Frank Voncken

A hydrogenosome with a genome

Some anaerobic protozoa and chytridiomycete fungi possess membrane-bound organelles known as hydrogenosomes. Hydrogenosomes are about 1 micrometre in diameter and are so called because they produce molecular hydrogen. It has been postulated that hydrogenosomes evolved from mitochondria by the concomitant loss of their respiration and organellar genomes,, and so far no hydrogenosome has been found that has a genome,. Here we provide evidence for the existence of a hydrogenosomal genome of mitochondrial descent, and show that the anaerobic heterotrichous ciliate Nyctotherus ovalis possesses a new type of nuclear-encoded ‘iron-only’ hydrogenase enzyme.

Hydrogenosomes: eukaryotic adaptations to anaerobic environments

Like mitochondria, hydrogenosomes compartmentalize crucial steps of eukaryotic energy metabolism; however, this compartmentalization differs substantially between mitochondriate aerobes and hydrogenosome-containing anaerobes. Because hydrogenosomes have arisen independently in different lineages of eukaryotic microorganisms, comparative analysis of the various types of hydrogenosomes can provide insights into the functional and evolutionary aspects of compartmentalized energy metabolism in unicellular eukaryotes.

Substrate Specificity, Localization, and Essential Role of the Glutathione Peroxidase-type Tryparedoxin Peroxidases in Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical cysteine homologues of the classical selenocysteine-containing glutathione peroxidases. Although one of the sequences, peroxidase III, carries both putative mitochondrial and glycosomal targeting signals, the proteins are detectable only in the cytosol and mitochondrion of mammalian bloodstream and insect procyclic T. brucei. The enzyme is a trypanothione/tryparedoxin peroxidase as are the 2 Cys-peroxiredoxins of the parasite. Hydrogen peroxide, thymine hydroperoxide, and linoleic acid hydroperoxide are reduced with second order rate constants of 8.7 × 104, 7.6 × 104, and 4 × 104m–1 s–1, respectively, and represent probable physiological substrates. Phosphatidylcholine hydroperoxide is a very weak substrate and, in the absence of Triton X-100, even an inhibitor of the enzyme. The substrate preference clearly contrasts with that of the closely related T. cruzi enzyme, which reduces phosphatidylcholine hydroperoxides but not H2O2. RNA interference causes severe growth defects in bloodstream and procyclic cells in accordance with the peroxidases being essential in both developmental stages. Thus, the cellular functions of the glutathione peroxidase-type enzymes cannot be taken over by the 2 Cys-peroxiredoxins that also occur in the cytosol and mitochondrion of the parasite.

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

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

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate‐lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E

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.

Hydrogenosomes: convergent adaptations of mitochondria to anaerobic environments

Hydrogenosomes are membrane-bound organelles that compartmentalise the final steps of energy metabolism in a number of anaerobic eukaryotes. They produce hydrogen and ATP. Here we will review the data, which are relevant for the questions: how did the hydrogenosomes originate, and what was their ancestor? Notably, there is strong evidence that hydrogenosomes evolved several times as adaptations to anaerobic environments. Most likely, hydrogenosomes and mitochondria share a common ancestor, but an unequivocal proof for this hypothesis is difficult because hydrogenosomes lack an organelle genome - with one remarkable exception (Nyctotherus ovalis). In particular, the diversity of extant hydrogenosomes hampers a straightforward analysis of their origins. Nevertheless, it is conceivable to postulate that the common ancestor of mitochondria and hydrogenosomes was a facultative anaerobic organelle that participated in the early radiation of unicellular eukaryotes. Consequently, it is reasonable to assume that both, hydrogenosomes and mitochondria are evolutionary adaptations to anaerobic or aerobic environments, respectively.

Mitochondrial carrier family inventory of Trypanosoma brucei brucei: identification, expression and subcellular localisation.

The mitochondrial carrier family (MCF) is a group of structurally conserved proteins that mediate the transport of a wide range of metabolic intermediates across the mitochondrial inner membrane. In this paper, an overview of the mitochondrial carrier proteins (MCPs) of the early-branching kinetoplastid parasite Trypanosoma brucei brucei is presented. Sequence analysis and phylogenetic reconstruction gave insight into the evolution and conservation of the 24 identified TbMCPs; for most of these, putative transport functions could be predicted. Comparison of the kinetoplastid MCP inventory to those previously reported for other eukaryotes revealed remarkable deviations: T. b. brucei lacks genes encoding some prototypical MCF members, such as the citrate carrier and uncoupling proteins. The in vivo expression of the identified TbMCPs in the two replicating life-cycle forms of T. b. brucei, the bloodstream-form and procyclic-form, was quantitatively assessed at the mRNA level by Northern blot analysis. Immunolocalisation studies confirmed that majority of the 24 identified TbMCPs is found in the mitochondrion of procyclic-form T. b. brucei.

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen‐producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N‐terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria‐bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N‐terminal leader sequences.

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

ABSTRACT Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.