Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.     

The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes

The human pathogen Trichomonas vaginalis lacks conventional mitochondria and instead contains divergent mitochondrial-related organelles. These double-membrane bound organelles, called hydrogenosomes, produce molecular hydrogen. Phylogenetic and biochemical analyses of hydrogenosomes indicate a common origin with mitochondria; however identification of hydrogenosomal proteins and studies on its metabolism have been limited. Here we provide a detailed proteomic analysis of the T. vaginalis hydrogenosome. The proteome of purified hydrogenosomes consists of 569 proteins, a number substantially lower than the 1,000-1,500 proteins reported for fungal and animal mitochondrial proteomes, yet considerably higher than proteins assigned to mitosomes. Pathways common to and distinct from both mitochondria and mitosomes were revealed by the hydrogenosome proteome. Proteins known to function in amino acid and energy metabolism, Fe-S cluster assembly, flavin-mediated catalysis, oxygen stress response, membrane translocation, chaperonin functions, proteolytic processing and ATP hydrolysis account for ～30% of the hydrogenosome proteome. Of the 569 proteins in the hydrogenosome proteome, many appear to be associated with the external surface of hydrogenosomes, including large numbers of GTPases and ribosomal proteins. Glycolytic proteins were also found to be associated with the hydrogenosome proteome, similar to that previously observed for mitochondrial proteomes. Approximately 18% of the hydrogenosomal proteome is composed of hypothetical proteins of unknown function, predictive of multiple activities and properties yet to be uncovered for these highly adapted organelles.     

Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis

Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental Km of L protein for the two H proteins were 2.6 microM and 3.7 microM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.     

A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.     

Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H₂-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.     

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria

A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.     

A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry

Matrix proteins of mitochondria, hydrogenosomes and mitosomes are typically targeted and translocated into their respective organelles using N-terminal presequences that are subsequently cleaved by a peptidase. Here we characterize a approximately 47 kDa metallopeptidase, from the hydrogenosome-bearing, unicellular eukaryote Trichomonas vaginalis, that contains the active site motif (HXXEHX(76)E) characteristic of the beta subunit of the mitochondrial processing peptidase (MPP) and localizes to hydrogenosomes. The purified recombinant protein, named hydrogenosomal processing peptidase (HPP), is capable of cleaving a hydrogenosomal presequence in vitro, in contrast to MPP which requires both an alpha and beta subunit for activity. T. vaginalis HPP forms an approximately 100 kDa homodimer in vitro and also exists in an approximately 100 kDa complex in vivo. Our phylogenetic analyses support a common origin for HPP and betaMPP and demonstrate that gene duplication gave rise to alphaMPP and betaMPP before the divergence of T. vaginalis and mitochondria-bearing lineages. These data, together with published analyses of MPPs and putative mitosomal processing peptidases, lead us to propose that the length of targeting presequences and the subunit composition of organellar processing peptidases evolved in concert. Specifically, longer mitochondrial presequences may have evolved to require an alpha/beta heterodimer for accurate cleavage, while shorter hydrogenosomal and mitosomal presequences did not.     

Beta-succinyl-coenzyme A synthetase from Trichomonas vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences.

We describe studies directed toward understanding the biogenesis and origin of the hydrogenosome, an unusual organelle found exclusively in certain anaerobic eukaryotes that lack mitochondria. Hydrogenosomes are involved in fermentative carbohydrate metabolism and are proposed to have arisen through conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. We cloned a gene encoding the beta subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (beta-SCS) and isolated the protein from Trichomonas vaginalis. The T. vaginalis beta-SCS gene encodes a protein with a calculated molecular mass of 43,980 Da that has 43% amino acid identity (65% similarity) with beta-SCS from Escherichia coli. The trichomonad protein partitions into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization within the organelle. The protein is encoded by a multigene family composed of at least three members. Amino-terminal sequencing of beta-SCS purified from T. vaginalis hydrogenosomes shows that the mature protein lacks the first nine amino acids encoded in the gene. This apparent amino-terminal leader sequence is strikingly similar to that of another hydrogenosomal protein and to mitochondrial presequences.     

A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.     

Iron hydrogenases and the evolution of anaerobic eukaryotes

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp

A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial-type AACs. Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.     

Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.     

Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.     

Fe-hydrogenase maturases in the hydrogenosomes of Trichomonas vaginalis

Assembly of active Fe-hydrogenase in the chloroplasts of the green alga Chlamydomonas reinhardtii requires auxiliary maturases, the S-adenosylmethionine-dependent enzymes HydG and HydE and the GTPase HydF. Genes encoding homologous maturases had been found in the genomes of all eubacteria that contain Fe-hydrogenase genes but not yet in any other eukaryote. By means of proteomic analysis, we identified a homologue of HydG in the hydrogenosomes, mitochondrion-related organelles that produce hydrogen under anaerobiosis by the activity of Fe-hydrogenase, in the pathogenic protist Trichomonas vaginalis. Genes encoding two other components of the Hyd system, HydE and HydF, were found in the T. vaginalis genome database. Overexpression of HydG, HydE, and HydF in trichomonads showed that all three proteins are specifically targeted to the hydrogenosomes, the site of Fe-hydrogenase maturation. The results of Neighbor-Net analyses of sequence similarities are consistent with a common eubacterial ancestor of HydG, HydE, and HydF in T. vaginalis and C. reinhardtii, supporting a monophyletic origin of Fe-hydrogenase maturases in the two eukaryotes. Although Fe-hydrogenases exist in only a few eukaryotes, related Narf proteins with different cellular functions are widely distributed. Thus, we propose that the acquisition of Fe-hydrogenases, together with Hyd maturases, occurred once in eukaryotic evolution, followed by the appearance of Narf through gene duplication of the Fe-hydrogenase gene and subsequent loss of the Hyd proteins in eukaryotes in which Fe-hydrogenase function was lost.     

Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.     

The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis

Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group.     

Acquisition of hydrogenosomal presequences: examples from Trichomonas vaginalis

Presequences play an important role in protein import into mitochondria-like organelles. Acquisition pathways have been revealed for some mitochondrial presequences, but little is known about hydrogenosomal presequences. Here we investigated the hydrogenosomal proteins of Trichomonas vaginalis and suggest that several hydrogenosomal presequences probably evolved from pre-existing sequences that were thereafter modified.     

Hydrogenosomal activity of Trichomonas vaginalis cultivated under different iron conditions

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.     

Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.