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• W2921813312 abstract "Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function. Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function. GABA neurons are a critical component of nervous systems across the animal kingdom from mammals (1Huang Z.J. Scheiffele P. GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development.Curr. Opin. Neurobiol. 2008; 18 (18513949): 77-8310.1016/j.conb.2008.05.008Crossref PubMed Scopus (80) Google Scholar, 2Krueger-Burg D. Papadopoulos T. Brose N. Organizers of inhibitory synapses come of age.Curr. Opin. Neurobiol. 2017; 45 (28460365): 66-7710.1016/j.conb.2017.04.003Crossref PubMed Scopus (42) Google Scholar) to simple invertebrates, such as Caenorhabditis elegans (3Schuske K. Beg A.A. Jorgensen E.M. The GABA nervous system in C. elegans.Trends Neurosci. 2004; 27 (15219740): 407-41410.1016/j.tins.2004.05.005Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Zhen M. Samuel A.D. C. elegans locomotion: small circuits, complex functions.Curr. Opin. Neurobiol. 2015; 33 (25845627): 117-12610.1016/j.conb.2015.03.009Crossref PubMed Scopus (100) Google Scholar). They provide essential inhibitory activity within neural circuits. In humans, various dysfunctions in GABA neurons and the imbalance between excitatory and inhibitory neurotransmission contribute to neurodevelopmental disorders (5Ramamoorthi K. Lin Y. The contribution of GABAergic dysfunction to neurodevelopmental disorders.Trends Mol. Med. 2011; 17 (21514225): 452-46210.1016/j.molmed.2011.03.003Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 6Ko J. Choii G. Um J.W. The balancing act of GABAergic synapse organizers.Trends Mol Med. 2015; 21 (25824541): 256-26810.1016/j.molmed.2015.01.004Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, understanding how GABA neuron function is regulated is critical for our understanding of nervous system function and disease. Much remains unknown about molecular mechanisms that preferentially affect GABAergic transmission. Core presynaptic machinery, such as synaptotagmin, the SNARE complex, and active zone proteins, influence both glutamatergic and GABAergic transmission (7Chapman E.R. How does synaptotagmin trigger neurotransmitter release?.Annu. Rev. Biochem. 2008; 77 (18275379): 615-64110.1146/annurev.biochem.77.062005.101135Crossref PubMed Scopus (420) Google Scholar, 8Südhof T.C. The presynaptic active zone.Neuron. 2012; 75 (22794257): 11-2510.1016/j.neuron.2012.06.012Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). A few post-synaptic regulators that preferentially or specifically affect GABAergic transmission are known, including Gephyrin, Neuroligin2, Slitrk3, and GARHLs (9Tretter V. Jacob T.C. Mukherjee J. Fritschy J.M. Pangalos M.N. Moss S.J. The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.J. Neurosci. 2008; 28 (18256255): 1356-136510.1523/JNEUROSCI.5050-07.2008Crossref PubMed Scopus (205) Google Scholar10Poulopoulos A. Aramuni G. Meyer G. Soykan T. Hoon M. Papadopoulos T. Zhang M. Paarmann I. Fuchs C. Harvey K. Jedlicka P. Schwarzacher S.W. Betz H. Harvey R.J. Brose N. et al.Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.Neuron. 2009; 63 (19755106): 628-64210.1016/j.neuron.2009.08.023Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 11Takahashi H. Katayama K. Sohya K. Miyamoto H. Prasad T. Matsumoto Y. Ota M. Yasuda H. Tsumoto T. Aruga J. Craig A.M. Selective control of inhibitory synapse development by Slitrk3-PTPδ trans-synaptic interaction.Nat. Neurosci. 2012; 15 (S1–S2) (22286174): 389-39810.1038/nn.3040Crossref PubMed Scopus (166) Google Scholar, 12Li J. Han W. Pelkey K.A. Duan J. Mao X. Wang Y.X. Craig M.T. Dong L. Petralia R.S. McBain C.J. Lu W. Molecular dissection of Neuroligin 2 and Slitrk3 reveals an essential framework for GABAergic synapse development.Neuron. 2017; 96 (29107521): 808-826.e810.1016/j.neuron.2017.10.003Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar13Yamasaki T. Hoyos-Ramirez E. Martenson J.S. Morimoto-Tomita M. Tomita S. GARLH family proteins stabilize GABAA receptors at synapses.Neuron. 2017; 93 (28279354): 1138-1152.e610.1016/j.neuron.2017.02.023Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In mammals, less is known about presynaptic GABA-specific regulators, but some proteins, such as synapsins, can differentially impact inhibitory transmission compared with excitatory transmission (14Feng J. Chi P. Blanpied T.A. Xu Y. Magarinos A.M. Ferreira A. Takahashi R.H. Kao H.T. McEwen B.S. Ryan T.A. Augustine G.J. Greengard P. Regulation of neurotransmitter release by synapsin III.J. Neurosci. 2002; 22 (12040043): 4372-438010.1523/JNEUROSCI.22-11-04372.2002Crossref PubMed Google Scholar, 15Gitler D. Takagishi Y. Feng J. Ren Y. Rodriguiz R.M. Wetsel W.C. Greengard P. Augustine G.J. Different presynaptic roles of synapsins at excitatory and inhibitory synapses.J. Neurosci. 2004; 24 (15601943): 11368-1138010.1523/JNEUROSCI.3795-04.2004Crossref PubMed Scopus (273) Google Scholar). In C. elegans, core presynaptic components play conserved roles in neurotransmission in the motor circuit, a model circuit with balanced excitatory cholinergic and inhibitory GABAergic neuron function (4Zhen M. Samuel A.D. C. elegans locomotion: small circuits, complex functions.Curr. Opin. Neurobiol. 2015; 33 (25845627): 117-12610.1016/j.conb.2015.03.009Crossref PubMed Scopus (100) Google Scholar, 16Barclay J.W. Morgan A. Burgoyne R.D. Neurotransmitter release mechanisms studied in Caenorhabditis elegans.Cell Calcium. 2012; 52 (22521667): 289-29510.1016/j.ceca.2012.03.005Crossref PubMed Scopus (23) Google Scholar). Like mammals, relatively few proteins are known that preferentially regulate presynaptic GABA function in C. elegans. Nonetheless, the worm motor circuit has proven valuable for identifying molecules that regulate GABA neuron function. Examples include the NPR-1 neuropeptide Y receptor, the SEK-1 MAP2K, the F-box protein MEC-15, and the anaphase-promoting complex (17Vashlishan A.B. Madison J.M. Dybbs M. Bai J. Sieburth D. Ch'ng Q. Tavazoie M. Kaplan J.M. An RNAi screen identifies genes that regulate GABA synapses.Neuron. 2008; 58 (18466746): 346-36110.1016/j.neuron.2008.02.019Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Sun Y. Hu Z. Goeb Y. Dreier L. The F-box protein MEC-15 (FBXW9) promotes synaptic transmission in GABAergic motor neurons in C. elegans.PLoS One. 2013; 8 (23527112)e5913210.1371/journal.pone.0059132Crossref PubMed Scopus (10) Google Scholar19Kowalski J.R. Dube H. Touroutine D. Rush K.M. Goodwin P.R. Carozza M. Didier Z. Francis M.M. Juo P. The anaphase-promoting complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction.Mol. Cell Neurosci. 2014; 58 (24321454): 62-7510.1016/j.mcn.2013.12.001Crossref PubMed Scopus (22) Google Scholar). Recently, we showed the HECT family ubiquitin ligase EEL-1 (enhancer of EfL-1) is expressed broadly in the nervous system but preferentially affects GABAergic presynaptic transmission in the motor circuit of C. elegans (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). At present, it is unknown how EEL-1 regulates GABAergic presynaptic transmission. Our interest in exploring this question was heightened by extensive genetic links between the EEL-1 ortholog HUWE1 (HECT, UBA, and WWE domains containing protein 1) and intellectual disability. These include HUWE1 copy number increases (21Orivoli S. Pavlidis E. Cantalupo G. Pezzella M. Zara F. Garavelli L. Pisani F. Piccolo B. Xp11.22 microduplications including HUWE1: case report and literature review.Neuropediatrics. 2016; 47 (26587761): 51-56PubMed Google Scholar) and missense loss-of-function mutations that cause Juberg-Marsidi-Brooks syndrome and non-syndromic X-linked intellectual disability (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 22Moortgat S. Berland S. Aukrust I. Maystadt I. Baker L. Benoit V. Caro-Llopis A. Cooper N.S. Debray F.G. Faivre L. Gardeitchik T. Haukanes B.I. Houge G. Kivuva E. Martinez F. et al.HUWE1 variants cause dominant X-linked intellectual disability: a clinical study of 21 patients.Eur. J. Hum. Genet. 2018; 26 (29180823): 64-7410.1038/s41431-017-0038-6Crossref PubMed Scopus (53) Google Scholar, 23Friez M.J. Brooks S.S. Stevenson R.E. Field M. Basehore M.J. Adès L.C. Sebold C. McGee S. Saxon S. Skinner C. Craig M.E. Murray L. Simensen R.J. Yap Y.Y. Shaw M.A. et al.HUWE1 mutations in Juberg-Marsidi and Brooks syndromes: the results of an X-chromosome exome sequencing study.BMJ Open. 2016; 6 (27130160)e00953710.1136/bmjopen-2015-009537Crossref PubMed Scopus (34) Google Scholar). To determine how EEL-1 regulates GABAergic transmission, we performed affinity purification proteomics using C. elegans to identify EEL-1 binding proteins. The most prominent EEL-1 binding protein we identified was OGT-1 (O-linked β-N-acetylglucosamine (O-GlcNAc) transferase 1), a conserved glycosyltransferase that modifies protein function in the cytosol, nucleus, and mitochondria (24Kreppel L.K. Blomberg M.A. Hart G.W. Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.J. Biol. Chem. 1997; 272 (9083067): 9308-931510.1074/jbc.272.14.9308Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 25Levine Z.G. Walker S. The biochemistry of O-GlcNAc transferase: which functions make it essential in mammalian cells?.Annu. Rev. Biochem. 2016; 85 (27294441): 631-65710.1146/annurev-biochem-060713-035344Crossref PubMed Scopus (112) Google Scholar26Hart G.W. Nutrient regulation of signaling and transcription.J. Biol. Chem. 2019; 294 (30626734): 2211-223110.1074/jbc.AW119.003226Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In mammals, OGT is expressed in the brain and localizes to presynaptic terminals (27Cole R.N. Hart G.W. Cytosolic O-glycosylation is abundant in nerve terminals.J. Neurochem. 2001; 79 (11739622): 1080-1089Crossref PubMed Scopus (161) Google Scholar, 28Akimoto Y. Comer F.I. Cole R.N. Kudo A. Kawakami H. Hirano H. Hart G.W. Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex.Brain Res. 2003; 966 (12618343): 194-20510.1016/S0006-8993(02)04158-6Crossref PubMed Scopus (69) Google Scholar). Despite prominent OGT-mediated O-GlcNAcylation of synaptic proteins (29Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2006; 5 (16452088): 923-93410.1074/mcp.T500040-MCP200Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), the functional effects of OGT in the nervous system have only recently begun to be explored. OGT regulates mitochondrial motility in neurons (30Pekkurnaz G. Trinidad J.C. Wang X. Kong D. Schwarz T.L. Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase.Cell. 2014; 158 (24995978): 54-6810.1016/j.cell.2014.06.007Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and has been implicated in neurodegenerative disease (31Wani W.Y. Chatham J.C. Darley-Usmar V. McMahon L.L. Zhang J. O-GlcNAcylation and neurodegeneration.Brain Res. Bull. 2017; 133 (27497832): 80-8710.1016/j.brainresbull.2016.08.002Crossref PubMed Scopus (65) Google Scholar). In OGT conditional knockout mice, AgRP (agouti-related protein) and PVN (paraventricular nucleus) neurons are functionally impaired, leading to impacts on fat metabolism and feeding behavior, respectively (32Ruan H.B. Dietrich M.O. Liu Z.W. Zimmer M.R. Li M.D. Singh J.P. Zhang K. Yin R. Wu J. Horvath T.L. Yang X. O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat.Cell. 2014; 159 (25303527): 306-31710.1016/j.cell.2014.09.010Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 33Lagerlöf O. Slocomb J.E. Hong I. Aponte Y. Blackshaw S. Hart G.W. Huganir R.L. The nutrient sensor OGT in PVN neurons regulates feeding.Science. 2016; 351 (26989246): 1293-129610.1126/science.aad5494Crossref PubMed Scopus (93) Google Scholar). While glycosyltransferase activity is the most widely studied OGT activity, a much smaller body of work indicates that OGT can also act as a scaffold protein (25Levine Z.G. Walker S. The biochemistry of O-GlcNAc transferase: which functions make it essential in mammalian cells?.Annu. Rev. Biochem. 2016; 85 (27294441): 631-65710.1146/annurev-biochem-060713-035344Crossref PubMed Scopus (112) Google Scholar). At present, it is unknown whether this transferase-independent function of OGT has a role in the nervous system. To explore the biological relationship between OGT-1 and EEL-1, we expanded upon our proteomic finding that OGT-1 was a putative EEL-1 binding protein with several independent experimental approaches. We biochemically validated the interaction between OGT-1 and EEL-1 and showed that it occurs in C. elegans neurons in vivo. Importantly, this interaction was conserved as it also occurred between HUWE1 and OGT, the orthologous human proteins. Similar to EEL-1, OGT-1 was broadly expressed in the nervous system, including the cholinergic and GABAergic neurons of the motor circuit, and localized to presynaptic terminals in GABA neurons. Results from genetic analysis using an automated behavioral assay and pharmacological manipulation of the motor circuit showed that OGT-1 affects GABA neuron function. Similar phenotypic defects in GABA neuron function were previously observed in eel-1 mutants (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Furthermore, genetic results indicate that OGT-1 functions in parallel to EEL-1 in GABA neurons. Consistent with this, OGT-1 and EEL-1 also act in parallel to affect locomotion. Findings with point mutations that impair catalytic activity show that OGT-1 functions independently of glycosyltransferase activity to affect GABA neuron function, whereas EEL-1 ubiquitin ligase activity is required. Thus, our study reveals the discovery of an OGT-1/EEL-1 protein complex that regulates GABA neuron function and provides the first evidence of a nonenzymatic OGT-1 function in the nervous system. Previously, we used a combination of electrophysiology and behavioral pharmacology to show that EEL-1 regulates GABAergic presynaptic transmission (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). To determine how EEL-1 regulates GABA transmission, we wanted to use affinity purification proteomics to identify EEL-1 binding proteins. As the first step in this process, we developed an automated platform for evaluating motor circuit function using aldicarb pharmacology. Once established, this assay would allow us to rapidly and quantitatively evaluate whether EEL-1 reagents are functional in vivo and suitable for affinity purification proteomics. The C. elegans motor circuit is composed of excitatory cholinergic and inhibitory GABAergic motor neurons that innervate body wall muscles to control contraction and relaxation, respectively (Fig. 1A). This balance of excitation and inhibition allows for coordinated sinusoidal movement of the body. A traditional pharmacological assay for assessing motor circuit function relies upon aldicarb, an inhibitor of acetylcholine esterase (AchE). 2The abbreviations used are: AchEacetylcholinesteraseAchacetylcholineIPimmunoprecipitationNMJneuromuscular junctionFDRfalse discovery rateLSDleast significant differenceANOVAanalysis of variance. By impairing AchE, aldicarb causes accumulation of Ach over time, which leads to excess muscle contraction and gradual paralysis (Fig. 1A). Traditionally, this is measured by assessing C. elegans paralysis while animals are on agar plates containing aldicarb. Aldicarb-induced paralysis on plates is usually assessed manually, but it has been automated (34Mahoney T.R. Luo S. Nonet M.L. Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay.Nat. Protoc. 2006; 1 (17487159): 1772-177710.1038/nprot.2006.281Crossref PubMed Scopus (178) Google Scholar, 35Ramot D. Johnson B.E. Berry Jr, T.L. Carnell L. Goodman M.B. The Parallel Worm Tracker: a platform for measuring average speed and drug-induced paralysis in nematodes.PLoS One. 2008; 3 (18493300)e220810.1371/journal.pone.0002208Crossref PubMed Scopus (212) Google Scholar). We developed an automated, liquid assay that uses MWT (Multi-Worm Tracker) to evaluate locomotion and aldicarb-induced paralysis (Fig. 1B). We simultaneously monitored 20 wells with 4 worms/well (Fig. 1B). We recorded 10 min of baseline movement, added a desired aldicarb dose, and recorded animal speed in response to drug. As expected, WT animals showed dose-dependent paralysis after aldicarb treatment (Fig. 1C). Compared with our experience with manual aldicarb assays on agar plates (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), this automated liquid assay increases throughput and has a large dynamic range that facilitates dose response analysis. acetylcholinesterase acetylcholine immunoprecipitation neuromuscular junction false discovery rate least significant difference analysis of variance. Mutants that have disrupted motor circuit function have altered aldicarb sensitivity (17Vashlishan A.B. Madison J.M. Dybbs M. Bai J. Sieburth D. Ch'ng Q. Tavazoie M. Kaplan J.M. An RNAi screen identifies genes that regulate GABA synapses.Neuron. 2008; 58 (18466746): 346-36110.1016/j.neuron.2008.02.019Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Mahoney T.R. Luo S. Nonet M.L. Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay.Nat. Protoc. 2006; 1 (17487159): 1772-177710.1038/nprot.2006.281Crossref PubMed Scopus (178) Google Scholar). Mutants with impaired cholinergic function accumulate Ach more slowly at the synapse when treated with aldicarb, which results in slower paralysis and resistance to aldicarb compared with WT animals. This is also the case for mutants that affect cholinergic and GABAergic function equally. There are two scenarios that lead to aldicarb resistance. The first is mutants with increased cholinergic function. The second is mutants that have preferentially disrupted inhibitory GABA function, which results in loss of relaxation and faster paralysis in the presence of aldicarb (Fig. 1A). To assess the performance of our automated aldicarb assay, we tested several aldicarb hypersensitive mutants: hypersensitive mutants with increased cholinergic transmission (goa-1 and slo-1), mutants that are defective in GABA biosynthesis (unc-25), and mutants that lack a GABA receptor subunit (unc-49) (17Vashlishan A.B. Madison J.M. Dybbs M. Bai J. Sieburth D. Ch'ng Q. Tavazoie M. Kaplan J.M. An RNAi screen identifies genes that regulate GABA synapses.Neuron. 2008; 58 (18466746): 346-36110.1016/j.neuron.2008.02.019Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 36Wang Z.W. Saifee O. Nonet M.L. Salkoff L. SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction.Neuron. 2001; 32 (11738032): 867-88110.1016/S0896-6273(01)00522-0Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). We also evaluated eel-1 (zu462) deletion mutants, which we previously showed are hypersensitive to aldicarb due to defects in GABAergic presynaptic transmission (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Consistent with prior studies, these mutants were hypersensitive in automated aldicarb assays (Fig. 1D and Figs. S1 and S2A). Hypersensitivity in eel-1 mutants was rescued by an integrated transgene that expressed EEL-1 using the native eel-1 promoter (Fig. 1D and Fig. S2A). It is unclear why the EEL-1 transgene did not fully rescue. This could be because the eel-1 promoter we designed is not ideal for EEL-1 expression, or EEL-1 is not expressed at optimal levels by the integrated multicopy transgene we used. Nonetheless, these results indicate that we have developed an automated liquid aldicarb assay that rapidly and quantitatively assesses motor circuit function. This approach allowed us to assess aldicarb hypersensitivity and rescue in eel-1 mutants. Thus, this assay is suitable for functional evaluation of EEL-1 constructs used for proteomics. The next step toward EEL-1 affinity purification proteomics was to generate an eel-1 protein null allele and evaluate this mutant in automated aldicarb assays. A protein null allele is particularly important to ensure that transgenic EEL-1 used for affinity purification proteomics is not competing with endogenous EEL-1 protein or EEL-1 fragments. We generated bgg1, an eel-1 protein null, using Mos1-mediated deletion to eliminate the entire eel-1 coding sequence, including the HECT ubiquitin ligase domain (Fig. 2, A and B). Importantly, eel-1 (bgg1) mutants were hypersensitive to aldicarb (Fig. 2C), similar to eel-1 (zu462) mutants (Fig. 1D). Next, we used automated aldicarb assays to evaluate rescue for EEL-1 constructs tagged with affinity purification tags. Several transgenes were generated that fused different constructs to a GS (protein G and streptavidin-binding protein) tag. GS was fused to WT EEL-1 or EEL-1 point-mutated at a critical residue (C4144A) required for E3 ubiquitin ligase activity (Fig. 2B) (37Zhao X. Heng J.I. Guardavaccaro D. Jiang R. Pagano M. Guillemot F. Iavarone A. Lasorella A. The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein.Nat. Cell Biol. 2008; 10 (18488021): 643-65310.1038/ncb1727Crossref PubMed Scopus (206) Google Scholar). We refer to the catalytically inactive point mutant as EEL-1 LD (ligase-dead). GS-tagged GFP served as a negative control. All transgenes were driven by the native eel-1 promoter and expressed in the eel-1 (bgg1) protein null background. There were two reasons we included the EEL-1 LD transgene: 1) it remains unclear whether EEL-1 effects on aldicarb sensitivity and GABA transmission are mediated by EEL-1 ubiquitin ligase activity, and 2) we wanted to evaluate whether the EEL-1 LD can biochemically “trap” and enrich EEL-1 ubiquitination substrates in proteomic experiments. As shown in Fig. 2C, GS::EEL-1 significantly rescued aldicarb hypersensitivity of eel-1 (bgg1) mutants. Similar to untagged EEL-1 (Fig. 1D), GS::EEL-1 only partially rescued eel-1. In contrast, rescue was not observed with GS::EEL-1 LD or GS::GFP (Fig. 2C). Previous work showed that locomotion, the behavioral output of the motor circuit, is also impaired in eel-1 mutants (20Opperman K.J. Mulcahy B. Giles A.C. Risley M.G. Birnbaum R.L. Tulgren E.D. Dawson-Scully K. Zhen M. Grill B. The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development.Cell Rep. 2017; 19 (28445732): 822-83510.1016/j.celrep.2017.04.003Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Consistent with this, eel-1 (bgg1) null mutants had defective baseline locomotion while swimming in liquid. Locomotion defects were partially rescued by GS::EEL-1, but not GS::EEL-1 LD or GS::GFP (Fig. S3). Taken together, these results support several conclusions. First, the eel-1 protein null allele, bgg1, is hypersensitive to aldicarb and impairs locomotion in liquid. Second, the GS::EEL-1 affinity purification reagent is functional and rescues eel-1 (bgg1). Finally, failure of GS::EEL-1 LD to rescue defects in eel-1 (bgg1) mutants indicates that EEL-1 ubiquitin ligase activity is required for motor circuit function and locomotion. Our strategy for EEL-1 affinity purification proteomics is portrayed in Fig. 2D. Transgenic animals expressing GS::EEL-1, GS::EEL-1 LD, or GS::GFP (negative control) on an eel-1 (bgg1) protein null background were grown in large-scale liquid culture, harvested, and frozen in liquid nitrogen. Frozen animals were cryomilled in liquid nitrogen–cooled cylinders to obtain micron–scale grindates that facilitated rapid lysis and protein extraction. Whole worm lysates were applied to IgG-Dynabeads to affinity capture protein complexes containing GS-tagged target proteins. Sample quality was assessed and optimized using two parameters: 1) immunoblotting (1% of sample) to confirm GS-tagged target proteins were successfully purified (Fig. S4) and 2) silver staining (9% of sample) to evaluate sample purity and estimate the total amount of purified target (Fig. S4). Using this approach, affinity purification procedures were extensively optimized to obtain as much GS target as possible, while also ensuring that the GS::GFP negative control was as clean as possible compared with GS::EEL-1 and GS::EEL-1 LD test samples. The majority of each sample (90%) was run on SDS-PAGE and subjected to in-gel trypsin digestion, and peptides were identified by LC-MS/MS (Fig. 2D). The most prominent species present in all samples were the affinity purification targets, GS::EEL-1, GS::EEL-1 LD, or GS::GFP (Fig. 2," @default.
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• W2921813312 title "A complex containing the O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 regulates GABA neuron function" @default.
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