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In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis, glucose 6-phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We show here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, ( i ) fructose-1,6-bisphosphatase (FBPase), the canonical enzyme performing this reaction, ( ii ) sedoheptulose-1,7-bisphosphatase (SBPase), and ( iii ) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyze the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13 C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas an approximately 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we show for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is possibly due to the supramolecular organization of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channeling.

In trypanosomes, HK and GK, as well as most other enzymes involved in glycolysis and glycerol metabolism, are located in peroxisome-related organelles, named glycosomes, which show limited or no nucleotide exchange with the cytosol on a metabolic timescale. [...]consumption and production of ATP are tightly balanced within the organelle [11], with each ATP molecule required to supply GK and HK being regenerated by phosphoenolpyruvate carboxykinase (PEPCK, step 11) and pyruvate phosphate dikinase (PPDK, step 15) in PCF glycosomes (Fig 1A and 1B). [...]the limitation of the glycosomal ATP pool available to glycosomal kinases offers a situation where a significant excess of one kinase (here GK) can theoretically abolish the metabolic flux through another one (here HK). Unfortunately, with the exception of amino acids [5], the metabolite content in the midgut and other organs of the tsetse has not been studied so far. [...]investigation should be done to determine whether glycerol plays a role in the biology of trypanosomes in the insect vector. Because of the intronless and polycistronic expression nature of trypanosome genes, differential expression of glycolytic enzymes should be controlled posttranscriptionally [21].

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

Trypanosoma brucei , a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that ( i ) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, ( ii ) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and ( iii ) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

Cardiolipin (CL) is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes including oxidative phosphorylation (OXPHOS). To function properly CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for the Barth syndrome (BTHS), presumably because of a diminished OXPHOS capacity. Here we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ) of the Barth syndrome. The rescuing activity involves the recovery of a normal expression of key components that sustain oxidative phosphorylation, including the cytochrome c and complexes IV and III, that are strongly down regulated in taz1Δ yeast. Interestingly, overexpressing Odc1p was shown previously to rescue also yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in the hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxidicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial disease including BTHS.

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein.

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.