A novel neuron-specific regulator of the V-ATPase in Drosophila.

The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. Here, we studied CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, we observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, we observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, we show that CG31030 knockdown induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, our data indicate that CG31030, which we renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release.Significance StatementIn this study, we provide evidence that a previously uncharacterized Drosophila protein, CG31030, is necessary for fly survival and expressed specifically in neurons, where it interacts with constitutive and accessory subunits of the V-ATPase. Physiologically, we show that this protein is required for synaptic vesicle acidification and to ensure proper synaptic transmission at the neuromuscular junction. This implies that CG31030, alias VhaAC45RP, is a novel synaptic protein essential to nervous system functioning and neurotransmitter release. This work therefore provides a new step towards a more exhaustive understanding of the regulations of neuronal V-ATPase and their potential repercussions on synaptic transmission and neurological diseases.