Compartment-specific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are essential to glycolysis, the major route of carbohydrate breakdown in eukaryotes. In animals and other heterotrophic eukaryotes, both enzymes are localized in the cytosol; in photosynthetic eukaryotes, GAPDH and TPI exist as isoenzymes that function in the glycolytic pathway of the cytosol and in the Calvin cycle of chloroplasts. Here, we show that diatoms--photosynthetic protists that acquired their plastids through secondary symbiotic engulfment of a eukaryotic rhodophyte--possess an additional isoenzyme each of both GAPDH and TPI. Surprisingly, these new forms are expressed as an TPI-GAPDH fusion protein which is imported into mitochondria prior to its assembly into a tetrameric bifunctional enzyme complex. Homologs of this translational fusion are shown to be conserved and expressed also in nonphotosynthetic, heterokont-flagellated oomycetes. Phylogenetic analyses show that mitochondrial GAPDH and its N-terminal TPI fusion branch deeply within their respective eukaryotic protein phylogenies, suggesting that diatom mitochondria may have retained an ancestral state of glycolytic compartmentation that existed at the onset of mitochondrial symbiosis. These findings strongly support the view that nuclear genes for enzymes of glycolysis in eukaryotes were acquired from mitochondrial genomes and provide new insights into the evolutionary history (host-symbiont relationships) of diatoms and other heterokont-flagellated protists.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Critical Analysis of Eukaryotic Phylogeny: A Case Study Based on the HSP70 Family

Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species; and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes, likely after the mitochondrial endosymbiosis.     

Fungal hydrogenosomes contain mitochondrial heat-shock proteins

At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

Hydrogenosomes and Mitosomes: Conservation and Evolution of Functions1

ABSTRACT. The field studying unusual mitochondria in microbial eukaryotes has come full circle. Some 10–15 years ago it had the evangelical task of informing the wider scientific community that not all eukaryotes had mitochondria. Advances in the field indicated that although some protists might not have mitochondria, the presence of genes of mitochondrial ancestry suggested their lineage once had. The subsequent discovery of mitochondrial compartments in all supposedly amitochondriate protists studied so far indicates that all eukaryotes do have mitochondria indeed. This assertion has fuelled novel eukaryotic origin theories and weakened others. But what do we know about these unusual mitochondria from anaerobic protists? Have they all converged onto similar roles? Iron–sulphur cluster assembly is often hailed as the unifying feature of these organelles. However, the iron–sulphur protein that is so important that a complete organelle is being maintained has not been identified. Is it to be expected that all unusual mitochondria perform the same physiological role? These organelles have been found in numerous protists occupying different ecological niches. Different selection pressures operate on different organisms so there is no reason to suspect that their mitochondria should all be the same.     

Characterization of Trichomonad Species and Strains by PCR Fingerprinting

ABSTRACT. The random amplified polymorphic DNA (RAPD) technique was used for phylogenetic analysis of trichomonads, for intraspecies genealogical study of Trichomonas vaginalis strains, and for assessment of intrastrain polymorphism in Trichomonas vaginalis . The phylogenetic tree for 12 trichomonad species showed certain discrepancies with current models of trichomonad evolution. However, it shows that RAPD traits retain phylogenetically relevant information. The results of intraspecies analyses of 18 Trichomonas vaginalis strains suggested some concordance between the genetic relationship of strains and their geographic origin. They also suggested a concordance between the strain genetic relationships and the resistance to metronidazole. A concordance was also found with respect to the severity of disease observed in donor patients but not with the results of laboratory virulence assays. No concordance was found between genetic relationship of strains and strain infection with a dsRNA Trichomonas vaginalis virus (TVV). The latter suggests that TVV might be transmitted horizontally among Trichomonas vaginalis populations. The identity of RAPD patterns of clones isolated from in vitro cultures and those of the cultures reisolated independently from the same patient within a period of six weeks suggests that individual Trichomonas vaginalis strains are not polymorphic and that the RAPD patterns are stable. Therefore, the RAPD technique seems useful for addressing various clinically relevant issues.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

Electron microscope observations on the interaction of Mycoplasma fermentans with Trichomonas vaginalis

Cultures of Trichomonas vaginalis were found to be contaminated with Mycoplasma fermentans. By means of electron microscopy the interaction between the prokaryotic organisms and the trichomonads was examined. Cells of M. fermentans were observed in the medium; some of them were attached to the surface of the trichomonads and others were observed in membrane-bounded vacuoles of trichomonads. They were also present in the ground substance of the cytoplasm. The mycoplasmas divided by binary fission like other prokaryotes. The most obvious change occurring in the infected trichomonad cells was an increase in number of vacuoles containing mycoplasmas.     

Organelle origins and ribosomal RNA

As the detailed molecular biology of organelle genomes has unfolded, there has been a general acceptance of the view that plastids and mitochondria are of endosymbiotic, eubacterial origin. Plastid genes are strikingly similar to their eubacterial (particularly cyanobacterial) counterparts in sequence, organization, and mode of expression, and such features strongly support the hypothesis that the plastid and its genome were derived in evolution from a blue-green alga-like endosymbiont. Mitochondria, on the other hand, are problematic: mitochondrial genes are organized and expressed in remarkably diverse ways in the different major groups of eukaryotes, and in no case are these features particularly characteristic of either bacterial or nuclear genomes. There is, however, clear evidence derived from gene sequence supporting the eubacterial ancestry of mitochondria, and some of the most compelling data have come from analyses of mitochondrial ribosomal RNA (rRNA). Plant mitochondrial rRNA genes diverge in sequence at a particularly slow rate, and these genes have proven to be especially supportive of the endosymbiont hypothesis, pointing to an origin of mitochondria from within the alpha subdivision of the purple bacteria. Ribosomal RNA sequences provide a basis for the construction of global phylogenetic trees that probe the evolutionary history of organelles, and that address the question of whether mitochondria and plastids are monophyletic or polyphyletic in origin. Such studies raise the possibility that the rRNA genes of plant mitochondria originated separately from the mitochondrial rRNA genes of other eukaryotes.     

Phylogeny of Trichomonads Inferred from Small-Subunit rRNA Sequences

ABSTRACT. Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to other early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.     

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.     

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria

Microsporidia are small (1–20 μm) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2] and [3], and are reported to lack mitochondria [2] and [4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2] and [4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5], [6], [7], [8] and [9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.     

Studies on Trichomonads. II. The Metabolism of a Trichomonas batrachorum-type Flagellate from the Cecum of Swine

SYNOPSIS. Certain aspects of the metabolism of a Trichomonas batrachorum -type flagellate from the cecum of swine were studied. This trichomonad (1) oxidized glucose, mannose, maltose, sucrose, and inulin, (2) was incapable of oxidizing Krebs' cycle intermediates, (3) possessed peroxide-splitting capabilities, (4) was inhibited by only iodoacetate and arsenite, and (5) formed acid(s) aerobically. Although there was no effect on oxygen uptake, pyruvate and lactate increased the anaerobic evolution of gas(es). In addition to CO₂, other gas, not absorbed by KOH, was produced anaerobically.
Compared with other porcine trichomonads, the metabolism of this small trichomonad resembles most closely that of the large cecal trichomonad, T. suis. However, the smaller trichomonad had a generally lower respiratory rate, a slightly lower optimal pH, and failed to oxidize fructose, galactose, lactose, raffinose, and trehalose.     

Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships

Sequences of putative fructose-1,6-bisphospate aldolases (FBA) in five amitochondriate unicellular eukaryotes, the diplomonads Giardia intestinalis (published earlier) and Spironucleus barkhanus, the pelobiont Mastigamoeba balamuthi,the entamoebid Entamoeba histolytica, and the parabasalid Trichomonas vaginalis all belong to Class II of FBAs and are highly similar to each other (>48% amino acid identity). The five protist sequences, however, do not form a monophyletic group. Diplomonad FBAs share a most recent common ancestor, while FBAs of the three other protist species are part of a lineage that also includes sequences from a few eubacteria (Clostridium difficile, Treponema pallidum, Chlorobium tepidum). Both clades are part of the Type B of Class II aldolases, a complex that contains at least three additional lineages (subgroups) of enzymes. Type B enzymes are distant from Type A Class II aldolases, which consists of a number of bacterial and fungal enzymes and also contains the cytosolic FBA of Euglena gracilis. Class II aldolases are not homologous to Class I enzymes, to which animal and plant enzymes belong. The results indicate that amitochondriate protists acquired their FBAs from separate and different sources, involving lateral gene transfer from eubacteria, than did all other eukaryotes studied so far and underscore the complex composition of the glycolytic machinery in unicellular eukaryotes.     

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.     

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.     

Live imaging of mitosomes and hydrogenosomes by HaloTag technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.