The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

Ariadne-1 (Ari-1) is an essential E3 ubiquitin-ligase whose neuronal substrates are yet to be identified. We have used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics to identify putative Ari-1 substrates in Drosophila heads. Sixteen candidates met the established criteria. Amongst those, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF). Using an in vivo GFP pulldown approach, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons. The interaction results in the monoubiquitination of Comt/NSF. We also report that Ari-1 loss of function mutants display a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants is enhanced in a Ca2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. The distinct Ari-1 mutant phenotypes in spontaneous versus evoked release indicate that NSF activity discriminates the two corresponding protein ensembles that mediate each mode of release. Our results, thus, provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.