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.

• MeSH
• financování organizované MeSH
• genetická transkripce MeSH
• konzervovaná sekvence MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• organely metabolismus   MeSH
• proteiny vázající železo genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Trichomonas vaginalis genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata 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
• antitrichomonádové látky farmakologie   metabolismus   MeSH
• financování organizované MeSH
• léková rezistence fyziologie   MeSH
• metronidazol farmakologie   metabolismus   MeSH
• organely enzymologie   metabolismus   MeSH
• Trichomonas vaginalis růst a vývoj   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata 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
• biologický transport fyziologie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• gelová chromatografie MeSH
• membránové proteiny chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• organely metabolismus   MeSH
• poriny metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Trichomonas vaginalis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.

• MeSH
• anaerobióza genetika   MeSH
• Archamoebae enzymologie   genetika   metabolismus   MeSH
• duplikace genu * MeSH
• energetický metabolismus genetika   MeSH
• enzymy genetika   izolace a purifikace   MeSH
• molekulární evoluce * MeSH
• organely enzymologie   genetika   metabolismus   MeSH
• přenos genů horizontální * MeSH
• struktury buněčné membrány genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tail-anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N-terminal domain that performs various functions and a single transmembrane domain (TMD) near the C-terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C-terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen-poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C-tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage-specific differences.

• MeSH
• multienzymové komplexy metabolismus   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• organely metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• anaerobióza MeSH
• genetické vektory genetika   MeSH
• Giardia lamblia cytologie   genetika   MeSH
• hydrolasy genetika   MeSH
• ligandy MeSH
• mitochondrie metabolismus   MeSH
• molekulární zobrazování metody   MeSH
• organely metabolismus   MeSH
• protozoální proteiny genetika   MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• reportérové geny genetika   MeSH
• Trichomonas vaginalis cytologie   genetika   MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• adenosintrifosfát farmakologie   MeSH
• difosfáty metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• fosfofruktokinasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• organely účinky léků   metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• transport proteinů účinky léků   MeSH
• Trichomonas vaginalis účinky léků   enzymologie   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• energetický metabolismus MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• organely metabolismus   ultrastruktura   MeSH
• oxidace-redukce MeSH
• proteom metabolismus   MeSH
• proteomika MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• regulace genové exprese MeSH
• shluková analýza MeSH
• síra metabolismus   MeSH
• Trichomonas vaginalis genetika   metabolismus   MeSH
• železo metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• ferredoxiny genetika   chemie   izolace a purifikace   metabolismus   MeSH
• financování organizované MeSH
• flavinmononukleotid metabolismus   MeSH
• kyslík metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• organely enzymologie   genetika   chemie   MeSH
• oxidoreduktasy genetika   chemie   izolace a purifikace   metabolismus   MeSH
• protozoální proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• Trichomonas vaginalis enzymologie   genetika   chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import.

• MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH