bioRxiv preprint doi: https://doi.org/10.1101/2020.01.23.916619; this version posted January 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release Juanma Ramírez1, Miguel Morales2#, Nerea Osinalde3, Imanol Martínez-Padrón2 Ugo Mayor1,4,* and Alberto Ferrús2,* From the 1Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, UPV/EHU, Leioa 48940, Bizkaia, Spain; 2Cajal Institute, CSIC, Madrid 28002, Spain; 3Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, UPV/EHU, Vitoria-Gasteiz 01006, Araba, Spain; 4Ikerbasque, Basque Foundation for Science, Bilbao 48013, Bizkaia, Spain Running title: Ubiquitinated synaptic NSF #Molecular Cognition Laboratory, Biophysics Institute UPV/EHU-CSIC, Barrio Sarriena, Leioa 48940, Bizkaia, Spain *To whom the correspondence should be addressed: Alberto Ferrús: Cajal Institute, CSIC, Madrid 28002, Spain; [email protected]; Ugo Mayor: Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, UPV/EHU, Leioa 48940, Bizkaia, Spain; [email protected]. Keywords: Ariadne-1, Drosophila, E3 ubiquitin ligase, neurotransmitter release, NSF, synapse, ubiquitination ABSTRACT mechanism to regulate NSF activity in the synapse Ariadne-1 (Ari-1) is an essential E3 ubiquitin- through Ari-1-dependent ubiquitination. ligase whose neuronal substrates are yet to be identified. We have used an in vivo ubiquitin biotinylation strategy coupled to quantitative Neurotransmitter release is mediated by a set proteomics to identify putative Ari-1 substrates in of protein-protein interactions that include the N- Drosophila heads. Sixteen candidates met the ethylmaleimide sensitive factor (NSF), soluble established criteria. Amongst those, we identified NSF attachment proteins (SNAPs) and SNAP Comatose (Comt), the homologue of the N- receptors (SNAREs) (1). These proteins assemble ethylmaleimide sensitive factor (NSF). Using an in into a tripartite complex in order to elicit synaptic vivo GFP pulldown approach, we validate vesicle fusion, which is formed by one synaptic Comt/NSF to be an ubiquitination substrate of Ari- vesicle membrane SNARE protein (v-SNARE), 1 in fly neurons. The interaction results in the Synaptobrevin, and two plasma membrane monoubiquitination of Comt/NSF. We also report SNARE proteins (t-SNAREs), Syntaxin and the that Ari-1 loss of function mutants display a lower 25-kDa synaptosome-associated protein (2). rate of spontaneous neurotransmitter release due to Following vesicle fusion, the tripartite SNARE failures at the pre-synaptic side. By contrast, complex disassembles by the activities of NSF and evoked release in Ari-1 mutants is enhanced in a SNAPs. Free t-SNAREs from the plasma 2+ Ca dependent manner without modifications in membrane can then participate in new priming the number of active zones, indicating that the reactions, while the v-SNAREs can be probability of release per synapse is increased in incorporated into recycled synaptic vesicles (3). these mutants. The distinct Ari-1 mutant These interactions, also routinely used for phenotypes in spontaneous versus evoked release intracellular vesicle trafficking in all cell types, are indicate that NSF activity discriminates the two conserved across species (4), including Drosophila corresponding protein ensembles that mediate (5). each mode of release. Our results, thus, provide a Deviations on the rate of neurotransmitter release are at the origin of multiple neural 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.23.916619; this version posted January 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ubiquitinated synaptic NSF diseases, including Parkinson’s disease (6). Under 26). Remarkably, this approach can be efficiently physiological conditions, the leucine-rich repeat applied to identify neuronal E3 ligase substrates serine/threonine-protein kinase 2 (LRRK2) (28, 29). The second methodology, in contrast, phosphorylates NSF to enhance its ATPase favours the isolation of GFP-tagged proteins under activity, which facilitates the disassembly of the denaturing conditions to further characterize their SNARE complex (7–9). However, the most ubiquitination pattern under the presence or common Parkinson’s disease mutation in LRRK2 absence of an E3 ligase (30, 31). causes an excess of kinase activity (10) that Here we have combined the bioUb strategy interferes with the vesicle recycling (11). with the overexpression of Ari-1 and identified 16 Similarly, α-Synuclein, another Parkinson’s putative neuronal substrates of Ari-1. Among disease protein (12), alters neurotransmitter release those, we focused on Comatose (Comt), the fly by preventing the v-SNARE vesicle-associated NSF orthologue (32), due to its relevance in membrane protein (VAMP)-2, also known as normal and pathological function of the synapse. Synaptobrevin-2, from joining the SNARE By the isolation of GFP-tagged Comt from complex cycle (13). Correct neural functioning, Drosophila photoreceptor neurons overexpressing therefore, requires delicate regulation in vesicle Ari-1, we confirmed Comt/NSF as an Ari-1 trafficking. This regulation can be achieved by ubiquitin substrate, and showed that it is mostly post-translational modifications, such as monoubiquitinated. Furthermore, we reported that ubiquitination. In fact, ubiquitination of certain Ari-1 loss of function mutants displayed lower rate proteins can affect their activity or lifespan (14, of spontaneous neurotransmitter release, but 15). At the pre-synaptic side, for example, enhanced evoked release, due to failures at the increased neurotransmitter release correlates with pre-synaptic side. These defects in the mutants are decreased protein ubiquitination (16). Similarly, compatible with a deregulation of Com/NSF acute pharmacological proteasomal inhibition activity. Altogether, our data show that Ari-1 causes rapid strengthening of neurotransmission regulates neurotransmitter release by controlling (17). Comt/NSF activity through ubiquitination. Ariadne 1 (Ari-1) is an E3 ubiquitin-ligase, first identified in Drosophila (18), from a Results conserved gene family defined by two C3HC4 Ring Generation of flies for the identification of fingers separated by a C6HC in-Between-Rings neuronal Ari-1 substrates domain, the RBR motif (19). Ari-1 had been We generated bioari flies in order to screen, in described to be essential for neuronal an unbiased manner, for putative substrates of the development, and its mutants reported to exhibit E3 ligase Ari-1. These flies overexpress in reduced eye rhabdomere surface and endoplasmic photoreceptor neurons (using the GMR-Gal4 reticulum, as well as aberrant axonal pathfinding driver) both an untagged version of Ari-1 protein bio (18). However, despite its importance, no neuronal (Fig. 1A) and the ( Ub)6-BirA construct (Fig. substrates have been reported so far. Only three 1B), which is the one that provides the Ari-1 substrates have been postulated, either in biotinylatable ubiquitins and the BirA enzyme to cultured cells or in vitro (20–22), while three the system. The rationale for using GMR-Gal4 is Parkin substrates were reported to interact with that it has been shown to be a suitable driver for Ari-1 in COS-1 cells (23). For this reason, with the ubiquitin proteomics experiments from mature aim to identify neuronal Ari-1 substrates, we took neurons, in comparison with other neuronal advantage of two methodologies developed by our drivers tested (26). bioUb flies only overexpressing bio bio lab (24). The first one, the Ub strategy, allows the ( Ub)6-BirA construct were used as control. the identification of hundreds of ubiquitinated Overall, expression levels of biotinylated ubiquitin proteins from neuronal tissues (25, 26). The were similar in both genotypes, as indicated by a system relies on the overexpression of a tagged Western blot performed with biotin antibody using ubiquitin that bears a 16 amino-acid long whole head extracts (see input fraction of Fig. biotinylatable peptide (25, 27), which can be 1C). Similarly, the amount of ubiquitinated biotinylated by the E.coli biotin holoenzyme material eluted from biotin pulldowns on both fly synthetase enzyme (BirA) in neurons in vivo (25, 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.23.916619; this version posted January 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ubiquitinated synaptic NSF lines was also similar (see elution fraction of Fig. did not change significantly either; this included 1C). Avidin (AVD) and proteins known to be Higher levels of Ari-1 enzyme in bioari flies endogenously conjugated with biotin (ACC: should result on an enhanced ubiquitination of acetyl-CoA carboxylase, PCB: pyruvate Ari-1 substrates relative to bioUb flies. Therefore, carboxylase) (25, 34), which serve as internal quantitative proteomic experiments were carried controls to determine the correct processing of the out, following a previously described work-flow biotin pulldowns (Fig. 3, marked in blue). (28, 29), to decipher the ubiquitinated proteome Out of the 1452 proteins quantified (see bioUb from both bioari and bioUb