Extramitochondrial localization of NADH-fumarate reductase in trypanosomatids

doi:10.1016/S1096-4959(02)00094-5

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology

Volume 133, Issue 1, September 2002, Pages 23-27

https://doi.org/10.1016/S1096-4959(02)00094-5 Get rights and content

Abstract

Trypanosoma brucei procyclic trypomastigotes and T. cruzi epimastigotes (both Tulahuen and Y strains) were permeabilized by incubation with increasing amounts of digitonin, causing enzymes to be released from different intracellular compartments. After 10 min incubation with digitonin, the cells were centrifuged and the activity of marker enzymes (aspartate-dependent malic enzyme for cytoplasm, hexokinase for glycosomes and either isocitrate dehydrogenase or citrate synthase for mitochondria) was analyzed in the supernatant. The results were compared with the release of NADH-fumarate reductase in order to determine if this enzyme was preferentially released with a specific intracellular marker. Fumarate reductase was released at lower digitonin concentration than those required to either release isocitrate dehydrogenase or citrate synthase. Similarly, Leishmania donovani promastigotes (S-2 strain) were exposed to a single concentration of digitonin (200 μM) but in this case we monitored the release of fumarate reductase and hexokinase, while monitoring the mitochondrial membrane potential (using safranine O). Again, substantial fumarate reductase and hexokinase activities were released without loss of mitochondrial membrane potential indicating that part of the enzyme was released while the inner mitochondrial membrane remained intact. These results suggest that, in the three species of trypanosomatids the enzyme fumarate reductase is, at least in part, located outside the mitochondrial matrix.

Introduction

Trypanosomatids are parasitic protozoa causing several diseases including Chagas’ disease, sleeping sickness and a variety of leishmaniases in humans, as well as nagana in cattle. Intermediate metabolism in these parasites shows substantial differences with their mammalian counterpart, not only when comparing species but also when comparing different stages within a given species (Boveris et al., 1986, Clarkson et al., 1989, Chen et al., 2001, Denicola-Seoane et al., 1992, Fairlamb and Opperdoes, 1986, Hill, 1976, Santhamma and Bhaduri, 1995, Turrens, 1989).

Several studies have shown that a mitochondrial respiratory chain of trypanosomatids utilizes significant amounts of succinate as the primary electron donor (Turrens, 1989, Denicola-Seoane et al., 1992). In addition, trypanosomes excrete succinate to the extracellular environment, particularly under anaerobic conditions, suggesting that this metabolite may become a terminal sink of reducing equivalents (Flynn and Bowman, 1973, Cazzulo, 1992). Given this dual role of succinate in trypanosomatids we have proposed that interfering with succinate production should be lethal to these cells (Turrens et al., 1999, Turrens, 1999). What makes this hypothesis more attractive is that one of the succinate-producing enzymes, NADH-fumarate reductase, is absent in mammalian cells but has been identified in trypanosomes and various species of Leishmania and Plasmodium sp. (Boveris et al., 1986, Chen et al., 2001, Fry and Beesley, 1991). Thus, this enzyme may provide a unique target against the diseases caused by these parasites.

Since the enzyme NADH-fumarate reductase was first identified in trypanosomatids, It has been generally accepted that was confined to the mitochondrial matrix (Klein et al., 1975). Several reasons led to this assumption. First, the enzyme reverts a step in the Krebs cycle, and generates succinate, which is a mitochondrial substrate. Second, although this enzyme is not an integral protein this enzyme is mostly membrane bound (Mracek et al., 1991, Christmas and Turrens, 2000) and therefore its activity is in part associated with mitochondrial markers, which contaminate all membrane fractions (Boveris et al., 1986). This study was designed to determine whether fumarate reductase is confined to the mitochondrial matrix or is present in other intracellular compartments. The results show that in three different species of trypanosomatids T. brucei procyclic trypomastigotes, T. cruzi epimastigotes (Tulahuen and Y strains) and L. donovani promastigotes (S-2 strain) the enzyme NADH-fumarate reductase is located, at least in part, in an extramitochondrial compartment.

Section snippets

Cell cultures and processing

Trypanosoma brucei procyclic trypomastigotes were grown at 28 °C, in SDM-79 medium JRH Biosciences, Lenexa, KS, supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin 50 units/ml. The cells were harvested in a buffer containing 0.23 M mannitol, 0.07 M sucrose, 0.1 mM EDTA and 10 mM Tris–HCl (pH 7.4) buffer A.

T. cruzi epimastigotes (Y and Tulahuen strains) were grown at 28 °C in a liquid medium consisting of brain-heart infusion 33 g/l, disodium hydrogen phosphate 4

Results

The specific activity of NADH-fumarate reductase in homogenates of T. brucei procyclic trypomastigotes was 82±9 munits/mg protein (n=13) while in the Tulahuen and Y strains of T. cruzi epimastigotes the specific activities were 17±5 (n=15) and 15±3 munits/mg protein (n=4), respectively.

In order to estimate the intracellular localization of fumarate reductase, T. brucei procyclic trypomastigotes and T. cruzi epimastigotes were permeabilized with digitonin as described in Section 2. The

Discussion

The results shown in this report indicate that when either T. brucei procyclic trypomastigotes, T. cruzi epimastigotes or L. donovani promastigotes were permeabilized with digitonin, the enzyme NADH-fumarate reductase is released at lower digitonin concentrations than those required to permeate the inner mitochondrial membrane. Our experiments do not rule out that a proportion of fumarate reductase may be located in the mitochondrial matrix, since the release of fumarate reductase has not

Acknowledgements

This work was supported by a PHS grants # R15 AI/32703-01 and R15 AI/OD39692-01to J.F.T. The authors thank Dr Roberto Docampo for his helpful comments and for his help with the experiments involving L. donovani.

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Glycosomal membrane proteins and lipids from Leishmania mexicana
2015, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
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Fig. 1A shows that differential centrifugation resulted in a subcellular (small granular) fraction that was enriched in glycosomes since it had a specific activity of the glycosomal marker HK markedly higher than each of the other fractions obtained. The large granular fraction is known to be enriched in mitochondria, as confirmed by the data of Fig. 1B, because it contained major parts of the MDH, FR and ALAT activities which, previously have been reported to be present in the mitochondrion of Leishmania spp. as well as in glycosomes (FR, MDH) and cytosol (MDH, ALAT) (Denicola et al., 2002; Leroux et al., 2006; Marciano et al., 2009). Phosphoglucose isomerase (PGI) is a glycosomal enzyme, but known to be readily released from the organelles during purification, explaining the high activity found in the cytosolic fraction (Fig. 1A).
Constituents of the glycosomal membrane from Leishmania mexicana should play a critical role in the coordination of metabolic processes occurring in the cytosol and those compartmentalized within glycosomes. We have made an inventory of glycosomal membrane-associated proteins using approaches specific for enriching both integral and peripheral membrane proteins. Surprisingly, 70% of the proteins were recovered in the hydrophobic fraction of membranes solubilized with Triton X-114, while 20% were present in the soluble fraction obtained upon treatment with Na₂CO₃. 14 major polypeptides, ranging in molecular weight from 65 to 16 kDa, were found to be associated with the membrane, nine of them behaving as integral membrane proteins. Assessment of their topology in the membrane indicated that the polypeptides of 56, 50, 46 and 32 kDa have no domains exposed to the cytosol. The 50 kDa protein is the most abundant one of the glycosomal membrane, where it is peripherically located at the matrix face. The major phospholipids of glycosomal membranes are phosphatidyl-ethanolamine, phosphatidyl-choline and phosphatidyl-serine, with smaller proportions of sphingomyelin and phosphatidyl-inositol. The sterols found were of 5-dehydroepisterol, ergosta-5,7,24(24¹)-trien-3β-ol, and also their precursors, consistent with the notion that these organelles are involved in de novo biosynthesis of sterols in trypanosomatids.
Trypanosoma cruzi: Multiple nucleoside diphosphate kinase isoforms in a single cell
2008, Experimental Parasitology
Citation Excerpt :
Pyruvate kinase and hexokinase activities were determined using a lactic dehydrogenase and glucose-6-phosphate dehydrogenase coupled assay, respectively (Cazzulo et al., 1989; Caceres et al., 2003). NADP+-dependent isocitrate dehydrogenase activity was determined as previously described (Denicola et al., 2002). All assays were performed at least in triplicates and data are representative of at least three independent experiments.
Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml⁻¹. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.
Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei
2008, Journal of Biological Chemistry
Citation Excerpt :
Electrons are then transferred via the quinone pool and the respiratory chain to molecular oxygen. Although hypothetical, this model is supported by the presence in the mitochondrion of the procyclic trypanosomes of both an FRD activity encoded by FRDm1 (12) and an SDH activity (53). Clearly, the maintenance of the mitochondrial redox balance in procyclic trypanosomes is an open question that requires further comprehensive analyses of each working hypothesis.
The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of ¹³C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F₀/F₁-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F₀/F₁-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.
An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi
2006, Biochimica et Biophysica Acta - General Subjects
Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.
The malate dehydrogenase isoforms from Trypanosoma brucei: Subcellular localization and differential expression in bloodstream and procyclic forms
2006, International Journal for Parasitology
Citation Excerpt :
Citrate is then exported to the cytosol where it is cleaved into acetyl-CoA, required for fatty acid synthesis, and oxaloacetate (van Weelden et al., 2003, 2005). The latter is converted to malate by the cytosolic MDH and then to pyruvate by an aspartate-activated NADP-linked malic enzyme (Denicola et al., 2002). A similar malic enzyme is present in the cytosol of T. cruzi epimastigotes (Cannata et al., 1979).
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.
A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei
2005, Journal of Biological Chemistry
Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of ¹³C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.

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Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology

Abstract

Introduction

Section snippets

Cell cultures and processing

Results

Discussion

Acknowledgements

References (22)

Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi

Mol. Biochem. Parasitol.

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

FEMS Microbiol. Lett.

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

J. Biol. Chem.

Succinate-dependent metabolism in Trypanosoma cruzi epimastigotes

Mol. Biochem. Parasitol.

Electron transport systems in kinetoplastida

Biochim. Biophys. Acta

Hexokinase. I. Brain

Methods Enzymol.

Characterization of the respiratory chain of Leishmania donovani promastigotes

Molec. Biochem. Parasitol.

Comment response on: Differences in Energy Metabolism Between Trypanosomatidae—Tielens et al

Parasitol. Today

Mercaptopyridine-N-oxide, a NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

FEMS Microbiol. Lett.

Two forms of ‘malic’ enzyme with different regulatory properties in Trypanosoma cruzi

Biochem. J.

Aerobic fermentation of glucose by trypanosomatids

Fed. Am. Soc. Exp. Biol. J.

Cited by (11)

Glycosomal membrane proteins and lipids from Leishmania mexicana

Trypanosoma cruzi: Multiple nucleoside diphosphate kinase isoforms in a single cell

Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi

The malate dehydrogenase isoforms from Trypanosoma brucei: Subcellular localization and differential expression in bloodstream and procyclic forms

A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei