Oxidative phosphorylation and substrate level phosphorylation catalyzed by succinyl-CoA synthetase found in the citric acid and the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle contribute to mitochondrial ATP synthesis in procyclic Trypanosoma brucei. The latter pathway is specific for trypanosome but also found in hydrogenosomes. In organello ATP production was studied in wild-type and in RNA interference cell lines ablated for key enzymes of each of the three pathways. The following results were obtained: 1) ATP production in the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle was directly demonstrated. 2) Succinate dehydrogenase appears to be the only entry point for electrons of mitochondrial substrates into the respiratory chain; however, its activity could be ablated without causing a growth phenotype. 3) Growth of procyclic T. brucei was not affected by the absence of either a functional citric acid or the acetate: succinate CoA transferase/succinyl-CoA synthetase cycle. However, interruption of both pathways in the same cell line resulted in a growth arrest. In summary, these results show that oxygen-independent substrate level phosphorylation either linked to the citric acid cycle or tied into acetate production is essential for growth of procyclic T. brucei, a situation that may reflect an adaptation to the partially hypoxic conditions in the insect host.Trypanosoma brucei is a unicellular parasite responsible for human sleeping sickness and nagana in cattle. It cycles between the bloodstream of a mammalian host and the digestive tract of the tsetse fly. During transmission, T. brucei differentiates into different life cycle stages characterized by distinct morphologies, surface proteins, and energy metabolisms.The energy metabolism of the long slender bloodstream forms in the vertebrate host is based on glycolysis, which is localized in a specialized organelle, called the glycosome. Long slender bloodstream forms have a mitochondrion whose volume is much reduced when compared with the one in the other life cycle stages. Furthermore, since the cytochromes and many citric acid cycle enzymes are missing, it is not capable of performing oxidative phosphorylation (1, 2).The procyclic form in the midgut of the fly, on the other hand, is characterized by a large mitochondrion. It has a complete citric acid cycle and a fully functional respiratory chain (3).Energy production in procyclic cells is mainly based on the mitochondrion. There are three partially overlapping pathways in which ATP can be produced (indicated as types I-III in Fig.

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA Met , whereas the only other trypanosomal tRNA Met , the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs Met carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA Met is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA Met becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA Met is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA Met formyltransferases, it has about twice the mass of any of these proteins. mitochondrial translation ͉ initiator tRNA Met ͉ formylation ͉ mitochondrial tRNA import ͉ methionyl-tRNA Met formyltransferase

The protozoan Trypanosoma brucei has a single mitochondrion and lacks an apoptotic machinery. Here we show that expression of the proapoptotic protein Bax in T. brucei causes the release of cytochrome c, the depolarization of the mitochondrial membrane potential and mitochondrial fission. However, in contrast to mammalian cells, the three events are temporally well separated. The release of cytochrome c from the intermembrane space precedes mitochondrial fission, showing that it does not depend on mitochondrial fragmentation. Furthermore, halting Bax expression allows some cells to recover even after mitochondrial fission, the last recorded event, went to completion, indicating that all three Bax-induced events are, in principle, reversible.