The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F₁F₀‐ATPase

Abstract

Bloodstream forms of Trypanosoma hrueei were found to maintain a significant membrane potential across their mitochondrial inner membrane (dym) in addition to a plasma membrane potential (d yp). Significantly, the dy, was selectively abolished by low concentrations of specific inhibitors of the FIFn-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondria1 membrane potential is generated and maintained exclusively by the electrogenic translocation of H +, catalysed by the mitochondrial FIFo-ATPase at the expense of ATP rather than by the mitochondrial electrontransport chain present in T. brucei. Consequently, bloodstream forms of T . brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F,F,-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh,P+) and was shown to have a value of approximately 150mV. When the value for the dym is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondria1 proton-motive force serves a role in the directional transport of ions and rnctabolites across thc promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclearencoded protein into the promjtochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. hrucei.

Non‐cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

“…absence of glucose and presence of 2-oxoglutarate, a mitochondrial transmembrane potential could not be generated by hydrolysis of ATP produced by glycolysis (or by phosphagens since trypanosomes do not store phosphagens). Although the measured magnitude of the potential in long slender forms is similar to potentials measured in other cells (Nolan and Voorheis, 1992), this does not exclude the possibility that it is differentially regulated between the two bloodstream forms as suggested by other recent studies of rhodamine 123 uptake (Divo et al, 1993). These potentials were measured at a non-physiological temperature (30°C) and the actual magnitude of the potentials may be dependent upon temperature (Ter Kuile et al, 1992).…”

“…A recent paper by Nolan and Voorheis (1992) shows that the mitochondrion of bloodstream trypanosomes is energized by the mitochondrial FIFO-ATPase acting in the direction of ATP hydrolysis and utilizing ATP generated from glycolysis. These data, also supported by other workers (Vercesi et al, 1992), are not inconsistent with the work presented here.…”

Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

“…We found cells that were morphologically stumpy and dykinetoplastid in the population at high frequency and further demonstrated that these cells specifically lacked a significant mitochondrial membrane potential as assessed by Mitotracker staining. This suggests a disruption of the function of the F1F0 ATPase, believed to be responsible for mitochondrial import in T. brucei (Nolan and Voorheis, 1992). Thus, the morphological and biochemical events of slender to stumpy transition can occur in the absence of the kinetoplast despite the fact that stumpy cells normally up-regulate transcripts for mitochondrial proteins and exhibit some metabolic adaptation to the procyclic form.…”

“…To establish that these cells did not retain kDNA-dependent mitochondrial activity, the population was stained using Mitotracker. This detects mitochondrial membrane potential, which in bloodstream forms requires protein subunits encoded in both the nucleus and kinetoplast (Nolan and Voorheis, 1992). Figure 6C shows that those cells with an obvious kinetoplast demonstrated a prominent mitochondrial staining with Mitotracker, indicating an extant mitochondrial membrane potential.…”

“…To distinguish these possibilities, wild-type (kDNAϩ) stumpy cells were differentiated to procyclic forms in the presence of either 2 mM KCN or 1.25 g/ml oligomycin. These drug regimes inhibit the cytochrome oxidase complex (our unpublished observations) and F1F0 ATPase (Nolan and Voorheis, 1992), respectively, and thereby mimic two metabolic consequences of the absence of kDNA. On exposure of wild-type (kDNAϩ) cells to these treatments we observed that 2 mM KCN had no effect upon the events of differentiation: the cells gained procyclin ( Figure 9A), reentered into a cell cycle with equivalent kinetics to untreated cells ( Figure 9B) and repositioned their kinetoplast ( Figure 9C).…”