BACKGROUND: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. RESULTS: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. CONCLUSIONS: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

• Keywords
• Anaerobiosis, Cardiolipin, ERMES, Endoplasmic reticulum, Hydrogenosomes, Structure, Trichomonas vaginalis,
• MeSH
• Anaerobiosis MeSH
• Endoplasmic Reticulum * metabolism   MeSH
• Phospholipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Mitochondria metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phospholipids MeSH
• Membrane Proteins * MeSH

BACKGROUND: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. RESULTS: We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. CONCLUSIONS: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

• Keywords
• Arginine deiminase, Carbamate kinase, Metamonada, Ornithine transcarbamylase, Phylogeny, Preaxostyla, Protists,
• MeSH
• Archaea metabolism   MeSH
• Arginine metabolism   MeSH
• Diplomonadida enzymology   MeSH
• Eukaryota classification   genetics   metabolism   MeSH
• Phylogeny MeSH
• Hydrolases metabolism   MeSH
• Metabolic Networks and Pathways * MeSH
• Evolution, Molecular * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• arginine deiminase MeSH Browser
• Hydrolases MeSH

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

• MeSH
• Adenosine Triphosphate pharmacology   MeSH
• Diphosphates metabolism   MeSH
• Ferredoxins metabolism   MeSH
• Phosphofructokinases chemistry   metabolism   MeSH
• Phylogeny MeSH
• Mitochondria drug effects   metabolism   MeSH
• Molecular Sequence Data MeSH
• Organelles drug effects   metabolism   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Saccharomyces cerevisiae drug effects   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Protein Transport drug effects   MeSH
• Trichomonas vaginalis drug effects   enzymology   MeSH
• Hydrogen metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Diphosphates MeSH
• Ferredoxins MeSH
• Phosphofructokinases MeSH
• Hydrogen MeSH

Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

• MeSH
• Energy Metabolism MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Organelles metabolism   ultrastructure   MeSH
• Oxidation-Reduction MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Gene Expression Regulation MeSH
• Cluster Analysis MeSH
• Sulfur metabolism   MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH
• Protozoan Proteins MeSH
• Sulfur MeSH
• Iron MeSH

All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

• MeSH
• Eukaryota genetics   metabolism   MeSH
• Gene Expression MeSH
• Glycine Decarboxylase Complex H-Protein genetics   metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Multienzyme Complexes genetics   metabolism   MeSH
• Organelles metabolism   MeSH
• Amino Acid Oxidoreductases genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Transferases genetics   metabolism   MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• glycine cleavage system MeSH Browser
• Glycine Decarboxylase Complex H-Protein MeSH
• Mitochondrial Proteins MeSH
• Multienzyme Complexes MeSH
• Amino Acid Oxidoreductases MeSH
• Transferases MeSH
• Carrier Proteins MeSH

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.

• MeSH
• Anaerobiosis MeSH
• Genetic Vectors genetics   MeSH
• Giardia lamblia cytology   genetics   MeSH
• Hydrolases genetics   MeSH
• Ligands MeSH
• Mitochondria metabolism   MeSH
• Molecular Imaging methods   MeSH
• Organelles metabolism   MeSH
• Protozoan Proteins genetics   MeSH
• Recombinant Fusion Proteins genetics   MeSH
• Genes, Reporter genetics   MeSH
• Trichomonas vaginalis cytology   genetics   MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Ligands MeSH
• Protozoan Proteins MeSH
• Recombinant Fusion Proteins MeSH

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.

• MeSH
• Biological Transport physiology   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Chromatography, Gel MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Organelles metabolism   MeSH
• Porins metabolism   MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Trichomonas vaginalis metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Porins MeSH
• Protozoan Proteins MeSH

Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen.

• MeSH
• Ferredoxins chemistry   genetics   isolation & purification   metabolism   MeSH
• Flavin Mononucleotide metabolism   MeSH
• Oxygen metabolism   MeSH
• Molecular Sequence Data MeSH
• Organelles chemistry   enzymology   genetics   MeSH
• Oxidoreductases chemistry   genetics   isolation & purification   metabolism   MeSH
• Protozoan Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Trichomonas vaginalis chemistry   enzymology   genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ferredoxins MeSH
• Flavin Mononucleotide MeSH
• Oxygen MeSH
• Oxidoreductases MeSH
• Protozoan Proteins MeSH

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes.

• MeSH
• Antitrichomonal Agents metabolism   pharmacology   MeSH
• Drug Resistance physiology   MeSH
• Metronidazole metabolism   pharmacology   MeSH
• Organelles enzymology   metabolism   MeSH
• Trichomonas vaginalis drug effects   growth & development   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antitrichomonal Agents MeSH
• Metronidazole MeSH

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas, proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.

• MeSH
• Biological Transport, Active MeSH
• Ferredoxins genetics   metabolism   MeSH
• Giardia lamblia genetics   metabolism   ultrastructure   MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Organelles metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Iron-Sulfur Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Trichomonas vaginalis genetics   metabolism   ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Comparative Study MeSH
• Names of Substances
• Ferredoxins MeSH
• Iron-Sulfur Proteins MeSH
• Protozoan Proteins MeSH
• Recombinant Proteins MeSH