Bioenergetic consequences of FoF1–ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei

Abstract

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei , the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the F o F 1 –ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome F o F 1 –ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of F o F 1 –ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased F o F 1 –ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei F o F 1 –ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of F o F 1 –ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of F o F 1 –ATP synthase loss in insect versus mammalian forms of the parasite.

Redesigned and reversed: architectural and functional oddities of the trypanosomal ATP synthase

“…3D). However, the reinforcement of the interaction between the F 1 head and the peripheral stalk is unlikely the only role of p18, because the protein is essential for the integrity of the F 1 -ATPase (Gahura et al ., 2018 b ), in contrast to the peripheral stalk components OSCP (Hierro-Yap et al ., 2021) and ATPTB2/d-subunit (Subrtova et al ., 2015). In E. gracilis , the extensions of OSCP and d-subunit compensate for the reduction of the b-subunit, the major constituent of the peripheral stalk in bacteria and mitochondria of mammals and yeast.…”

“…This represents a novel and unique role of the F-ATPase connecting this enzyme to the regulation of the glycolytic pathway, which is intimately linked to respiration via the alternative oxidase (Bakker et al ., 1999). Despite these fundamental roles, BSF trypanosomes can withstand a loss of approximately 90% of the membrane-bound F-ATPase complexes with no obvious deleterious effect on their viability in culture nor on the mitochondrial membrane potential in live cells (Hierro-Yap et al ., 2021). We speculate that the long-term tolerance of reduced levels of F-ATPase complexes might be favourable for the emergence of trypanosomes lacking mitochondrial genome, as it might provide the time frame for the parasites to gain key nuclear mutation(s) in the F-ATPase that enable(s) generation of mitochondrial membrane potential in the absence of mitochondrial-encoded a-subunit.…”

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector

“…Secondly, the L262Pγ mutation causes a less profound defect in F1FO-ATP synthase function than the loss of Tb1. This suggests that, whereas the F1FO-ATP synthase is nearly completely disrupted in the absence of Tb1 [98,99], the F1Fo complex retains some functionality in L262P/L262Pγ cells as these cells can still generate enough ATP mitochondrially to survive in these conditions.…”

“…In Tb1 -/Tb1cells, however, the near-complete loss of Fo from ATP synthase complexes [98,99] might cause sustained hyperpolarisation of the inner mitochondrial membrane as the ΔΨm generated by complex III and IV cannot be dissipated by proton movement through the Fo moiety [121][122][123]. Hyperpolarisation would interfere with metabolite exchange and mitochondrial protein import and cause ROS generation and enhanced oxidative stress, thus damaging cells in a multitude of ways [124].…”

“…). Tb1 (systematic TriTrypDB ID Tb927.10.520) is the largest T.brucei FO subunit and required to maintain the intact F1Fo complex structure and ATP synthase activity; however, it is not required for F1-ATPase activity[98,99]. Like other FO subunits, the gene becomes dispensable in BSF T. brucei in the presence of an L262Pγ allele, which enabled us to generate mutants that completely lack FO but possess all kDNA-encoded genes (SFig 2).Three independent Tb1 null clones (Tb1 -/Tb1 -#1, #2, #3) were used in this study.…”