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Patricia J. Johnson - One of the best experts on this subject based on the ideXlab platform.
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The Trichomonas vaginalis Hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes.
International journal for parasitology, 2011Co-Authors: Rachel E Schneider, April M. Shiflett, Sabrina D. Dyall, Mark T Brown, Richard D Hayes, Yongming Xie, Joseph A Loo, Patricia J. JohnsonAbstract: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.
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Mitochondrion-related organelles in eukaryotic protists.
Annual Review of Microbiology, 2010Co-Authors: April M. Shiflett, Patricia J. JohnsonAbstract:The discovery of mitochondrion-type genes in organisms thought to lack mitochondria led to the demonstration that Hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as Hydrogenosomes, mitosomes, or mitochondrion-like organelles, are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite their diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. This review examines the metabolism and biogenesis of these organelles in divergent unicellular microbes, with a focus on parasitic protists.
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Genetic evidence for a mitochondriate ancestry in the 'amitochondriate' flagellate Trimastix pyriformis.
PLOS ONE, 2008Co-Authors: Vladimir Hampl, Patricia J. Johnson, Jeffrey D. Silberman, Alexandra Stechmann, Sara Diaz-trivino, Andrew J. RogerAbstract:Most modern eukaryotes diverged from a common ancestor that contained the α-proteobacterial endosymbiont that gave rise to mitochondria. The ‘amitochondriate’ anaerobic protist parasites that have been studied to date, such as Giardia and Trichomonas harbor mitochondrion-related organelles, such as mitosomes or Hydrogenosomes. Yet there is one remaining group of mitochondrion-lacking flagellates known as the Preaxostyla that could represent a primitive ‘pre-mitochondrial’ lineage of eukaryotes. To test this hypothesis, we conducted an expressed sequence tag (EST) survey on the preaxostylid flagellate Trimastix pyriformis, a poorly-studied free-living anaerobe. Among the ESTs we detected 19 proteins that, in other eukaryotes, typically function in mitochondria, Hydrogenosomes or mitosomes, 12 of which are found exclusively within these organelles. Interestingly, one of the proteins, aconitase, functions in the tricarboxylic acid cycle typical of aerobic mitochondria, whereas others, such as pyruvate:ferredoxin oxidoreductase and [FeFe] hydrogenase, are characteristic of anaerobic Hydrogenosomes. Since Trimastix retains genetic evidence of a mitochondriate ancestry, we can now say definitively that all known living eukaryote lineages descend from a common ancestor that had mitochondria.
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proteins of the glycine decarboxylase complex in the Hydrogenosome of trichomonas vaginalis
Eukaryotic Cell, 2006Co-Authors: Mandira Mukherjee, Mark T Brown, Andrew G Mcarthur, Patricia J. JohnsonAbstract: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.
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identification and biochemical characterization of serine hydroxymethyl transferase in the Hydrogenosome of trichomonas vaginalis
Eukaryotic Cell, 2006Co-Authors: Mandira Mukherjee, Mark T Brown, Stuart A Sievers, Patricia J. JohnsonAbstract:Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.
Marlene Benchimol - One of the best experts on this subject based on the ideXlab platform.
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The Hydrogenosome as a drug target.
Current Pharmaceutical Design, 2008Co-Authors: Marlene BenchimolAbstract:: Hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms. In Trichomonas Hydrogenosomes measure between 200 to 500 nm, but under drug treatment they can reach 2 microm. Like mitochondria Hydrogenosomes: (1) are surrounded by two closely apposed membranes and present a granular matrix: (2) divide in three different ways: segmentation, partition and the heart form; (3) they may divide at any phase of the cell cycle; (4) produce ATP; (5) participate in the metabolism of pyruvate formed during glycolysis; (6) are the site of molecular hydrogen formation; (7) present a relationship with the endoplasmic reticulum; (8) incorporate calcium; (9) import proteins post-translationally; (10) present cardiolipin. However, there are differences, such as: (1) absence of genetic material, at least in trichomonas; (2) lack a respiratory chain and cytochromes; (3) absence of the F(0)-F(1) ATPase; (4) absence of the tricarboxylic acid cycle; (5) lack of oxidative phosphorylation; (6) presence of peripheral vesicles. Hydrogenosomes are considered an excellent drug target since their metabolic pathway is distinct from those found in mitochondria and thus medicines directed to these organelles will probably not affect the host-cell. The main drug used against trichomonads is metronidazole, although other drugs such as beta-Lapachone, colchicine, Taxol, nocodazole, griseofulvin, cytochalasins, hydroxyurea, among others, have been used in trichomonad studies, showing: (1) flagella internalization forming pseudocyst; (2) dysfunctional Hydrogenosomes; (3) Hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid; (4) intense autophagy in which Hydrogenosomes are removed and further digested in lysosomes.
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the Hydrogenosome peripheral vesicle similarities with the endoplasmic reticulum
Tissue & Cell, 2008Co-Authors: Marlene BenchimolAbstract:The Hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. The Hydrogenosome of Tritrichomonas foetus, a cattle parasite, is a spherical organelle that presents a peripheral vesicle the origin and behavior of which is poorly known. In this article it is reported an ultrastructural and microanalytical study using energy dispersive X-ray analysis, 3D reconstruction and cytochemistry of the Hydrogenosome peripheral vesicle and then compare the results with the endoplasmic reticulum and the nuclear envelope of T. foetus. Similarities between the Hydrogenosome peripheral vesicle and the ER are presented. This study included: (1) the detection of ER enzymes by cytochemistry, such as glucose-6-phosphatase, IDPase, acid phosphatase and Ca(2+) -ATPase; (2) elemental composition by X-ray microanalysis and the mapping of calcium, phosphorus and oxygen in both ER and Hydrogenosome peripheral vesicle; (3) freeze-fracture; (4) TEM of routine and cryofixed cells by high-pressure freezing and freeze-substitution; (5) 3D reconstruction, (6) monoclonal antibody anti-trichomonads ER; and (6) other cytochemical techniques that detects ER, such as the ZIO and lectins. We found a similar composition of the tested enzymes and other elements present in the ER when compared with the Hydrogenosome's peripheral vesicle. It was concluded that, like mitochondria, Hydrogenosome presents relationships with the ER, especially the peripheral vesicle.
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structure of the Hydrogenosome
2007Co-Authors: Marlene BenchimolAbstract:Hydrogenosomes are very interesting organelles found in nonmitochondrial organisms. They display similarities and differences with mitochondria. Hydrogenosomes are spherical or slightly elongated organelles, although very elongated Hydrogenosomes are also found. They measure between 200 and 1 000 nm, but under stress conditions can reach 2 μm. Hydrogenosomes divide in three different ways, like mitochondria: segmentation, partition, and the heart form. They may divide at any phase of the cell cycle. Nucleoid or electron-dense deposits are not considered part of the normal structure of the Hydrogenosome. Hydrogenosomes are surrounded by two closely apposed membranes and present a granular matrix. Hydrogenosomes have one or multiple peripheral vesicles, which incorporate calcium. The peripheral vesicle can be isolated from the hydrogenosomal matrix and is considered a distinct hydrogenosomal compartment. Dysfunctional Hydrogenosomes are removed by an autophagic process and further digested in lysosomes.
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the effect of drugs on cell structure of tritrichomonas foetus
Parasitology Research, 2004Co-Authors: Rodrigo Madeiro Da Costa, Marlene BenchimolAbstract:The effects of the microtubule affecting drugs taxol, nocodazole and colchicine on the cell cycle and ultrastructure of Tritrichomonas foetus, a protist parasite of cattle, were studied. Alterations in the cytoskeleton, motility and organellar ultrastructure were followed using anti-tubulin antibodies and fluorescence microscopy, scanning- and transmission-electron microscopy. Flow cytometry was also used to analyze the effect of the drugs on the cell cycle. T. foetus was treated with 10 μM taxol, 15 μM nocodazole or 1.5 mM colchicine for 12 h. The first effect observed was pseudocyst formation and alterations in cell motility. The cell cycle was affected and the cells have blocked cytokinesis, but not karyokinesis. The behavior of Golgi, Hydrogenosomes and vacuoles was analyzed. The following effects were seen following drug treatments: (1) cell motility was altered and flagella internalized; (2) microtubules of the pelta-axostyle complex were not depolymerized and the axostyle assumed a curved form; (3) Hydrogenosomes were of abnormal size and shape; (4) cells became multinucleate; (5) the division process was blocked in cytokinesis; (6) autophagic vacuoles containing a large amount of microtubules were seen; (7) axoneme organization was altered; (8) zoids were formed; (9) signs of cell death, such as membrane blebbing, were observed.
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hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial tritrichomonas foetus
Histochemistry and Cell Biology, 2003Co-Authors: Cinthya A Guimaraes, Rafael M Mariante, Rafael Linden, Marlene BenchimolAbstract:Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the Hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H2O2 leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H2O2 treatment. The Hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.
Aloysius G M Tielens - One of the best experts on this subject based on the ideXlab platform.
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the mitochondrion of euglena gracilis
Advances in Experimental Medicine and Biology, 2017Co-Authors: Verena Zimorski, Jaap J Van Hellemond, Cessa Rauch, Aloysius G M Tielens, William MartinAbstract:In the presence of oxygen, Euglena gracilis mitochondria function much like mammalian mitochondria. Under anaerobiosis, E. gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. Some components (enzymes and cofactors) of Euglena’s anaerobic energy metabolism are found among the anaerobic mitochondria of invertebrates, others are found among Hydrogenosomes, the H2-producing anaerobic mitochondria of protists.
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Acetate:succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis: identification and characterization.
Journal of Biological Chemistry, 2007Co-Authors: Koen W. A. Van Grinsven, Mark van der Giezen, William Martin, Aloysius G M Tielens, Silke Rosnowsky, Susanne W. H. Van Weelden, Simone Pütz, Jaap J. Van Hellemond, Katrin HenzeAbstract: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.
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the anaerobic chytridiomycete fungus piromyces sp e2 produces ethanol via pyruvate formate lyase and an alcohol dehydrogenase e
Molecular Microbiology, 2004Co-Authors: Brigitte Boxma, Frank Voncken, Jaap J Van Hellemond, Anna Akhmanova, Martijn A Huynen, Sander Jannink, Theo A Van Alen, Susanne W H Van Weelden, Guenola Ricard, Aloysius G M TielensAbstract:Anaerobic chytridiomycete fungi possess Hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among Hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the Hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of Hydrogenosome has to be revisited.
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a divergent adp atp carrier in the Hydrogenosomes of trichomonas gallinae argues for an independent origin of these organelles
Molecular Microbiology, 2004Co-Authors: Joachim Tjaden, Aloysius G M Tielens, Brigitte Boxma, Martijn A Huynen, Ilka Haferkamp, Johannes H P HacksteinAbstract: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.
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multiple origins of Hydrogenosomes functional and phylogenetic evidence from the adp atp carrier of the anaerobic chytrid neocallimastix sp
Molecular Microbiology, 2002Co-Authors: Frank Voncken, Anna Akhmanova, Aloysius G M Tielens, Brigitte Boxma, Martijn A Huynen, Joachim Tjaden, Ilka Haferkamp, Fons J. Verbeek, Ekkehard H Neuhaus, G D VogelsAbstract: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.
William Martin - One of the best experts on this subject based on the ideXlab platform.
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the mitochondrion of euglena gracilis
Advances in Experimental Medicine and Biology, 2017Co-Authors: Verena Zimorski, Jaap J Van Hellemond, Cessa Rauch, Aloysius G M Tielens, William MartinAbstract:In the presence of oxygen, Euglena gracilis mitochondria function much like mammalian mitochondria. Under anaerobiosis, E. gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. Some components (enzymes and cofactors) of Euglena’s anaerobic energy metabolism are found among the anaerobic mitochondria of invertebrates, others are found among Hydrogenosomes, the H2-producing anaerobic mitochondria of protists.
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conservation of transit peptide independent protein import into the mitochondrial and hydrogenosomal matrix
Genome Biology and Evolution, 2015Co-Authors: Sriram G Garg, William Martin, Jan Tachezy, Verena Zimorski, Petr Rada, Jan Stolting, Sven B GouldAbstract: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.
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knockout of the abundant trichomonas vaginalis hydrogenosomal membrane protein tvhmp23 increases Hydrogenosome size but induces no compensatory up regulation of paralogous copies
FEBS Letters, 2013Co-Authors: Xavier Pereira Bras, William Martin, Verena Zimorski, Kathrin Bolte, Uwe G Maier, Sven B GouldAbstract:Abstract The Trichomonas vaginalis genome encodes up to 60 000 genes, many of which stem from genome duplication events. Paralogous copies thus accompany most T. vaginalis genes, a phenomenon that limits genetic manipulation. We characterized one of the parasite’s most abundant hydrogenosomal membrane proteins, TvHMP23, which is phylogenetically distinct from canonical metabolite carriers, and which localizes to the inner hydrogenosomal membrane as shown through sub-organellar fractionation and protease protection assays. Knockout of Tvhmp23 through insertion of the selectable neomycin marker led to a size increase of Hydrogenosomes, the first knockout-induced phenotypes reported for Trichomonas, but no growth impairment. The transcriptional response of its four paralogous copies then analyzed revealed that they are not up-regulated, and hence do not compensate for the Tvhmp23 knockout.
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Acetate:succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis: identification and characterization.
Journal of Biological Chemistry, 2007Co-Authors: Koen W. A. Van Grinsven, Mark van der Giezen, William Martin, Aloysius G M Tielens, Silke Rosnowsky, Susanne W. H. Van Weelden, Simone Pütz, Jaap J. Van Hellemond, Katrin HenzeAbstract: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.
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euglena gracilis rhodoquinone ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions
Journal of Biological Chemistry, 2004Co-Authors: Meike Hoffmeister, Anita Van Der Klei, Carmen Rotte, Koen W A Van Grinsven, Jaap J Van Hellemond, Katrin Henze, Alexander G. G. M. Tielens, William MartinAbstract:Abstract Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1β subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1α, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP+ oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of Hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and Hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of Hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.
Jan Tachezy - One of the best experts on this subject based on the ideXlab platform.
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anaerobic peroxisomes in mastigamoeba balamuthi
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Tien Le, Ivan Hrdý, Petr Rada, Vojtěch Žárský, Eva Nývltova, Karel Harant, Marie Vancova, Zdeněk Verner, Jan TachezyAbstract:The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, Hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from Hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.
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Localisation of TvTom40-2 in the hydrogenosomal outer membrane.
2019Co-Authors: Abhijith Makki, Marian Novotný, Petr Rada, Vojtěch Žárský, Sami Kereïche, Lubomír Kováčik, Tobias Jores, Doron Rapaport, Jan TachezyAbstract:(A) HA-tagged TvTom40-2 and malic enzyme (hydrogenosomal matrix protein) were visualised using mouse α-HA (green) and rabbit α-malic enzyme (red) antibodies, respectively. The nucleus was stained with DAPI (blue). (B) Localisation and topology of TvTom40-2 in T. vaginalis subcellular fractions. Immunoblot analysis of the whole cell lysate, cytoplasm, Hydrogenosomes, Hydrogenosomes treated with proteinase K, and Hydrogenosomes treated with proteinase K in the presence of Triton X-100 using antibodies against HA, Fdx1 (hydrogenosomal matrix protein), and cytosolic malic enzyme. (C) BN-PAGE immunoblots of digitonin-lysed hydrogenosomal extract from the strain expressing HA-tagged TvTom40-2. The samples were probed with α-HA antibody. BN-PAGE, blue native PAGE; Cyt, cytoplasm; cytME, cytoplasmic malic enzyme; DIC, differential interference contrast; Fdx, ferredoxin; H/PK, Hydrogenosomes treated with proteinase K; H/TX/PK, Hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Hyd, Hydrogenosomes; Lys, lysate; TOM, translocase of the outer membrane; TvTom, T. vaginalis TOM.
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Localisation and topology of the TA proteins.
2019Co-Authors: Abhijith Makki, Marian Novotný, Petr Rada, Vojtěch Žárský, Sami Kereïche, Lubomír Kováčik, Tobias Jores, Doron Rapaport, Jan TachezyAbstract:(A) Immunoblot analysis of TA proteins in T. vaginalis subcellular fractions using α-V5 and α-αSCS (hydrogenosomal matrix protein) antibodies. Total cell lysates, cytoplasm, Hydrogenosomes, Hydrogenosomes treated with either proteinase K, or Hydrogenosomes treated with proteinase K and Triton X-100 isolated from the strains expressing V5-tagged Tom36, Tom46, Homp38, Homp19, and Tom22-like protein. (B) Double transfectants expressing HA-tagged TvTom40-2 along with one of the V5-tagged proteins, Tom36, Tom46, Homp38, Homp19 or Tom22-like protein were visualised using mouse α-HA/α-mouse Abberior STAR 580 (green) and rabbit α-V5/α-rabbit Abberior STAR 635p (red) antibodies. Scale bar, 1 μm. αSCS, α-subunit of succinyl CoA synthetase; CoA, coenzyme A; Cyt, cytoplasm; H/PK, Hydrogenosomes treated with proteinase K; H/TX/PK, Hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Homp, hydrogenosomal outer membrane protein; Hyd, Hydrogenosomes; Lys, lysate; TA, tail-anchored; TOM, translocase of the outer membrane; TvTom, T. vaginalis TOM.
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TvTom40-2 is involved in hydrogenosomal protein import.
2019Co-Authors: Abhijith Makki, Marian Novotný, Petr Rada, Vojtěch Žárský, Sami Kereïche, Lubomír Kováčik, Tobias Jores, Doron Rapaport, Jan TachezyAbstract:(A) Autoradiograph showing a time-dependent in vitro import of 35S-Met-labeled TvFdx1-DHFR into Hydrogenosomes. (B) Autoradiograph showing the in vitro import of 35S-Met-labeled TvFdx1-DHFR into Hydrogenosomes in either the absence (−) or the presence (+) of MTX, followed by proteinase K (+) treatment. (C) Autoradiograph showing the eluates for the TvTom40-2-HA coIP following the in vitro import of 35S-Met-labeled TvFdx1-DHFR into Hydrogenosomes isolated from a strain expressing both TvTom40-2-HA and Tom36-V5 either in the presence (+) or the absence (−) of MTX. (D) Immunoblot of the same eluates as in panel C using α-HA, α-V5, and α-Sam50 antibodies. coIP, co-immunoprecipitation; DHFR, dihydrofolate reductase; Fdx, ferredoxin; HA, human influenza hemagglutinin; In, input; MTX, methotrexate; PK, proteinase K; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TvTom, T. vaginalis TOM.
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TA proteins and Sam50 associated with TvTom40-2 are present in a high molecular weight complex.
2019Co-Authors: Abhijith Makki, Marian Novotný, Petr Rada, Vojtěch Žárský, Sami Kereïche, Lubomír Kováčik, Tobias Jores, Doron Rapaport, Jan TachezyAbstract:(A) Digitonin-lysed extracts of Hydrogenosomes isolated from the recombinant strains expressing both HA-tagged TvTom40-2 and one of the V5-tagged proteins, Tom36, Tom46, Homp38, Homp19, or Tom22-like protein were subjected to IP using α-HA antibody. Eluates from the IPs were probed for the presence of HA-tagged TvTom40-2, V5-tagged candidate proteins, and Sam50 under both crosslinking and native conditions using α-HA, α-V5, and polyclonal α-Sam50 antibodies, respectively. (B) BN-PAGE immunoblots of digitonin-lysed hydrogenosomal extracts from the strains expressing HA-tagged and V5-tagged proteins as indicated. BN-PAGE, blue native PAGE; HA, human influenza hemagglutinin; Homp, hydrogenosomal outer membrane protein; In, input; IP, immunoprecipitation; Sam, sorting and assembly machinery; TA, tail-anchored; TOM, translocase of the outer membrane; TvTom, T. vaginalis TOM.
