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.
Highlights
Fusion, the tripartite SNAP receptor (SNARE) complex disassembles by the activities of N-ethylmaleimide sensitive factor (NSF) and Soluble NSF attachment proteins (SNAP) [3]
We reported that Ariadne 1 (Ari-1) loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side
We found a reduction of biotin and GFP signal in whole extracts of ari-1OE/comt flies (Fig. S1A, input and Fig. S1B), which led to a reduction in the amount of eluted GFP-Comt protein from Ari-1 overexpressing flies compared with comt flies (Fig. S1A, elution original washing buffer (WB))
Summary
Fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs [3]. The system relies on the overexpression of a tagged ubiquitin that bears a 16 amino acid long biotinylatable peptide [25, 27], which can be biotinylated by the Escherichia coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo [25, 26] This approach can be efficiently applied to identify neuronal E3 ligase substrates [28, 29]. We reported that Ari-1 loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side. These defects in the mutants are compatible with a deregulation of Com/NSF activity. Our data show that Ari regulates neurotransmitter release by controlling Comt/NSF activity through ubiquitination
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