The ADP/ATP carrier and its relationship to oxidative phosphorylation in ancestral protist trypanosoma brucei.

The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.

Direct silylation of Trypanosoma brucei metabolites in aqueous samples and their GC-MS/MS analysis.

A simple two-step method for the derivatization of polar compounds (lactate, alanine, glycerol, succinate and glucose) using hexamethyldisilazane (HMDS) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was developed. This method allows direct derivatization of aqueous samples wihout sample pretreatment. The method was used for the analysis of the metabolites of the unicellular organism Trypanosoma brucei. The limits of detection by GC-MS/MS analysis were in the range of 0.02 mg L(-1) for glucose to 0.85 mg L(-1) for lactate.

Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits.

The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.

Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability.

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.