The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.
Author Summary African trypanosomes (Trypanosoma brucei and related subspecies) are unicellular parasites that cause the devastating disease of African sleeping sickness in man and nagana in livestock. Both of these diseases are lethal, killing thousands of people each year and causing major economical complications in the developing world, thus affecting the lives of millions. Furthermore, available drugs are obsolete, difficult to administer and have many undesirable side-effects. Therefore, there is a reinvigorated effort to design new drugs against these parasites. From the pharmacological perspective, unique metabolic processes and protein complexes with singular structure, composition and essential function are of particular interest. One such remarkable protein complex is the mitochondrial F0F1-ATP synthase/ATPase. Here we show that F0F1-ATP synthase complex is essential for viability of procyclic T. brucei cells and it possesses unique and novel subunits. The three F0F1-ATP synthase subunits that were tested were shown to be crucial for the structural integrity of the F0F1-ATP synthase complex and its activities. The compositional and functional characterization of the F0F1-ATP synthase in T. brucei represents a major step towards deciphering the unique and essential properties of the respiratory chain of both an early diverged eukaryote and a lethal human parasite.