The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
| العنوان: | The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF |
|---|---|
| المؤلفون: | Ugo Mayor, Imanol Martínez-Padrón, Juanma Ramirez, Miguel Morales, Nerea Osinalde, Alberto Ferrús |
| المساهمون: | Ministerio de Economía y Empresa (España) |
| المصدر: | Addi: Archivo Digital para la Docencia y la Investigación Universidad del País Vasco The Journal of Biological Chemistry Addi. Archivo Digital para la Docencia y la Investigación instname Digital.CSIC. Repositorio Institucional del CSIC |
| بيانات النشر: | American Society for Biochemistry and Molecular Biology, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | 0301 basic medicine, t-SNARE, plasma membrane SNARE protein, Mutant, Vesicular Transport Proteins, ARIH1, human Ariadne-1, Membrane Fusion, Synaptic Transmission, Biochemistry, Ariadne-1, Synapse, chemistry.chemical_compound, Ubiquitin, synapse, Drosophila Proteins, Monoubiquitination, Neurotransmitter, NSF, N-Ethylmaleimide-Sensitive Proteins, Neurons, Neurotransmitter Agents, SNARE, SNAP receptor, biology, Ari-1, Drosophila Ariadne-1, TEVC, two-electrode voltage clamp conditions, drosophila, NMJ, neuromuscular junctions, Cell biology, Ubiquitin ligase, Drosophila melanogaster, Phenotype, Comt, Comatose, Vesicle-associated membrane protein, E3 ubiquitin ligase, Biotinylation, SNAP, Soluble NSF attachment proteins, Drosophila, Synaptic Vesicles, Research Article, Ubiquitin-Protein Ligases, v-SNARE, vesicle membrane SNARE protein, ubiquitination, RRP, readily releasable pool, LRRK2, Leucine-rich repeat Serine/Threonine-protein kinase 2, 03 medical and health sciences, NSF synapse, mEJC, mini excitatory junctional current, Animals, Molecular Biology, 030102 biochemistry & molecular biology, LFQ, label-free quantification, Cell Biology, NSF, N-ethylmaleimide sensitive factor, VAMP, vesicle-associated membrane protein, 030104 developmental biology, chemistry, neurotransmitter release, DTT, dithiothreitol, Mutation, Synapses, EJP, excitatory junctional potential, biology.protein, LC-MS/MS, liquid chromatography with tandem mass spectrometry, Carrier Proteins |
| الوصف: | 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 Cadependent 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. This research was funded by grants BFU2015-65685 and PGC2018-094630-B-100 from the Spanish Ministry of Economy to A. F. and grant SAF2016-76898-P from the Spanish Ministry of Economy cofinanced with FEDER funds to U. M. J. R. was supported with a postdoctoral research fellowship from the University of the Basque Country (UPV/EHU). |
| وصف الملف: | application/pdf |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::75375988a0ecd8a0071aa6eab5d124cd http://hdl.handle.net/10261/260697 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....75375988a0ecd8a0071aa6eab5d124cd |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |