A complex containing the O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 regulates GABA neuron function

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.

Introduction

GABA neurons are a critical component of nervous systems across the animal kingdom from mammals (1, 2) to simple invertebrates, such as Caenorhabditis elegans (3, 4). 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 (5, 6). 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 (7, 8). A few post-synaptic regulators that preferentially or specifically affect GABAergic transmission are known, including Gephyrin, Neuroligin2, Slitrk3, and GARHLs (9–13). 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 (14, 15).

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 (4, 16). 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 (17–19).

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 (20). 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 (21) and missense loss-of-function mutations that cause Juberg-Marsidi-Brooks syndrome and non-syndromic X-linked intellectual disability (20, 22, 23).

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 (24–26).

In mammals, OGT is expressed in the brain and localizes to presynaptic terminals (27, 28). Despite prominent OGT-mediated O-GlcNAcylation of synaptic proteins (29), the functional effects of OGT in the nervous system have only recently begun to be explored. OGT regulates mitochondrial motility in neurons (30) and has been implicated in neurodegenerative disease (31). 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 (32, 33). 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 (25). 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 (20). 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.

Results

Measuring C. elegans motor circuit function using an automated aldicarb assay

Previously, we used a combination of electrophysiology and behavioral pharmacology to show that EEL-1 regulates GABAergic presynaptic transmission (20). 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).² 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 (34, 35). 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 (20), this automated liquid assay increases throughput and has a large dynamic range that facilitates dose response analysis.

Figure 1.

Automated behavioral assay shows that eel-1 mutants are hypersensitive to aldicarb. A, depicted is the C. elegans motor circuit composed of excitatory cholinergic and inhibitory GABAergic motor neurons. Balance of contraction and relaxation is required for normal movement (left). Application of aldicarb, an AchE inhibitor, leads to excess cholinergic transmission and paralysis (middle). Mutants with impaired GABAergic transmission, such as eel-1, are hypersensitive to aldicarb and paralyze faster (right). B, schematic showing automated aldicarb assay with MWT. 20 wells are monitored simultaneously. Shown is an image of a single well with four animals (right). MWT plots depict animal swimming over a 1-min timeframe with and without aldicarb treatment (below). C, MWT analysis of aldicarb dose response for WT animals. Shown is the mean of multiple wells for each dose (n = 5–20 wells/dose); significance was determined using two-way ANOVA (dose versus time). D, eel-1 (zu462) mutants are hypersensitive to aldicarb, and transgenic expression of EEL-1 rescues aldicarb hypersensitivity. Shown are means (n = 20 wells/genotype). Inset, mean speed (line) and speed in each well (circles) at the indicated time for each genotype. Comparisons between genotypes represent pairwise two-way ANOVAs. Comparisons in the inset represent Fisher's LSD post hoc test (see Fig. S2A for further statistical analysis). ***, p < 0.001.

Mutants that have disrupted motor circuit function have altered aldicarb sensitivity (17, 34). 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) (17, 36). We also evaluated eel-1 (zu462) deletion mutants, which we previously showed are hypersensitive to aldicarb due to defects in GABAergic presynaptic transmission (20). 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.

Functional assessment of EEL-1 affinity purification proteomic reagents

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).

Figure 2.

Affinity purification proteomics from C. elegans identifies OGT-1 as a putative EEL-1 binding protein. A, schematic of eel-1 gene and eel-1 (bgg1) null allele generated by MosDel. Shown are exons (black), promoter (white), UTRs (gray), and introns (gaps connected by lines). B, schematic of EEL-1 protein with annotated domain of unknown function (DUF), ubiquitin-associated domain (UBA), and HECT ubiquitin ligase domain (HECT). Highlighted is the residue required for catalytic activity (purple). C, automated aldicarb assay showing that eel-1 (bgg1) null mutants are hypersensitive to aldicarb compared with WT. Aldicarb hypersensitivity is rescued by GS::EEL-1 but not GS::EEL-1 LD. GS::GFP is negative control. Shown are means of multiple wells for each genotype (n = 10–30 wells with 4 worms/well). The inset shows mean speed (line) and individual data points at the indicated time. D, work flow for affinity purification proteomics from C. elegans. E, example of LC-MS/MS spectrum identifying OGT-1 peptide sequence from GS::EEL-1 sample. F, comparison of total peptide spectra from four proteomic experiments for proteins identified in GS::EEL-1 sample compared with GS::GFP sample. The dashed line denotes 2× enrichment above GS::GFP negative control. G, comparison of normalized total peptide spectra from four proteomic experiments for GS::EEL-1 LD compared with GS::EEL-1. Highlighted in gold are proteins found exclusively in or enriched in EEL-1 LD. The dashed line denotes the point of equivalency between GS::EEL-1 and GS::EEL-1 LD. H, summary of EEL-1 affinity purification proteomics data. OGT-1 is identified in GS::EEL-1 and GS::EEL- LD but absent in GS::GFP. OGT-1 was identified at similar levels in GS::EEL-1 and GS::EEL-1 LD when samples were normalized to the amount of EEL-1 target. For C, comparisons between genotypes represent pairwise two-way ANOVAs. Comparisons in the inset represent Fisher's LSD post hoc test. For H, significance was determined using Student's t test. ***, p < 0.001; ns, not significant (p > 0.05).

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) (37). 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 (20). 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.

C. elegans proteomics identifies OGT-1 as a putative EEL-1 binding protein

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, F and H). Across four independent proteomic experiments, we identified 23 other proteins that were exclusive to, or enriched ≥2-fold in, the GS::EEL-1 or GS::EEL-1 LD samples compared with GS::GFP (Fig. 2F and Table S1). 13 proteins were either unique to WT GS::EEL-1 or present in both WT GS::EEL-1 and GS::EEL-1 LD samples (Fig. 2, F and G) and Table S1). These proteins represented putative EEL-1 binding proteins. 10 proteins were exclusive to or enriched in the GS::EEL-1 LD sample, which suggested that these proteins could be putative ubiquitination substrates (Fig. 2G and Table S1).

Two putative EEL-1 binding proteins were the most prominent: the glycosyltransferase OGT-1 (Fig. 2, E–H) and UBQ-1 (ubiquitin 1) (Fig. 2, F–H). It was not surprising that UBQ-1 co-purified with EEL-1 for two reasons: 1) HECT family E3 ubiquitin ligases, such as EEL-1, bind directly to ubiquitin and transfer it to substrates (38), and 2) EEL-1 contains a UBA-like domain, which binds polyubiquitin chains. Proteomics with C. elegans EEL-1 has not been performed before, and no EEL-1 binding proteins have previously been reported. Also, none of these putative EEL-1 binding proteins and putative EEL-1 ubiquitination targets have been described for the EEL-1 ortholog HUWE1 in prior studies (37–43).

In contrast to UBQ-1, the cytosolic and nuclear O-GlcNAc transferase OGT-1 was a novel, interesting putative EEL-1 binding protein (Fig. 2, E–H). OGT-1 spectra were found exclusively in GS::EEL-1 and GS::EEL-1 LD samples with sequence coverage of 27 and 41%, respectively (Fig. S5). To determine whether there was an increased number of OGT-1 peptides associated with EEL-1 LD compared with WT EEL-1, we normalized the total number of OGT-1 peptides to the total number of EEL-1 target peptides. Importantly, this was feasible because we performed four blinded, independent proteomic experiments. Normalization indicated that OGT-1 was present at similar levels and not significantly enriched in EEL-1 LD samples compared with WT EEL-1 samples (Fig. 2, G and H). These results suggest that OGT-1 is more likely to be an EEL-1 binding protein than an EEL-1 ubiquitination substrate, although they do not rule out the possibility that OGT-1 is ubiquitinated by EEL-1. Because OGT-1 potentially forms a complex with EEL-1, we prioritized investigating the biological relationship between these two proteins further.

OGT-1 is a conserved EEL-1 binding protein that forms a protein complex in neurons

To validate EEL-1 binding to OGT-1, we used co-immunoprecipitation (co-IP) from C. elegans. Transgenic worms were generated that express a fusion protein of GFP and EEL-1 (GFP::EEL-1) alone or in combination with FLAG epitope–tagged OGT-1 (FLAG::OGT-1). Transgenes were expressed using the rgef-1 promoter, which drives expression in the worm nervous system. When FLAG::OGT-1 was precipitated from whole worm lysates, we observed robust coprecipitation of GFP::EEL-1 (Fig. 3A). Similarly, FLAG::OGT-1 coprecipitated with GFP::EEL-1 but not GFP alone when co-IP was performed in the opposite orientation (Fig. S6).

Figure 3.

OGT-1 is a conserved EEL-1 binding protein that interacts with EEL-1 in neurons. A, GFP::EEL-1 was expressed alone or with FLAG::OGT-1 in the nervous system of C. elegans. Co-IP from transgenic worm lysates shows that GFP::EEL-1 binds FLAG::OGT-1. B, co-IP showing human GFP::HUWE1 binds to human FLAG::OGT when expressed in HEK 293 cells. Shown are representative images from three or more independent experiments.

To determine whether OGT-1 is a conserved EEL-1 binding protein, we tested whether this interaction occurs between HUWE1 and OGT, the sole human orthologs of EEL-1 and OGT-1. We observed co-IP of FLAG-OGT with human GFP-HUWE1 from transfected HEK 293 cells (Fig. 3B). These results indicate that EEL-1 binds OGT-1 in neurons in vivo and that this interaction is conserved between human HUWE1 and OGT.

OGT-1 is expressed in the motor circuit and localizes to GABA presynaptic terminals

We previously showed that eel-1 is expressed broadly in the nervous system, including both cholinergic and GABAergic motor neurons (20). Because proteomic and biochemical results indicate that EEL-1 and OGT-1 form a protein complex (Figs. 2 and 3), we wanted to evaluate ogt-1 expression in the nervous system.

Previous work using an OGT-1 translational GFP reporter showed that OGT-1 is widely expressed in intestine, hypodermis, and neurons (44). To confirm this expression pattern and further characterize ogt-1 neuronal expression, we generated transgenic animals that express an ogt-1 transcriptional GFP reporter. We observed ogt-1 expression broadly in the nervous system, as well as in pharyngeal muscle, intestine, and vulva (Fig. 4A). To determine whether ogt-1 is expressed in the motor circuit, we coexpressed the ogt-1 transcriptional GFP reporter with markers for either cholinergic (P_unc-129 mCherry) or GABAergic (P_unc-25 mCherry) motor neurons. We observed coexpression of GFP with mCherry in both cholinergic (Fig. 4B) and GABAergic motor neurons (Fig. 4C). Thus, ogt-1 is expressed in both cholinergic and GABAergic neurons of the C. elegans motor circuit, similar to eel-1.

Figure 4.

OGT-1 is expressed in cholinergic and GABAergic motor neurons and localizes to GABAergic presynaptic terminals. A, representative epifluorescent images showing that GFP driven by the ogt-1 promoter (P_ogt-1 GFP) is broadly expressed inside and outside the nervous system. B and C, schematic and representative confocal images showing that GFP expressed using the ogt-1 promoter (P_ogt-1 GFP) coexpresses with markers for cholinergic (B) and GABAergic (C) motor neurons. D, schematic and representative confocal images showing that mCherry::OGT-1 colocalizes with synaptic vesicle marker SNB-1::GFP at presynaptic terminals of GABAergic motor neurons. Shown are maximum intensity z-projection (left) and single z-slices (right) from the indicated region (blue box). All scale bars are 20 μm except for the one in the right panel of D, which is 5 μm.

We previously showed that EEL-1 is localized to presynaptic terminals in GABAergic motor neurons (20). Therefore, we evaluated where OGT-1 localizes in GABAergic motor neurons. To do so, we generated transgenic extrachromosomal arrays that use a GABA neuron promoter to express mCherry::OGT-1. This transgenic array was generated on a background that carried a second integrated transgene that expressed the synaptic vesicle marker SNB-1 (Synaptobrevin-1) fused to GFP (SNB-1::GFP) in GABA neurons. We found that mCherry::OGT-1 colocalized with SNB-1::GFP at presynaptic terminals of GABA motor neurons (Fig. 4D). This indicates that OGT-1 is present at presynaptic terminals in GABA neurons, similar to what was previously observed for EEL-1.

OGT-1 functions in GABA neurons to affect motor circuit function

Prior work using both electrophysiology and pharmacological approaches showed that EEL-1 regulates GABAergic presynaptic transmission, thereby impacting motor circuit function (20). Because OGT-1 binds EEL-1 (Figs. 2 and 3A and Fig. S6), is expressed in GABAergic motor neurons (Fig. 4C), and localizes to GABAergic presynaptic terminals like EEL-1 (Fig. 4D), we tested how OGT-1 affects motor circuit function using the automated aldicarb assay described earlier (Fig. 1). We evaluated two ogt-1 mutants, ok430 and ok1474. These are both large insertion/deletions that introduce premature stop codons upstream of the glycosyltransferase domain and are likely null alleles (45, 46). Both ogt-1 (ok430) and ogt-1 (ok1474) mutants were mildly hypersensitive to aldicarb (Fig. 5, A and C and Figs. S2B and S7). Aldicarb hypersensitivity was rescued by Mos1-mediated single-copy insertion (MosSCI) of OGT-1 using the pan-neuronal rab-3 promoter (Fig. 5A and Figs. S2B and S7). Single-copy expression of OGT-1 in GABA neurons using the unc-47 promoter also significantly rescued aldicarb hypersensitivity defects in ogt-1 (ok430) mutants (Fig. 5A and Fig. S2B). Weaker rescue with the GABA neuron promoter compared with the pan-neuronal driver might occur for two reasons. The GABA-specific promoter we used might not express OGT-1 at appropriate levels for rescue. Alternatively, OGT-1 could function in other types of neurons to contribute to aldicarb hypersensitivity. Nevertheless, these results show that OGT-1 functions in GABA neurons to regulate motor circuit function in C. elegans.

Figure 5.

ogt-1 functions in GABA neurons to regulate aldicarb sensitivity and enhances eel-1. Shown are automated aldicarb assays with speed normalized to the buffer-only control group for each genotype. A, ogt-1 (ok430) mutants are hypersensitive to a moderate aldicarb dose (64 μm) compared with WT. Aldicarb hypersensitivity is rescued by transgenic expression of OGT-1 pan-neuronally or in GABA neurons. Shown are means of multiple wells for each genotype (n = 29–30 wells, 4 worms/well). Inset, mean speed (line) and individual data points at the indicated time. B, eel-1 (bgg1) mutants are hypersensitive to low aldicarb dose (8 μm) compared with WT, and hypersensitivity is enhanced in ogt-1; eel-1 double mutants. Shown are means (n = 10–15 wells). C, dose response showing that ogt-1 mutants are hypersensitive to moderate and high doses of aldicarb. Aldicarb hypersensitivity is enhanced in ogt-1; eel-1 double mutants across doses. Shown are mean ± S.E. for normalized speed 35 min after aldicarb exposure (n = 5–15 wells). D, OGT-1 expressed in neurons rescues enhanced aldicarb hypersensitivity of ogt-1; eel-1 double mutants. Shown are means (n = 30 wells for each genotype, 1 worm/well due to ogt-1; eel-1 low brood size). E, automated analysis of swimming speed for indicated genotypes. Swimming defects in eel-1 and ogt-1 single mutants are enhanced in ogt-1; eel-1 double mutants (n = 30–65 wells). Inset, mean speed (line) and individual data points at the indicated time. Comparisons between genotypes represent pairwise two-way ANOVAs. Comparisons in the inset represent Fisher's LSD post hoc test (see Fig. S2B for further statistical analysis). **, p < 0.01; ***, p < 0.001; ns, not significant (p > 0.05).

ogt-1 and eel-1 function in parallel to regulate motor circuit function and locomotion

We showed that OGT-1 functions in GABAergic motor neurons to regulate motor circuit function as assessed by aldicarb pharmacology (Fig. 5A). Because this result is similar to prior findings with EEL-1 (20), we investigated the genetic relationship between ogt-1 (ok430) and eel-1 (bgg1) mutants using automated aldicarb assays. Interestingly, eel-1; ogt-1 double mutants showed enhanced aldicarb hypersensitivity compared with eel-1 single mutants at low aldicarb dose (8 μm) (Fig. 5B). Enhanced hypersensitivity was observed across aldicarb dose response for eel-1; ogt-1 double mutants compared with eel-1 mutants (Fig. 5C). To ensure that enhanced aldicarb hypersensitivity was not due to effects on locomotion, speed was normalized to no drug controls for each genotype for these experiments. Enhanced hypersensitivity to aldicarb displayed by eel-1; ogt-1 double mutants was rescued by using MosSCI to express OGT-1 in the nervous system (Fig. 5D). These results indicate that eel-1 and ogt-1 function in parallel in neurons to affect aldicarb sensitivity.

To further assess how genetic interactions between ogt-1 and eel-1 influence motor circuit function, we analyzed locomotion using MWT to monitor swimming speed. ogt-1 mutants had a mild defect in swim speed compared with WT animals, and eel-1 mutants were moderately impaired (Fig. 5E). Importantly, eel-1; ogt-1 double mutants showed enhanced decreases in swim speed compared with eel-1 single mutants (Fig. 5E). Genetic enhancer effects between ogt-1 and eel-1 were also observed when brood size and viability were evaluated (Fig. S8). Thus, OGT-1 and EEL-1 function in parallel to affect multiple phenotypes with prominent effects on motor circuit function and locomotion.

OGT-1 does not affect synapse formation in GABA motor neurons

Next, we evaluated a straightforward hypothesis that might explain how OGT-1 and EEL-1 function in parallel to regulate GABA neuron function: Do OGT-1 and EEL-1 function in parallel to affect synapse formation in GABA neurons? To test this, we analyzed the distribution and pairing of pre- and postsynaptic markers at GABAergic neuromuscular junctions (NMJs). As a presynaptic marker, we used an integrated transgenic array that expresses SNB-1::GFP in GABA motor neurons (Fig. 6A). To postsynaptically label GABA synapses, we used a single-copy transgene that expresses the GABA receptor subunit UNC-49 fused to TagRFP (UNC-49::RFP) (Fig. 6A). Results from confocal microscopy indicated that pairing of pre- and postsynaptic terminals was normal in ogt-1 (ok430) mutants and eel-1 (bgg1) mutants (Fig. 6B). Similarly, pre- and postsynaptic pairing was normal in eel-1; ogt-1 double mutants (Fig. 6B). Quantitation of the number of SNB-1::GFP puncta showed no defects in single mutants or eel-1; ogt-1 double mutants (Fig. 6C). These results demonstrate that synapse formation is not impaired at GABAergic NMJs of eel-1; ogt-1 double mutants and rules this out as an explanation for enhanced defects in aldicarb hypersensitivity and locomotion in these double mutants.

Figure 6.

GABA synapse formation is not impaired in eel-1; ogt-1 double mutants. A, schematic of GABA NMJs in C. elegans dorsal cord. SNB-1::GFP marks GABA presynaptic terminals and GABA receptor subunit, and UNC-49::RFP marks postsynaptic terminals. B, representative confocal images showing colocalization of presynaptic SNB-1::GFP and postsynaptic UNC-49::RFP is normal for all indicated genotypes. C, quantitation indicates that the number of SNB-1::GFP puncta is not changed for any genotype. Shown are mean ± S.E. (error bars) (n = 38–44 worms). Significance was tested using ANOVA. ns, not significant (p > 0.05). Scale bar, 20 μm.

OGT-1 glycosyltransferase activity is dispensable for motor circuit function

To begin deciphering how OGT-1 affects motor circuit function, we tested whether OGT-1 glycosyltransferase activity is involved. To do so, we evaluated whether point mutations in OGT-1 that impair glycosyltransferase activity can rescue aldicarb hypersensitivity of ogt-1 null mutants. We tested two point mutants, OGT-1 K957M and H612A, that affect conserved residues known to completely abolish and strongly impair glycosyltransferase activity of OGT-1 orthologs, respectively (47–49). Interestingly, we observed similar strong rescue when MosSCI was used to pan-neuronally express WT OGT-1, OGT-1 K957M, or OGT-1 H612A (Fig. 7, A and B). These results demonstrate that OGT-1 functions independent of enzymatic glycosyltransferase activity to regulate motor circuit function.

Figure 7.

OGT-1 glycosyltransferase activity is not required for motor circuit function. Shown are automated aldicarb assays with speed normalized to buffer-only control group for each genotype. Aldicarb hypersensitivity in ogt-1 (ok430) mutants is significantly rescued by pan-neuronal expression of two OGT-1 point mutants that lack catalytic glycosyltransferase activity: OGT-1 K957M (A) and OGT-1 H612A (B) (n = 30 wells, 4 worms/well). C, oga-1 (ok1207) mutants have similar aldicarb hypersensitivity as WT animals. Note that ogt-1 (ok430) and rab-3 (js49) mutants are included as controls for aldicarb hypersensitivity and resistance, respectively. D, dose response showing that oga-1 mutants have similar responses to aldicarb as WT animals. Shown are mean ± S.E. (error bars) for normalized speed 45 min after aldicarb exposure (n = 20 wells). Comparisons between genotypes represent pairwise two-way ANOVAs. Comparisons in the inset represent Fisher's LSD post hoc tests. ***, p < 0.001; ns, not significant (p > 0.05).

As an independent approach to address this question, we asked whether loss of OGA-1 (O-GlcNAcase 1), which has opposing enzymatic activity to OGT-1 (46), affects motor circuit function. If motor circuit function is affected by O-GlcNAcylation, oga-1 mutants would be expected to have the opposite phenotype to ogt-1 mutants. In the case of automated aldicarb assays, this would be resistance to aldicarb paralysis rather than hypersensitivity displayed by ogt-1 mutants (Fig. 5, A and C). To ensure that our automated aldicarb assay can accurately evaluate aldicarb resistance, we tested rab-3 mutants, which are mildly resistant to aldicarb (34). Indeed, rab-3 mutants were resistant to aldicarb compared with WT animals in our automated liquid assay (Fig. 7, C and D). In contrast, oga-1 null mutants had similar aldicarb sensitivity to WT animals across a range of doses (Fig. 7, C and D). Taken together, these results with catalytically inactive OGT-1 point mutants and oga-1 mutants suggest that OGT-1 does not rely upon glycosyltransferase activity to affect motor circuit function.

Discussion

OGT-1 forms a protein complex with EEL-1 and affects GABA neuron function

We previously showed that the EEL-1 E3 ubiquitin ligase, a gigantic protein broadly expressed in the nervous system, plays an important role in motor circuit function by regulating presynaptic GABAergic transmission (20). We now provide evidence that EEL-1 ubiquitin ligase activity affects aldicarb sensitivity and locomotion, and therefore is likely to be involved in this process (Fig. 2C and Fig. S3).

To begin further deciphering how EEL-1 regulates GABAergic transmission, we used affinity purification proteomics from C. elegans to identify EEL-1 binding proteins. This approach identified the OGT-1 O-GlcNAc transferase as a prominent EEL-1 binding protein (Fig. 2, E–H). Biochemical results from C. elegans indicate that OGT-1 binds EEL-1 in neurons in vivo (Fig. 3A and Fig. S6). Like EEL-1, OGT-1 is expressed broadly in the nervous system, including the motor circuit, and localizes to GABAergic presynaptic terminals (Fig. 4). Results from an automated pharmacological assay showed that OGT-1 functions in GABAergic motor neurons to affect motor circuit function (Fig. 5A and Fig. S2B). Interestingly, our genetic results show that defects in motor circuit function are enhanced in ogt-1; eel-1 double mutants (Fig. 5, B–D). Consistent with this, enhanced defects in locomotion were also observed in eel-1; ogt-1 double mutants (Fig. 5E). Taken together, our results and prior findings support the conclusion that OGT-1 and EEL-1 form a complex and function in parallel to affect GABA neuron function, thereby influencing the motor circuit and locomotion (Fig. 8). In the future, more extensive physiological and cellular studies will be useful to further test this model. Nonetheless, it is noteworthy that our findings are the first example of OGT-1 or its orthologs forming a complex with a ubiquitin ligase to affect a common process in any system.

Figure 8.

OGT-1 and EEL-1 form a protein complex and regulate GABA neuron function in C. elegans motor circuit. Shown is a summary of our findings that OGT-1 and EEL-1 form a complex in vivo in C. elegans neurons. OGT-1 and EEL-1 are expressed in both cholinergic and GABAergic motor neurons but function in parallel to regulate GABA neuron function.

Two observations suggest that OGT-1 may not be ubiquitinated by EEL-1. First, EEL-1 affinity purification proteomics was done with two EEL-1 constructs, WT EEL-1 and EEL-1 LD (Fig. 2). Because EEL-1 LD cannot ubiquitinate targets, it potentially enriches ubiquitination substrates. Consistent with this, we detected several proteins that were exclusive to or enriched in EEL-1 LD samples, which represent putative ubiquitination substrates (Fig. 2G and Table S1). In contrast, OGT-1 was not significantly enriched in normalized EEL-1 LD samples (Fig. 2, G and H). The second argument is based on genetics. We observed that aldicarb hypersensitivity was enhanced in ogt-1; eel-1 double mutants (Fig. 5, B–D), which indicates that ogt-1 and eel-1 function in parallel to regulate GABA neuron function. If EEL-1 ubiquitinates OGT-1, we would expect a genetic relationship reflective of same pathway genetics, such as suppression or no increased effects in double mutants, neither of which occurred. Thus, our findings with affinity purification proteomics and loss-of-function genetics do not rule out the possibility that OGT-1 is ubiquitinated by EEL-1 but do suggest that this is unlikely in the context of GABA neuron function.

At present, the function of C. elegans OGT-1 and mammalian OGT in the nervous system remains minimally explored. Our results show that OGT-1 is expressed broadly throughout the nervous system, including both excitatory cholinergic and inhibitory GABAergic neurons in the motor circuit (Fig. 4, A–C). This suggests that OGT-1 could have wide-ranging functions in the nervous system and could affect both excitatory and inhibitory motor neuron function. However, ogt-1 mutants are hypersensitive to aldicarb, and hypersensitivity is rescued by OGT-1 expression in GABA neurons (Fig. 5A and Fig. S2B). Furthermore, aldicarb hypersensitivity in ogt-1 mutants is enhanced by eel-1, a known regulator of presynaptic GABAergic transmission (20). This suggests that the OGT-1/EEL-1 complex is likely to preferentially affect GABA neuron function. Consistent with this, a prior study in mammals showed that OGT regulates the activity of GABAergic AgRP neurons (32). Moving forward, it will be important to address whether the OGT-1/EEL-1 complex is a conserved regulator of GABA neuron function and determine how this complex regulates GABA neuron function.

As an initial foray into addressing this question, we show that OGT-1 does not require enzymatic glycosyltransferase activity to regulate motor circuit function. This is supported by results using two different conserved mutations that impair OGT-1 glycosyltransferase activity, one of which completely abolishes enzymatic activity (Fig. 7, A and B). Consistent with this, motor circuit function was not affected by OGA-1, which removes O-GlcNAc and opposes the activity of OGT-1 (Fig. 7, C and D). While prior studies have shown that OGT-1 has nonenzymatic activities, such as acting as a scaffold protein, these remain understudied (25). Importantly, it is unknown whether OGT has nonenzymatic functions in the nervous system. Our results using an in vivo model system now suggest that OGT-1 functions independent of glycosyltransferase activity and in a complex with EEL-1 to regulate GABA neuron function.

Interestingly, many of our observations on the OGT-1/EEL-1 complex share similarities with prior findings on the OGT/mSin3A complex that represses transcription (50). In both cases, OGT glycosyltransferase activity is dispensable, and OGT acts in parallel to the enzymatic activity of its binding partner. Thus, our work and this prior study highlight core biochemical principles at play in OGT complexes that act independently of glycosyltransferase activity.

Implications of the OGT/HUWE1 complex in cell biology and disease

Our discovery of the OGT-1/EEL-1 complex could have broad implications within the nervous system, outside the nervous system, and across species. We found that the physical interaction between OGT-1 and EEL-1 is conserved between the human orthologs, OGT and HUWE1 (Fig. 3B). Thus, the interaction between OGT and HUWE1 is conserved in human cells and could be functionally relevant across species. Consistent with our findings, two prior affinity purification proteomic studies with HEK 293 cells hinted at the possibility of an OGT/HUWE1 complex (51, 52). We now show that an OGT/HUWE1 complex exists, the interaction between OGT and HUWE1 is evolutionarily conserved, and these molecules have functional genetic interactions in C. elegans that affect GABA neuron function and locomotion.

Consistent with our results, previous studies that examined OGT or HUWE1 independently indicate that these molecules converge on several cellular functions as well as during disease. Both OGT and HUWE1 (also called Arf-BP1/Mule) are implicated in oncogenesis and regulate the transcription factor Myc (53, 54). In the nervous system, both HUWE1 and OGT affect neural progenitor proliferation (37, 40, 55, 56) and mitochondrial function (30, 57–59). Moreover, genetic changes in both HUWE1 and OGT are linked to intellectual disability. Human genetic studies have shown that both increases in copy number and missense mutations in HUWE1 result in intellectual disability (21–23). Because some disorder-associated point mutations in HUWE1 are loss-of-function, it is likely that both increased and reduced HUWE1 function are associated with intellectual disability (20). In recent human genetic studies, three different mutations in OGT were suggested to cause intellectual disability (60, 61). Thus, our discovery of a conserved, functionally relevant OGT-1/EEL-1 complex not only informs our understanding of how GABA neuron function is regulated in a model circuit, but could have important implications for oncogenesis, neural progenitor proliferation, mitochondrial function, and intellectual disability.

Experimental procedures

C. elegans genetics and transgenics

C. elegans were maintained using standard protocols (62) and were cultivated at 23 °C for experiments unless noted otherwise. WT strain was N2 isolate. Mutant alleles included goa-1 (n363), slo-1 (js379), unc-25 (e156), unc-49 (e407), unc-119 (ed3), pha-1 (e2123), eel-1 (zu462), eel-1 (bgg1), ogt-1 (ok430), ogt-1 (ok1474), oga-1 (ok1207), rab-3 (js49). Transgenic alleles included ttTi16881 [Mos1], ttTi5605 [Mos1], bggIs16 [P_eel-1 EEL-1], bggIs31 [P_eel-1 GS::EEL-1], bggIs32 [P_eel-1 GS::GFP], bggIs33 [P_eel-1 GS::EEL-1(C4144A)], bggSi2 [P_rab-3 OGT-1], bggSi19 [P_unc-47 OGT-1], bggSi21 [P_rab-3 OGT-1 H612A], bggSi22 [P_rab-3 OGT-1 K957M], juIs1 [P_unc-25 SNB-1::GFP], krSi2 [P_unc-49 UNC-49B::TagRFP]. For experiments with eel-1; ogt-1 double mutants, eel-1 was balanced using tmC25 [tmIs1241] to maintain the strain because of defects in brood size and viability (Fig. S8). As controls, WT animals and single mutants were also balanced. Homozygous unbalanced strains were selected for at least two generations before testing to avoid maternal effects.

Construction details for transgenic alleles are described in Table S2 and were made using standard procedures (63, 64). Plasmid details and sequences are available upon request.

Automated assays for aldicarb and swim speed

Synchronized young adults (20 h after L4) were loaded into 20 wells (4 animals/well) with 20 μl of assay buffer (0.01% Tween 20 in M9). Baseline behavior was recorded for 10 min, 10 μl of aldicarb was added at the desired concentration, and recording continued. Behavioral recordings were taken using MWT (65) with a Toshiba-Teli Ultra high-resolution 12 megapixel CMOS sensor camera-link camera (CSC12M25BMP19-01B) and Qioptiq Rodagon-F 50-mm f/2.8 lens (0703-089-000-24).

Custom-written scripts calculated the mean speed every minute per well. For Figs. 1 and 2, speed was normalized to baseline (10 min of swimming in the absence of aldicarb) for each genotype. For Fig. 5, speed was normalized to no aldicarb controls for the length of the experiment to rule out effects of a given genotype on locomotion. This was particularly important for eel-1; ogt-1 double mutants that displayed enhanced defects in both aldicarb paralysis (Fig. 5, B–D) and swimming speed in the absence of aldicarb (Fig. 5E). Note paralysis threshold was defined as at or below 0.02 mm/s. For locomotion analysis in Fig. 5E, speed across all time points was normalized to the first 5 min of the assay for each genotype.

C. elegans affinity purification proteomics

Animals were liquid-cultured for 4 days (66), harvested by centrifugation, separated on 30% sucrose flotation, and washed three times. Packed worms were resuspended in an equal volume of 50 mm NaCl and frozen as pellets in liquid N₂. Pellets were cryomilled (Retsch) with protease inhibitors (Complete, Roche Applied Science) and lysed in 3× volume lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1.5 mm MgCl₂, 10% glycerol, 0.1% Nonidet P-40, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 1.5× Halt Protease Inhibitor Mixture (Thermo Scientific), 1 mm sodium orthovanadate, 5 mm sodium fluoride, 1 mm sodium molybdate). Lysate was rotated for 10 min at 4 °C and centrifuged at 20,000 × g for 10 min.

Volume of lysate required for 200 mg of total protein was incubated with 400 μl of IgG-coated Dynabeads (Invitrogen) for 1 h at 4 °C. Following affinity capture, beads were washed five times with lysis buffer. Purification quality was assessed by immunoblotting 1% of sample (anti-SBP antibody, Sigma-Aldrich) and silver staining 9% of the sample (Thermo Scientific). The remaining 90% of the sample was run on SDS-PAGE, subject to in-gel trypsin digestion, and run on LC-MS/MS. Prior to MS analysis, pooled peptides were acidified, desalted with a Zip-Tip C18 column, dried, and resuspended in 100 μl of 0.1% formic acid. 13 μl of sample was used per MS run. Samples were analyzed using an Orbitrap Fusion^TM Tribrid^TM mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1000 system. Peptides were eluted on an analytical RP column (0.075 × 250-mm Acclaim PepMap RLSC nano Viper, Thermo Fisher Scientific), operating at 300 nl/min using the following gradient: 5–25% B for 40 min, 25–44% B for 20 min, 44–80% B in 10 s, 80% B for 5 min, 80–5% in 10 s, and 5% B for 20 min (solvent A: 0.1% formic acid (v/v); solvent B: 0.1% formic acid (v/v), 80% CH3CN (v/v) (Fisher)). The Orbitrap Fusion was operated in a data-dependent MS/MS mode using the 10 most intense precursors detected in a survey scan from 380 to 1,400 m/z performed at 120,000 resolution. Tandem MS was performed by HCD fragmentation with normalized collision energy of 30.0%.

Spectra were analyzed using Mascot (Matrix Science) and Sequest (Thermo Fisher Scientific). Mascot and Sequest were set up to search the C. elegans proteome (UniProt, May 2017, 27,483 entries). Analysis parameters included fragment ion mass tolerance of 20 ppm (Mascot) or 0.02 Da (Sequest) and parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified as a fixed modification, and deamidation of asparagine and glutamine and oxidation of methionine were specified as variable modifications.

Scaffold (Proteome Software) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >5.0% probability to achieve a false discovery rate (FDR) < 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at >98.0% probability to achieve an FDR <1.0% and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (67). EEL-1 binding proteins were identified using the following criteria: 1) the protein was detected in at least two of the four experiments; 2) the protein had 2× or more total spectra in the GS::EEL-1 or GS::EEL-1 LD samples compared with GS::GFP negative control; 3) ribosomal proteins were removed. Spectra were normalized by subtracting spectra from negative control (GS::GFP) and dividing by the target (EEL-1).

Biochemistry

For C. elegans biochemistry, animals were grown, processed, cryomilled, and lysed as discussed for proteomics. For biochemistry with HEK 293-T cells, cells were transfected with FLAG-OGT (pBG-GY845, 3 μg) and/or GFP-HUWE1 (pBG-GY896, 14 μg) using Lipofectamine 3000 (Invitrogen). OGT cDNA (NP_858059.1) was purchased (MGC, Dharmcon). HUWE1 cDNA was purchased (Addgene, catalog no. 37431), and two mutations were repaired to match HUWE1 (NP_113584.3). Cells were transfected initially with FLAG-OGT and 12 h later with GFP-HUWE1. 24 h after transfection, cells were lysed in buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10% glycerol, 0.1% Nonidet P-40, 1 mm DTT, and 1× HALT protease inhibitor mixture).

Lysates (10 mg of total protein C. elegans, 0.65 mg of total protein 293 cells) were incubated with primary antibody for 30 min and precipitated for 1 h (C. elegans) or 4 h (293 cells) with 10 μl of protein G–agarose (Roche Applied Science) at 4 °C. Antibodies used for IP included mouse monoclonal anti-FLAG (M2, Sigma-Aldrich) and anti-GFP (3E6, Invitrogen). Precipitates were boiled in sample buffer and run on 3–8% Tris acetate gels (Invitrogen). Gels were transferred overnight to polyvinylidene difluoride membranes and immunoblotted with either rabbit polyclonal anti-FLAG (Cell Signaling) or mouse monoclonal anti-GFP (Roche Applied Science) antibodies. Proteins were visualized using horseradish peroxidase–conjugated secondary antibodies (GE Healthcare Life Sciences, Fisher Scientific) and ECL (Supersignal West Femto or Pico, Thermo Scientific). Blots were imaged with X-ray film for C. elegans experiments and digitally imaged for 293 experiments (KwikQuant^TM Imager).

Microscopy

Epifluorescence microscopy was performed using a Leica CFR5000 with a ×40 magnification oil-immersion lens. Images were acquired using a CCD camera (Leica DFC345 FX). Confocal microscopy was performed using a Leica TCS SP8 MP confocal microscope system with a ×25 or ×40 water immersion lens. Settings that avoided bleed-through between different channels were confirmed when collecting coexpression and colocalization. For colocalization experiments, images were acquired in resonant mode.

Young adult animals were anesthetized and mounted on 2% agar for imaging. When colocalization was being evaluated, animals were anesthetized with 5 mm levamisole in M9 buffer. Epifluorescent microscopy was used to quantitate SNB-1::GFP puncta with animals anesthetized using 1% 1-phenoxy-2-propanol in M9 buffer.

Brood size and viability assay

Individual L4 larvae were placed on fresh plates every 12 h for 4 days at 20 °C. Brood size was total eggs laid per animal over 4 days. Eggs were cultivated at ∼23 °C for 3 days, and viability was measured as the percentage of eggs that developed to L4 stage or older.

Statistical analysis

For aldicarb and swim speed experiments, data were derived from multiple wells acquired across multiple independent experiments. Comparisons were made using two-way ANOVAs. If a significant effect or interaction was observed (p < 0.05), Fisher's LSD post hoc tests were performed to evaluate differences. Individual time points that best represented differences between genotypes were chosen for presentation as insets, but post hoc comparisons at all time points were also done (e.g. see Fig. S2). For proteomics, comparisons were made using Student's t test. For quantitation of SNB-1::GFP puncta, images were collected for each genotype from three independent experiments, and significance was tested using ANOVA. For brood size and viability, significance was assessed using ANOVA and Fisher's LSD post hoc tests.

Author contributions

A. C. G. designed, performed, and analyzed experiments using C. elegans. M. D. performed HEK 293 experiments. K. J. O. designed and generated eel-1 (bgg1), constructed plasmids, isolated transgenic strains, and advised on design and implementation of affinity purification proteomics. R. T. constructed plasmids and isolated mutant and transgenic strains. M. J. M. constructed plasmids. B. G. provided supervision, aided in experimental design, and oversaw project design and implementation. A. C. G. and B. G. wrote and edited the manuscript.