The Ubiquitin Ligase Ariadne-1 Regulates Neurotransmitter Release Via Ubiquitination of NSF

Abstract

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified sixteen candidates that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls 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. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination