Investigating the roles of F-ATP synthase subunits e and g in Drosophila melanogaster

Differently from yeast and mammals, Drosophila melanogaster does not show a mitochondrial permeability transition (PT), a process caused by the opening of the so-called permeability transition pore (PTP). The PTP is defined as a Ca2+-activated, high-conductance and unselective channel with a maximal conductance of 1.2 nS that allows ions and solutes up to 1.5 kDa to equilibrate across the inner membrane. PTP openings play a role in both Ca2+ homeostasis and cell death initiation. Conversely, Drosophila PTP appears to be specialized and operate uniquely as a selective Ca2+- release channel (CrC), that does not induce mitochondrial swelling and cell death. ATP synthase was recently demonstrated to mediate the PT in mammals and yeast and to generate a peculiar 53 pS channel in Drosophila that could represent the CrC. Genetic studies showed that the ablation of ATP synthase “accessory subunits” e and g dramatically affects PT occurrence in mammals and yeast, suggesting a primary role of these small proteins in PTP formation. To shed light on the roles of the two subunits in Drosophila, we generated knock-down (KD) lines for genes encoding either subunit e (ATPsynE) or g (ATPsynG) of ATP synthase. In vivo ubiquitous downregulation of each subunit causes a dramatic arrest in fly development at larval stage, impairs the dimerization and oligomerization states of ATP synthase and decreases mitochondrial respiration, yet the total amount of ATP is unaltered. Strikingly, the sensitivity to Ca2+ is decreased in both ATPsynE and ATPsynG KD mitochondria, which require higher matrix Ca2+ loads (1.5-fold and 3-fold, respectively) to induce the CrC. However, studies on Ca2+ dynamics in vivo revealed no major differences in mitochondrial basal Ca2+ levels, as well as in Ca2+ transients in motor neurons of flies carrying the specific downregulation of these two subunits in these cells, possibly due to compensatory mechanisms. Importantly, downregulation of both ATPsynE and ATPsynG only in muscle tissue leads to the development of adult flies which show compromised assembly and stability of the F-ATP synthase in muscle, characterized by decreased levels of oligomers and dimers, but also monomers and the accumulation of vestigial forms and of the F1 sector. Surprisingly, mitochondrial respiration is not affected, and the total ATP levels are comparable to those of control flies, although mitochondria of both KD models display altered cristae morphology. Altogether, our results confirm a key role of these two proteins in the formation of Drosophila channel and suggest that the phenotype of KD flies is not entirely due to bioenergetic defects but may also partially arise from a CrC-related Ca2+ dysregulation.