 mM HEPES, pH 7·2) and centrifuged at 5000 g for 90 min at 4 °C on Microcentrifuge Filters Ultrafree MC (Sigma) with the nominal molecular mass limits (NMML) of 5000 and 30 000 Da. The retentate was then washed twice with 200 µl NaCl-HEPES buffer. The washed retentates and the pooled ultrafiltrates were counted for 59Fe radioactivity. When DFO-chelatable iron was assayed, the chelator was added to the mixture at a final concentration of 500 µM and the samples were processed as described above.
Atomic absorption spectrophotometry.
Samples of cell fractions were diluted in cold PBS-chelex 100 (Sigma), sonicated and then analysed using a graphite furnace atomic absorption spectrophotometer model AS 800 (Perkin Elmer) at a wavelength of 248·3 nm with a 0·2 nm slit width and 20 mA lamp current. The following times and temperatures were used: injection at 80 °C; drying at 130 °C for 30 s with a 15 s ramp; charring at 1000 °C for 20 s with a 10 s ramp and atomization at 2450 °C for 3 s. The peak area was integrated for 10 s.
Amino acid sequencing.
The major hydrogenosomal iron-binding protein was cut out from a rehydrated native gradient gel, separated by SDS-PAGE and transferred to a PVDF membrane. The band of interest was excised and submitted for amino-terminal sequence determination using Edman degradation at the Department of Biochemistry, Charles University, Faculty of Science, Prague, Czech Republic.
Cloning and screening of the genomic library.
To obtain a probe for screening of a Tt. foetus genomic library (
ZAP II; Stratagene), specific primers were designed (5'-TTCGGATCAATCGTCGG-3' and 5'-TTGGAATGTAGCACCGT-3') based on a partial Tt. foetus ferredoxin sequence found in the GenBank database under accession no. AF312935. The PCR-amplified fragment was cloned into a pCR 2.1 vector (TOPO TA Cloning kit; Invitrogen). The insert was excised from the vector, gel-purified and labelled by means of a Random Primer DNA Labelling System (Gibco-BRL) with [
-32P]dATP. The sequences of positive clones were determined on both strands by primer walking. The consensus sequence of Tt. foetus ferredoxin (accession no. AF545472) was aligned to sequences of adrenodoxin-type ferredoxins from 42 taxa extracted from GenBank using CLUSTAL X (Thompson et al., 2000) and further edited using BIOEDIT (Hall, 1999). A sequence identity matrix was calculated based on the alignment from which all gaps were removed using BIOEDIT.
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a). To verify that this band represents a chelatable pool of iron, two iron chelators (Deferiprone and DFO) were tested. Addition of both chelators to the 59Fe-NTA-labelled cell lysate prior to electrophoresis resulted in concentration-dependent removal of the diffuse LIP band (Fig. 2). In the case of DFO, two residual bands appeared on the autoradiogram (Fig. 2) which corresponded to 59Fe-DFO complexes (Vyoral & Petrák, 1998a). In addition to LIP, lactoferrin-bound iron was observed in the cells incubated with 59Fe-lactoferrin, which formed a slowly migrating diffuse band of the same mobility as the cell-free control samples (Fig. 1c). When trichomonads were incubated with 59Fe-transferrin, the band corresponding to transferrin-bound iron was not observed in the cell lysates, although the parasites incorporated iron from transferrin into LIP (Fig. 1a). The latter observation is in agreement with an extracellular release of iron from transferrin without specific binding of transferrin to the cell surface (Tachezy et al., 1996).
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). Both parasites were able to incorporate iron from 59Fe-NTA into LIP efficiently. When the intestinal parasites were incubated with Fe-lactoferrin or Fe-transferrin, a considerably lower incorporation of iron into LIP was observed. To test whether incorporation of iron into LIP from Fe-NTA, lactoferrin and transferrin reflects the ability of the cells to cover their nutritional requirements from corresponding iron sources, we compared the effect of Fe-NTA, lactoferrin and transferrin on the growth of the parasite in vitro. All three iron sources stimulated growth of Tt. foetus, while growth of G. intestinalis and E. histolytica was only stimulated by Fe-NTA (Table 1) and other low-molecular-mass iron complexes (data not shown). These observations suggest that LIP represents a physiologically important pool of intracellular iron associated with cell growth.
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). Unlike the parasites, K562 cells incorporated a significant part of intracellular iron into ferritin (about 27 %). No major protein of parasite origin that binds iron with an intensity comparable to ferritin was observed in either Tt. foetus or the intestinal parasites (Fig. 1a).
Molecular mass estimation of LIP by ultrafiltration
59Fe-NTA-labelled Tt. foetus was homogenized and the soluble fraction was used for ultrafiltration. As is apparent from Table 2, 7·6 % of the total sample radioactivity appeared in the ultrafiltrate when a 5000 NMML filter was used, whereas 68·3 % of the radioactivity was detected in the 30 000 NMML ultrafiltrate. Less then 22 % of LIP-bound iron was present in the 30 000 retenate. Addition of 500 µM DFO to the control sample before ultrafiltration resulted in an increase of radioactivity in the 5000 NMML ultrafiltrate to 95·1 %, whereas radioactivity detected in the retentate corresponded to only 4·9 %. These results indicate that only a small part of LIP is associated with low-molecular-mass iron complexes (<5000 Da), while the majority of LIP consists of compounds corresponding to the molecular mass range 5000–30 000 Da.
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In other experiments, we investigated iron incorporation and its subcellular distribution when the trichomonads were incubated in a simple NaCl-HEPES buffer for 1 h with 59Fe-NTA. Four cell fractions were isolated (cytosol, hydrogenosomes, lysosomes and microsomes) and the incorporated radioactivity was determined for each fraction (Fig. 3). The majority of the iron (88·9 %) was present in the cytosol. The cytosolic iron concentration calculated from incorporated radioactivity was 304·7 pmol 59Fe (mg protein)-1. The hydrogenosomes possessed only 4·7 % of the total incorporated radioactivity; however, the iron concentration was the highest as compared to other cell compartments [360·4 pmol 59Fe (mg protein)-1]. Lysosomes and microsomes contained only 2·9 and 3·4 % of the total incorporated radioactivity, respectively. These results showed iron to be efficiently taken up and transported to the hydrogenosomes by Tt. foetus.
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a). In the cytosol, a prominent band designated ‘C’ and at least four additional bands containing 59Fe were observed. The majority of iron was present in LIP (about 62 %). The band pattern of the cytosolic fraction was similar to that observed in the whole-cell lysate. However, a remarkably different pattern was displayed by the hydrogenosomes. A prominent fast-migrating band (H-I) was observed within these organelles as well as eight other bands which differed in mobility from those found in the cytosol. In microsomes and lysosomes we observed only weak bands, which were at the detection limit of our assay. The H-I band, as well as other bands in the hydrogenosomes, correspond to hydrogenosomal FeS proteins such as PFOR, ferredoxin and hydrogenase. As these proteins are absent in metronidazole-resistant strains (Rasoloson et al., 2002), we compared iron incorporation into hydrogenosomes of a metronidazole-sensitive strain (Lub-1MIP) and its resistant derivative (Lub-MR100). Indeed, none of the eight bands present in the parent strain was observed in the resistant derivative (Fig. 4b).
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). Cytosolic band C appeared at the shortest time interval (3 min) and its intensity increased during prolonged incubation. Appearance of the hydrogenosomal H-I band was delayed from band C for at least 60 min. The intensity of the H-I band also increased in a time-dependent manner. In pulse–chase experiments, trichomonads were incubated for 10 min with 59Fe-NTA and then reincubated without radioactive iron for 0, 60 and 150 min. At each time point, the cells were fractionated and the cellular fractions were analysed as described above (Fig. 6). The intensity of all cytosolic bands increased during the cell reincubation, indicating that all observed cytosolic bands represent a final destination of iron transport. In hydrogenosomes, the intensity of the H-I band did not significantly change during reincubation. A remarkable increase in bound radioactivity was observed in the slower migrating band H-II. We also observed a single band (H-III), which was present after the 10 min incubation of cells with 59Fe-NTA and disappeared during cell reincubation. The kinetics of the H-III band suggest its role as an intermediate involved in iron transport.
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). The H-I-associated iron was rather tightly bound, as it was not removed by 500 µM DFO (Fig. 7). To estimate the molecular mass of the compound corresponding to the H-I band, the native gradient gel was rehydrated after autoradiography and stained by Coomassie brilliant blue. The band of corresponding mobility was cut-out and separated by SDS-PAGE which showed the presence of a 12 kDa protein (data not shown). This protein was submitted to Edman degradation to determine the N-terminal sequence. A sequence of 29 aa was obtained which matched exactly the N-terminal part of Tt. foetus ferredoxin as predicted from the partial gene sequence in GenBank. A complete Tt. foetus ferredoxin gene was subsequently obtained by screening of a Tt. foetus genomic library. Sequence analysis of the ferredoxin gene revealed an ORF of 327 bp, encoding 109 aa. The molecular mass and isoelectric point were 11 052 Da and 4·48, respectively. The N terminus of the predicted Tt. foetus ferredoxin contained a 13 aa extension similar to presequences known to target the proteins to hydrogenosomes in T. vaginalis (Bradley et al., 1997; Johnson et al., 1990). This extension was absent from the N-terminal sequence of the isolated peptide, indicating that the ferredoxin presequence had been processed within the Tt. foetus hydrogenosomes. Presence of a conserved CX5CX2CXnC pattern indicated that the ferredoxin belongs to the [2Fe–2S] adrenodoxin type found in other amitochondrial protists, mitochondria of eukaryotes and bacteria (Fig. 8). Comparison of the Tt. foetus ferredoxin with 42 sequences of adrenodoxin-type ferredoxins found in databases revealed the highest identity in a 66 aa overlap with the ferredoxin of T. vaginalis (58 %) and G. intestinalis (33 %), and with mitochondrial-type ferredoxin of Saccharomyces cerevisiae, Mus musculus and Caenorhabditis elegans (29–33 %). Significant similarity was also found in the proteobacterial ferredoxin of Acinetobacter sp. (33 %) and the cyanobacterium Mastigocladus laminosus (32 %). An alignment of selected homologues is shown in Fig. 8 (the complete alignment is available on request). These data show that a ferredoxin of the adrenodoxin type is a major iron-binding protein in the hydrogenosomes of Tt. foetus.
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DISCUSSION |
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The molecular basis and physiological significance of LIP has been an enigma for decades. It is believed to be an iron transport intermediate in iron acquisition from the cellular environment. It is also hypothesized to be involved in iron mobilization from its intracellular storage (Crichton, 1991). However, kinetics studies of LIP in K562 cells showed the chelatable compartment of cellular iron to be labelled with the kinetics of an end product (Vyoral & Petrák, 1998b), which argues against an intermediate character of LIP. It has been suggested that the ‘mobile’ iron forms low-molecular-mass complexes with nucleotides, nucleic acids, polyphosphates, amino acids, citrate or peptides (Jacobs, 1977; Bohnke & Matzanke, 1995; Weaver & Pollack, 1989). However, increasing evidence indicates that ‘mobile’ iron is (with low affinity) bound to compounds most likely to be proteins with a molecular mass greater than 20 000 Da (Petrák & Vyoral, 2001; Vyoral & Petrák, 1998a, b; Vyoral et al., 1992). This view is supported by our results using ultrafiltration of iron-labelled cytosol of Tt. foetus. It showed that 68·2 % of chelatable iron is bound to compounds with a molecular mass in the range of 5000–30 000 Da. Thus, it is likely that LIP in protists represents low-affinity iron associated with housekeeping proteins. The importance of LIP for cell function is supported by our observation that its presence correlated with the ability of cells to utilize various iron sources for their growth. The difference between LIP in protists and vertebrate cells is its abundance. In various human cell lines the LIP band corresponded to 5–30 % of cellular iron, while a greater part of intracellular iron is stored in ferritin (Vyoral et al., 1998). In protists, which do not possess ferritin, >60 % of iron appeared as chelation-sensitive LIP.
It is noteworthy that neither Tt. foetus nor other unicellular eukaryotes possesses ferritin. Higher eukaryotes rely on ferritin to scavenge and to store iron in the cytosol. Recently, ferritin has also been found in mitochondria (Levi et al., 2001), although the function of mitochondrial ferritin remains to be clarified. Two types of ferritin are present in bacteria, including the -proteobacteria (Andrews et al., 1991), which are considered to be the endosymbiotic ancestors of mitochondria and hydrogenosomes (Rotte et al., 2000; Dyall & Johnson, 2000). In three parasitic protists, we were unable to detect any protein of comparable mobility and iron-binding capacity with ferritin observed in K562 cells. The presence of a ferritin-encoding gene has not been reported in any unicellular eukaryote so far with the exception of the microsporidian parasite Encephalitozoon cuniculi (Katinka et al., 2001).
Hydrogenosomes represent an important destination of intracellular iron and this is supported by the following evidence: (i) iron is required for the catalytic centres of FeS proteins such as PFOR, ferredoxin and hydrogenase, which mediate key steps in hydrogenosomal pyruvate metabolism (Vaáová et al., 2001); (ii) hydrogenosomes efficiently accumulated iron when the trichomonads were incubated with 59Fe-NTA [360·4 pmol 59Fe h-1 (mg protein)-1]; and (iii) the steady-state concentration of iron in hydrogenosomes was about fourfold higher than that in cytosol as determined by atomic absorption spectrophotometry. The iron content in hydrogenosomes [54·4 nmol Fe (mg protein)-1] is unusually high. In yeast the concentrations of mitochondrial iron range from 0·512 to 4·9 nmol (mg mitochondrial protein)-1 when the organisms are grown on media containing 0·1–50 µM Fe (Li et al., 1999). A similar level of iron has been also found in mammalian mitochondria (Tangeras, 1985). Mitochondrial iron concentrations comparable to that in hydrogenosomes have only been observed in yeast mutants with altered iron homeostasis (Li et al., 1999).
The iron retained in hydrogenosomes consists of Triton X-100-soluble (26 %) and -resistant fractions (74 %). In yeast mitochondria, the Triton X-100-resistant fraction formed only a small proportion of total iron (5–12 %); however, it increased to 53–60 % in mutants with altered iron homeostasis (Li et al., 1999). It has been suggested that pelleted iron represents the iron pool associated with membrane lipids or protein aggregates (Li et al., 1999). However, its biological function is not known. The high proportion of Triton X-100-resistant iron in hydrogenosomes together with the absence of ferritin suggests that this iron may represent intrahydrogenosomal iron storage. The solubilized iron appeared as distinct bands on autoradiograms. It is likely that the bands correspond mainly to FeS proteins involved in pyruvate metabolism (PFOR, hydrogenase, ferredoxin) as they were absent in hydrogenosomes of metronidazole-resistant trichomonads. Decreased expression of hydrogenosomal proteins, including PFOR and ferredoxin, in metronidazole-resistant strains was recently reported in T. vaginalis (Rasoloson et al., 2002) and Tt. foetus (Land et al., 2001). The major iron-containing protein was isolated and identified as [2Fe–2S] ferredoxin. DFO, chelating non-specifically or weakly bound iron, did not remove radioactive iron associated with the labelled ferredoxin. This observation indicated that 59Fe was specifically incorporated into this protein, most likely into the chelation-resistant FeS centre. Moreover, chemical analysis of ferredoxin from a related organism, T. vaginalis, revealed approximately equal amounts of iron and acid-labile sulfur (Gorrell et al., 1984). The function of ferredoxin in hydrogenosomal metabolism is well established and the protein has been biochemically characterized (Marczak et al., 1983; Gorrell et al., 1984). It is considered as a major electron carrier providing reducing equivalents to hydrogenase, which results in the formation of molecular hydrogen (Kulda, 1999; Martin & Müller, 1998). Molecular analysis of T. vaginalis ferredoxin showed closest similarity to putidaredoxin of Pseudomonas putida and to a lesser extent to mitochondrial [2Fe–2S] ferredoxins (adrenodoxins) of vertebrates, which are components of mixed-function oxidase systems (Johnson et al., 1990). More recently, a different function was suggested for adrenodoxin-type ferredoxins. Genetic studies showed that adrenodoxin (Yah1p) is essential for the maturation of FeS proteins in yeast mitochondria (Lange et al., 2000). The homologues of adrenodoxins are ferredoxins encoded in the bacterial isc gene cluster together with other components of FeS cluster assembly machinery such as IscS, IscU and IscA (Takahashi & Nakamura, 1999). Adrenodoxins, as well as their bacterial homologues, form a group of [2Fe–2S] ferredoxins with a common cluster-binding pattern, CX5CX2CXnC (Bertini et al., 2002). As is apparent from primary structure analysis, [2Fe–2S] ferredoxin present in the amitochondrial protists Tt. foetus, T. vaginalis and G. intestinalis (Nixon et al., 2002) belongs to the adrenodoxin group (this study; Johnson et al., 1990; Land et al., 2002). The genes for a key component of FeS cluster assembly machinery, IscS, have been recently found both in trichomonad species and in G. intestinalis (Tachezy et al., 2001), indicating that common mechanisms of FeS protein biogenesis operate in mitochondrial as well as amitochondrial eukaryotes. Thus, it is likely that hydrogenosomal ferredoxin, in addition to its known metabolic function, is also involved in FeS cluster formation. The exact role of ferredoxin in this process is not known. Lange et al. (2000) suggested that ferredoxin may provide reducing equivalents for the formation of FeS cluster intermediates, which are pre-assembled on NifU/IscU or IscA proteins serving as a scaffold. Ollagnier-de-Choudens et al. (2001) showed that FeS intermediates formed on IscA are delivered to ferredoxin, and proposed that this process is a key step in the biosynthesis of the FeS cluster required for maturation of other cellular FeS proteins. The abundance of ferredoxin in Tt. foetus hydrogenosomes supports the latter possibility. It is tempting to speculate that ferredoxin represents a pool of pre-assembled FeS clusters that are provided during biogenesis of other hydrogenosomal FeS proteins.
This study raises a number of intriguing questions such as: (i) how is iron delivered to hydrogenosomes, and (ii) which species supply iron for FeS cluster formation in the organelles? The kinetic studies did not reveal any compound with the kinetics of a putative iron transporter in the cytosol. All cytosolic compounds were observed as distinct bands on autoradiograms with increasing intensity in a time-dependent manner. It is likely that these compounds represent the final destinations of iron transport. The putative intracellular iron transporter is either hidden in LIP or the iron might be transported in endosomal vesicles (Richardson et al., 1996). The hydrogenosomal H-III band was the only band with a transient kinetic pattern. Such kinetic behaviour would be typical for an iron transporter.
In conclusion, we have examined the iron-binding compounds in Tt. foetus cell fractions using native gradient PAGE followed by storage phosphorimaging. Our results demonstrate that hydrogenosomes represent an important destination in cellular iron transport and contain an unusually high iron concentration. We also determined ferredoxin to be a major iron-binding protein in these organelles. LIP appeared as a physiologically important iron pool, although its molecular basis remains enigmatic. The efficient iron accumulation and high concentrations of intracellular iron in Tt. foetus correspond to its high nutritional demands for this metal (Tachezy et al., 1996) and to the importance of iron for the virulence of this parasite (Kulda et al., 1998).
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ACKNOWLEDGEMENTS |
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ka (Faculty of Science, Charles University, Prague) for peptide microsequencing, and Dr A. Dancis (University of Pennsylvania, Philadelphia) and Dr M. Müller (The Rockefeller University, New York) for critical reading of the manuscript. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-counter and protein content was assayed according to the method of Lowry. Relative 59Fe radioactivity was plotted against cumulative percentage of protein recovered in each fraction. The order of the fractions from the left is: cytosol, lysosomes, hydrogenosomes and microsomes. NADH oxidase, acid phosphatase and NAD-dependent malate dehydrogenase (decarboxylating) were used as marker enzymes for cytosol, hydrogenosomes and lysosomes, respectively.
