The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

J Biol Chem. 2021 Jan-Jun:296:100408. doi: 10.1016/j.jbc.2021.100408. Epub 2021 Feb 11.

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 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down 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 presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with 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 and 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.

Keywords: Ariadne-1; Drosophila; E3 ubiquitin ligase; NSF; neurotransmitter release; synapse; ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / metabolism
  • Drosophila Proteins / metabolism*
  • Drosophila Proteins / physiology
  • Drosophila melanogaster
  • Membrane Fusion
  • Mutation
  • N-Ethylmaleimide-Sensitive Proteins / genetics
  • N-Ethylmaleimide-Sensitive Proteins / metabolism*
  • Neurons / metabolism
  • Neurotransmitter Agents / metabolism
  • Phenotype
  • Synapses / metabolism
  • Synaptic Transmission / genetics
  • Synaptic Vesicles / metabolism
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination
  • Vesicular Transport Proteins / metabolism

Substances

  • Ari-1 protein, Drosophila
  • Carrier Proteins
  • Drosophila Proteins
  • Neurotransmitter Agents
  • Ubiquitin
  • Vesicular Transport Proteins
  • comt protein, Drosophila
  • Ubiquitin-Protein Ligases
  • N-Ethylmaleimide-Sensitive Proteins