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

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 1...

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Authors: Ramírez, J., Morales, Miguel, Osinalde, N., Martínez-Padrón, I, Mayor, Ugo, Ferrús, Alberto
Format: article
Status:Published version
Publication Date:2021
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/260697
Online Access:http://hdl.handle.net/10261/260697
Access Level:Open access
Keyword:Ariadne-1
Drosophila
E3 ubiquitin ligase
Neurotransmitter release
NSF synapse
ubiquitination
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spelling The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSFRamírez, J.Morales, MiguelOsinalde, N.Martínez-Padrón, IMayor, UgoFerrús, AlbertoAriadne-1DrosophilaE3 ubiquitin ligaseNeurotransmitter releaseNSF synapseubiquitinationAriadne-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).American Society for Biochemistry and Molecular BiologyMinisterio de Economía y Empresa (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2022202220212022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/260697reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO//BFU2015-65685-Pinfo:eu-repo/grantAgreement/MINECO//PGC2018-094630-B-100info:eu-repo/grantAgreement/MINECO//SAF2016-76898-Phttp://dx.doi.org/10.1016/j.jbc.2021.100408Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2606972026-05-22T06:33:51Z
dc.title.none.fl_str_mv The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
title The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
spellingShingle The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
Ramírez, J.
Ariadne-1
Drosophila
E3 ubiquitin ligase
Neurotransmitter release
NSF synapse
ubiquitination
title_short The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
title_full The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
title_fullStr The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
title_full_unstemmed The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
title_sort The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
dc.creator.none.fl_str_mv Ramírez, J.
Morales, Miguel
Osinalde, N.
Martínez-Padrón, I
Mayor, Ugo
Ferrús, Alberto
author Ramírez, J.
author_facet Ramírez, J.
Morales, Miguel
Osinalde, N.
Martínez-Padrón, I
Mayor, Ugo
Ferrús, Alberto
author_role author
author2 Morales, Miguel
Osinalde, N.
Martínez-Padrón, I
Mayor, Ugo
Ferrús, Alberto
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Empresa (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Ariadne-1
Drosophila
E3 ubiquitin ligase
Neurotransmitter release
NSF synapse
ubiquitination
topic Ariadne-1
Drosophila
E3 ubiquitin ligase
Neurotransmitter release
NSF synapse
ubiquitination
description 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.
publishDate 2021
dc.date.none.fl_str_mv 2021
2022
2022
2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/260697
url http://hdl.handle.net/10261/260697
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO//BFU2015-65685-P
info:eu-repo/grantAgreement/MINECO//PGC2018-094630-B-100
info:eu-repo/grantAgreement/MINECO//SAF2016-76898-P
http://dx.doi.org/10.1016/j.jbc.2021.100408
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,812429

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