The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

Abstract

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. The origination event of an ADP/ATP carrier (AAC) was crucial in the evolution of the present-day mitochondrion that enabled it to become the powerhouse of the eukaryotic cell. Due to the activity of AAC, the energy-producing mitochondria can supply the cytosol with ATP molecules, which fuel most cellular reactions. AAC belongs to the well-defined family of mitochondrial (mt) carrier proteins that are located in the inner mt membrane and are involved in the transport of a wide range of metabolites (1). Under physiological aerobic conditions, AAC is responsible for the 1:1 counterexchange of mt ATP for cytosolic ADP (2). mt ATP is produced mainly by the evolutionarily conserved biochemical pathway of oxidative phosphorylation (OXPHOS), which employs respiratory complexes I through IV to convert the redox energy of various mt substrates into an electrochemical proton gradient (⌬m) across the mt inner membrane. Respiratory complexes I (NADH-ubiquinone oxidoreductase) and II (succinate-ubiquinone oxidoreductase) are responsible for the oxidation of reduced NADH and FADH 2 , respectively. The electrons derived from these biochemical processes continue along the electron transport chain as they are sequentially passed to coenzyme Q, complex III (ubiquinol-cytochrome c oxidoreductase), cytochrome c, complex IV (cytochrome c-O 2 oxid...

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

“…Thus it is plausible to speculate that a lack of ATP and decrease of Ψm will interfere with proper initiation of kDNA replication and result in cell cycle arrest. Similar results were observed upon RNAi knock-down of the ATP/ADP carrier in PF T. brucei cells, in which reduced ATP levels led to failure of the overall cell division machinery ( Gnipova et al., 2015 ). In contrast, a direct inhibition by bisphosphonium salts of kDNA replication, segregation and maintenance as their primary mode of action is far less likely, as (i) these compounds have a poor affinity to DNA as shown previously ( Dardonville et al., 2006 ) and in this study, (ii) CD38 and AHI-9 cause a decrease in Ψm in T. brucei cells that lack mitochondrial DNA (Gould, Schnaufer and De Koning, unpublished), and (iii) interference with kDNA replication, either by RNAi or drug treatment, usually leads to complete loss of kDNA ( Bruhn et al., 2011 , Hiltensperger et al., 2012 , Tyc et al., 2015 ).…”

AAC as a Potential Target Gene to Control <em>Verticillium dahliae</em>

“…By transforming fungal protoplasts in this way, we confirmed this hypothesis. Further support for this data comes from a previous study in which the silencing of a single functional AAC gene (TbAAC), in Trypanosoma brucei, by RNAi resulted in a severe growth defect, mainly due to reduced mitochondrial ATP synthesis [28]. Consistent with our RNAi results, the ∆VdAAC mutant had significant reduced colony diameter, conidia number and virulence as compared with Vd wt and ∆VdAAC-C.…”

Mitochondrial Gene Expression Is Responsive to Starvation Stress and Developmental Transition in Trypanosoma cruzi

“…Conversely, ETC complexes may actually experience a change in mitochondrion-encoded versus nucleus-encoded subunit ratios. Interestingly, at least one ETC protein is not physically associated with complex V in T. brucei as it is in other model systems ( 59 ). This demonstrates that increases in mitochondrial but not nuclear ETC proteins that could lead to ETC functional changes are possible without compromising the physical integrity of complex I to complex V.…”