Regulation of the trypanosoma brucei atp synthase β subunit

Mitochondrial activity in Trypanosoraa brucei is regulated through the life cycle of the organism. We have previously shown by western blot analysis and enzyme activity assays that the T. brucei ATP synthase is regulated throughout the life cycle of the organism. We have isolated and purified the T. brucei mitochondrial ATP synthase and have shown the F1 ATPase complex to be composed of five subunits: α: 55 kD, β: 42 kD, γ: 32 kD, δ: 22 kD, and ε: 17 kD. To gain an understanding of the complex regulation of this enzyme and the role it plays in mitochondriai function, key subunit components of this complex are now being studied. The β subunit is a primary target for this work because of its critical role as part of the catalytic site. Western blot analysis shows that the level of protein expression of the β subunit through the trypanosome life cycle is regulated in a manner comparable to that of the whole enzyme. The β gene was obtained by screening a T. brucei Agt11 eDNA library with PCR generated fragments obtained using primers for the miniexon and regions of homology. Southern blot analysis shows this to be a single copy gene. Northern blot analysis and quantitative RT-PCR shows that the β transcript is present at higher levels in the procyclic stage (greater than 5 fold) compared to both bloodstream stages. The relative amounts of transcript, do not entirely correlate with expression at the protein level, suggesting that a second level of regulation may occur postranscriptionally.