Hydrogenosomal Proteins Research Articles

Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins.

The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H(2), CO(2) and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic activity of the hydrogenosome and expression of hydrogenosomal enzymes were compared in Tritrichomonas foetus maintained under iron-rich (150 microM iron nitrilotriacetate) or iron-restricted (180 microM 2,2-dipyridyl) conditions in vitro. The activities of PFOR and hydrogenase, and also production of acetate and H(2), were markedly decreased or absent in iron-restricted trichomonads. Moreover, a decrease in activity of the hydrogenosomal malic enzyme, which is a non-Fe-S protein, was also observed. Impaired function of hydrogenosomes under iron-restricted conditions was compensated for by activation of the cytosolic pathway, mediating conversion of pyruvate to ethanol via acetaldehyde. This metabolic switch was fully reversible. Production of hydrogen by iron-restricted trichomonads was restored to the level of organisms grown under iron-rich conditions within 3 h after addition of 150 microM iron nitrilotriacetate. Protein analysis of purified hydrogenosomes from iron-restricted cells showed decreased levels of proteins corresponding to PFOR, malic enzyme and ferredoxin. Accordingly, these cells displayed decreased steady-state level and synthesis of mRNAs encoding PFOR and hydrogenosomal malic enzyme. These data demonstrate that iron is essential for function of the hydrogenosome, show its involvement in the expression of hydrogenosomal proteins and indicate the presence of iron-dependent control of gene transcription in Tt. foetus.

Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis

Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin.

The hydrogenosomal malic enzyme from the anaerobic fungus neocallimastix frontalis is targeted to mitochondria of the methylotrophic yeast hansenula polymorpha.

Hydrogenosomal proteins always contain an amino-terminal extension which is believed to be a hydrogenosomal targeting signal. In the anaerobic fungus Neocallimastix frontalis these putative targeting signals are 27 amino acids long, are enriched in Ala, Leu, Ser and Arg, and have an Arg at position -2 relative to amino-acid 1 of the mature protein. These features are typically observed in mitochondrial targeting signals. Here we show that the 27 amino-acid leader sequence of the hydrogenosomal malic enzyme of N. frontalis was capable of targeting the enzyme to mitochondria of the methylotrophic ascomycete yeast Hansenula polymorpha. The same protein without this leader sequence remained cytosolic. These data suggest a close relationship between the protein import machineries of mitochondria and hydrogenosomes in fungi and provide further support for the notion that these two organelles share a common evolutionary origin.

Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.

Primary structure and eubacterial relationships of the pyruvate:Ferredoxin oxidoreductase of the amitochondriate eukaryote Trichomonas vaginalis

In the eukaryotic unicellular organism Trichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex. This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane. PFO and its closely related homolog, pyruvate:flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria. The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups. To gain more insight into the evolutionary relationships of T. vaginalis PFO we determined the primary structure of its two genes (pfoA and pfoB). The deduced amino acid sequences showed 95% positional identity. Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved. The T. vaginalis PFOs were found to be homologous to eubacterial pyruvate:flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length. Lack of eubacterial PFO sequences precluded a comparison. pfoA and pfoB revealed a greater distance from related enzymes of Archaebacteria. The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein. This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the α-subunit of Trichomonas vaginalis hydrogenosomal succinyl CoA synthetase

The anaerobic, parasitic protist, Trichomonas vaginalis, is characterized by the absence of mitochondria and the presence of double membrane bound organelles called hydrogenosomes. Succinyl-cocnzyme A synthetase is a hydrogenosomal enzyme which catalyzes the formation of ATP via substrate-level phosphorylation. We have characterized genes encoding the α subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (SCS). The α-SCS of T. vaginalis is encoded by a multigene family composed of 3 similar genes that do not appear allelic. These 3 α-SCS genes encode a protein with a calculated molecular mass of approximately 32.5 kDa that has > 50% identity ( > 70% similarity) with α-SCSs from Escherichia coli, Thermus flavus, and rat liver mitochondria. Antibodies raised against recombinant T. vaginalis α-SCS expressed in bacteria were used to isolate α-SCS proteins from purified hydrogenosomes. These proteins partition into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization in the organelle. Amino-terminal sequencing of purified α-SCS proteins shows that mature proteins lack a short, leader sequence of 9 amino acids. These amino terminal sequences which are cleaved from T. vaginalis α-SCSs are similar to each other and to all other leader sequences identified on hydrogenosomal proteins.

Primary structure of the hydrogenosomal adenylate kinase of Trichomonas vaginalis and its phylogenetic relationships

Hydrogenosomal adenylate kinase of the amitochondriate protist, Trichomonas vaginalis, has been purified and the sequence of its 39 amino-terminal residues established. Based on this sequence and a conserved internal region of the enzyme, a probe was obtained by DNA polymerase chain reaction and used to isolate a genomic DNA clone containing the gene of this enzyme. This gene exists probably as a single copy in T. vaginalis and is not interrupted by introns. The open reading frame obtained codes for a large type adenylate kinase with a mature molecular mass of 24.5 kDa. The T. vaginalis enzyme is homologous with adenylate kinases of other eukaryotes and eubacteria. Strongly conserved parts and residues of the molecule are conserved also in this enzyme. Phylogenetic trees obtained with various methods placed the T. vaginalis adenylate kinase close to the point where the different subfamilies of this enzyme branch from each other, indicating that the T. vaginalis enzyme has no close relationship to any of these subfamilies and that it separated early from other adenylate kinases. The conceptual translation predicts the existence of an amino-terminal nonapeptide absent from the protein purified from hydrogenosomes, similar to the processed amino-terminal extensions of other hydrogenosomal proteins. These extensions have been considered as putative targeting and important signals.

Molecular cloning of hydrogenosomal ferredoxin cDNA from the anaerobic amoeboflagellate Psalteriomonas lanterna

Anaerobic parasitic and free living protozoa and anaerobic rumen fungi often contain a characteristic organelle, the hydrogenosome. Recently obtained molecular data show that hydrogenosomes in parasitic protozoa probably use a mitochondria-like protein targeting mechanism, whereas for hydrogenosomes in fungi a microbody-like mechanism is inferred. Here we present, to our knowledge, the first sequence data of a hydrogenosomal protein in a free-living anaerobic protozoan. It is shown that ferredoxin of the amoeboflagellate Psalteriomonas lanterna is similar to hydrogenosomal ferredoxin of the parasite Trichomonas vaginalis. We suggest that the two ferredoxins use similar organelle targeting mechanisms.

Biogenesis of the Hydrogenosome in the Anaerobic Protist Trichomonas vaginalis

Trichomonas vaginalis is a primitive protist that lacks mitochondria and peroxisomes. This aerotolerant anaerobe does, however, contain a specialized organelle involved in carbohydrate metabolism called the hydrogenosome. Hydrogenosomes are the site of fermentative oxidation of pyruvate, coupled to ATP production via substrate level phosphorylation. The organelle is surrounded by 2 membranes and appears to contain no genetic material. Hydrogenosomes are proposed to have arisen either through the conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. Our studies show that hydrogenosomal proteins are synthesized on free polyribosomes and are, thus, posttranslationally translocated into the organelle. The 2 hydrogenosomal proteins we have examined in detail, ferredoxin and the beta subunit of succinyl CoA synthetase, appear to be synthesized as larger precursors that contain an 8- or 9-amino acid amino-terminal extension, respectively. These apparent leader sequences are absent from the mature proteins found in hydrogenosomes. The biochemical properties of the amino-terminal extensions are similar to one another and to mitochondrial presequences. These observations are consistent with the hypotheses that the specificity of protein translocation into hydrogenosomes is conferred by a short amino-terminal signal sequence and that the mechanisms underlying translocation are similar to that of mitochondria. Using in vitro import assays, we have shown that this short leader sequence is absolutely required for association of hydrogenosomal proteins with the organelle and that this association is dependent on ATP, temperature, and the presence of intact organelles.

Influence of Metronidazole, CO, CO 2 , and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.

Trichomonas vaginalis hydrogenosomal proteins are synthesized on free polyribosomes and may undergo processing upon maturation

Trichomonas vaginalis hydrogenosomal proteins are synthesized on free polyribosomes and may undergo processing upon maturation