All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. To prepare primary neuron cultures, WT embryonic day 16 (E16) rat pups were removed from sacrificed Sprague Dawley mothers. Cortices were dissected from pup brains and incubated in dissection media with papain and DNase for 25 min at 37°C and then fully dissociated by gentle trituration. Neurons to be used for APEX2 labeling were then electroporated with the Rat Nucleofector Kit (Amaxa) with 4 μg pCAG_DGKθ-GFP-APEX2 and plated on poly-L-lysine (PLL)-coated 10 cm dishes in Neurobasal Plus medium supplemented with 5% horse serum, 50 U/mL penicillin, 50 U/mL streptomycin, 2 mM Glutamax and 2% b27 Plus at 10 e⁶ cells/dish. WT neurons were plated in the same media at 4 e⁶ cells/dish. At DIV4, all neurons were fed with serum-reduced (2% horse serum) media supplemented with fluoro-deoxyuridine (FDU) to prevent the proliferation of glial cells. Following this, neurons were fed about every 4 days with Neurobasal Plus medium supplemented with 50 U/mL penicillin, 50 U/mL streptomycin, 2 mM Glutamax, and 2% b27 plus. For most experiments, neurons were grown until DIV21 in a 37°C incubator (5%CO₂).

For immunofluorescence experiments, WT rat neurons were plated on PLL-coated 18 mm glass coverslips placed in the wells of a 12 well plate. On DIV19, neurons were transfected with a combination of 0.5 μg pCAG_DGKθ-GFP-APEX2, 0.5 μg LCK-myr-mCherry, or 0.5 μg pRK5-myc-DGKθ. To accomplish this, Lipofectamine 2000 (L2K) was diluted in Neurobasal media and incubated for 5 min. DNA was diluted in Neurobasal media and then combined at a 1:1 ratio with the L2K/Neurobasal dilution and incubated for 15 min at room temperature (RT). 1 mL of media was gently removed from each well of cells to be transfected and saved. The transfection mixture was then added dropwise to cells and incubated for about 13 min at 37°C, 5%CO₂. Following this incubation, the transfection media was aspirated and replaced with 1 mL of the saved media. The next day, the neurons were fixed for staining. Coverslips were washed 1× with phosphate-buffered saline (PBS) and then incubated with parafix (4% paraformaldehyde + 4% sucrose in PBS) for 15 min at RT and washed 3× with PBS. They were permeabilized for 10 min with 0.25% Triton-X in PBS and washed 3× with PBS. To reduce non-specific antibody binding, coverslips were incubated with 10% bovine serum albumin (BSA) at 37°C (5%CO₂) for 1 h. Coverslips were then incubated with primary antibodies diluted in 3% BSA overnight at 4°C. The next day, coverslips were washed 3× with PBS and incubated with secondary antibodies (goat conjugated Alexa Fluor 647, 568, or 488) diluted in 3% BSA in PBS +0.1% Triton-X (PBST) for 1 h at RT. Subsequently, they were washed 3× with PBS and mounted on glass slides with Fluoromount-G and stored at 4°C. Images were obtained with an 800-laser scanning confocal microscope (Zeiss) and images were analyzed with ImageJ.

All SDS-PAGE gels were performed with Bolt 8% Bis-Tris mini protein gels. Eluates from streptavidin pulldowns and CoIPs were loaded directly on gels. Input samples and other neuronal or HEK lysate samples were diluted with water and 4× Laemmli buffer with 10% β-mercaptoethanol and boiled for 5 min at 100°C before loading on gels. Proteins were transferred to nitrocellulose membranes with the Bio-Rad Mini Trans-Blot system and blocked with 3% BSA for 1 h. Blots were incubated with primary antibodies diluted in 3% BSA in PBST on a 4°C rocker overnight. The next day, blots were washed 3× with PBST and incubated with LICOR IRDye-conjugated secondary antibodies for 1 h at RT. Afterward blots were washed 3× with PBST before infrared imaging with an Odyssey Imaging System.

At DIV21, neurons expressing pCAG_DGKθ-GFP-APEX2 were incubated with aCSF supplemented with 500 μM biotin-phenol for 30 min at 37°C. Control neurons not incubated with biotin-phenol were incubated with non-supplemented aCSF for 30 min at 37°C. Immediately after incubation, aCSF was aspirated and 10 mL of 50 mM KCl in aCSF was added to each dish to induce depolarization for 30 s (non-depolarized neurons did not receive this treatment). Following depolarization, 1 mM H₂O₂ was added to each dish of cells for 1 min to catalyze the APEX2 labeling reaction. After 1 min, aCSF containing biotin-phenol and H₂O₂ was aspirated and quencher solution (10 mM sodium ascorbate, 5 mM Trolox, 10 mM sodium azide in DPBS) was added to each dish of cells to quench the labeling reaction. Neurons were washed with this solution 4×. Following the last wash, neurons were harvested in 1 mL RIPA containing protease inhibitors, each of the quenchers, and 50 mM TEABC. Each lysate was incubated on a rotator at 4°C for 20 min and then centrifuged at 17,000 × g for 5 min. The supernatant was transferred to a new tube and placed at −20°C until preparation for proteomic analysis.

Neurons (∼50 e⁶ cells total) from five 10 cm dishes were lysed in RIPA buffer. Proteins in lysates were reduced in 10 mM DTT at 56°C for 30 min and alkylated with 30 mM IAA at RT in the dark for 30 min prior to proteolysis with 1:20 dilution of 1 mg/mL trypsin (Worthington): protein in 50 mM TEABC buffer at 37°C on a shaker overnight. Following trypsin digestion, peptides were precipitated and acidified by adding 20% TFA in dH₂0 to pH4 and incubated at RT for 10 min. Subsequently, lysates were spun at 10,000 × g for 10 min at RT to pellet debris, undigested proteins, and membranes. The supernatant was loaded on a 30 mg Waters Oasis plate (WAT058951) to bind and desalt the peptides according to the manufacturer’s instructions. The peptides were eluted with 2× washes of 500 μL 60% trifluoroacetic acid and subsequently dried in a speed vacuum.

To prepare streptavidin beads, Pierce magnetic streptavidin beads were washed 3× with PBS. A 1:8 ratio was used (mL beads: mg protein). The dried peptides were resuspended completely in 1 mL RIPA and combined with beads. The peptide-bead mixture was incubated overnight on a 4°C rotator. The next day, the beads were washed 4× with 1 mL 1% SDS, 5× 1 mL 5% acetonitrile in dH₂0, and 3× 1 mL dH₂0. Washes were performed by pipetting with low retention tips and after each wash, samples were transferred to a new Eppendorf tube. After the last wash, biotinylated peptides were eluted from streptavidin beads with neat hexafluoroisopropanol (HFIP). For each sample, 600 μL HFIP was added to the tube, incubated for 5 min at RT, then placed on a magnetic rack and the liquid was pipetted off and transferred to a new tube. This was done a second time and the supernatants were combined into one tube for each sample and then dried in a speed vacuum. The dried peptides were kept in −20°C until mass spectrometry analysis.

Peptides were resuspended in 200 μL of 0.1% trifluoroacetic acid loaded and desalted on a Waters Oasis plate C18. After desalting with 0.1%TFA, peptides were eluted in basic (10 mM TEAB) in steps at 5, 10, 25, and 75% acetonitrile. Fractions were dried and rehydrated with 2% acetonitrile, 0.1% formic acid. Each peptide fraction was resuspended in 20 μL loading buffer (2% acetonitrile in 0.1% formic acid) and analyzed by reverse phase liquid chromatography interfaced with tandem mass spectrometry (LC/MSMS) using an Easy-LC 1200 HPLC system¹ interfaced with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Lumos, see text footnote 1). Peptides (20% each fraction) were loaded onto a C18 trap (S-10 μM, 120 Å, 75 μm × 2 cm, YMC, Japan) and subsequently separated on an in-house packed PicoFrit column (75 μm × 200 mm, 15 μ, +/−1 μm tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 μm, 120 Ų) using 2–90% acetonitrile gradient at 300 nl/min over 120 min. Eluting peptides were sprayed at 2.0 kV directly into the Lumos. Survey scans (full MS) were acquired from 350 to 1,400 m/z with data dependent monitoring with a 3 s cycle time. Each precursor individually isolated in a 1.2 Da window and fragmented using HCD activation collision energy 30 and 15 s dynamic exclusion, first mass being 120 m/z. Precursor and the fragment ions were analyzed at resolutions 120,000 and 50,000, respectively, with automatic gain control (AGC) target values at 4 e⁵ with 50 ms maximum injection time (IT) and 1 e⁵ with 54 ms maximum IT, respectively.

Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were processed in Proteome Discoverer (PD) software (v2.4, Thermo Scientific). All data were searched using Mascot (2.6.2³) against a custom database (191113 entries, including custom sequence with DGKθ-GFP-APEX2 protein plus Rattus) using trypsin as the enzyme, 2 missed cleavages, precursor and fragment tolerances or 6 ppm and 0.01 Da, respectively. Variable modifications included Asn and Gln deamidation, Met oxidation, and Cys carbamidomethylation as static modification. Target decoy PSM (peptide spectrum match) validator was used, and any low-scoring spectra were sent to a custom BYONIC search against SwissProt_Rat_032019_8060entries_DGK_Apex database. The enzyme was specified as trypsin, with 2 missed cleavages, precursor and fragment tolerances or 6 and 8 ppm, respectively. Total common modifications 2 max, rare modifications 1 max. Fixed modification of Carbamidomethyl / +57.021464 @ C, Dynamic Modifications of Oxidation / +15.994915 @ M | common1, Deamidated / +0.984016 @ N, Q | common1, Carbamyl / +43.005814 @ K, R | rare1, Acetyl / +42.010565 @ NTerm, Protein NTerm, K | common2, Acetyl / +42.010565 @ NTerm, Protein NTerm, K | common2, Phospho / +79.966331 @ S, T, Y | rare1, Trioxidation / +47.984744 @ W | rare1, Gln- > pyro-Glu / −17.026549 @ NTerm Q | common2, Formyl / +27.994915 @ NTerm, K, S, T | rare1, Dimethyl / +28.0313 @ K, R | rare1, Biotin Tyramide / +361.1460 @ Y | rare1, Biotin Tyramide_Plus1 / +362.149 @ Y | rare1.

Spectra and peptides with high confidence (1% FDR) were used for feature mapping and alignment, RT up to 10 min, Mass tolerance 10 ppm, min S/N threshold 5. Quantification calculated from precursor intensity, using unique peptides only. No scaling or normalization, protein abundance calculated from summed abundances, top 3, pair wise ratio based. Hypothesis test was set to t-test (background based).

For streptavidin pulldowns to confirm biotinylation of proteins, APEX2 labeled neuronal lysates were combined with magnetic streptavidin beads that had been washed 2× with RIPA. Following addition of the lysate to the beads, RIPA was added to the lysate-bead mixture to facilitate mixing and samples were incubated overnight on a 4°C rotator. The following day, the flow-through was pipetted off the magnetic beads and the beads were washed 2× 1 mL RIPA, 1× 1 mL 1 M KCl, 1× 1 mL 0.1 M Na₂CO₃, 1× 1 mL 2 M urea in 10 mM Tris–HCl pH8, 2× 1 mL RIPA. To elute biotinylated proteins from the beads, Laemmli sample buffer containing 6 mM biotin and 60 mM dithiothreitol (DTT) was added to the beads and incubated at 100°C for 15 min. Afterward, the supernatant was collected from the magnetic beads and loaded on gel for Western blot analysis.

All CoIPs were performed using HEK293FT cells. Cells were seeded on 10 cm dishes in DMEM supplemented with 10% fetal bovine serum and 1% Pen-Strep. Transfection was performed when the cells reached about 80% confluency. Lipofectamine 2000 (L2K) was diluted in Opti-MEM and incubated for 5 min at RT. 4 μg of pRK5-myc-DGKθ alone or 4 μg pRK5-myc-DGKθ plus 4 μg of each potential interactor DNA were diluted in Opti-MEM. The DNA and L2K were mixed at a 1:1 ratio and incubated for 15 min at RT. Afterward, the DNA/L2K mixture was added to cells dropwise and the cells were placed in the incubator (37°C, 5% CO₂) for 2 h. Then, the media containing the transfection mixture was aspirated completely from plates and fresh HEK cell media was replaced. The cells were harvested after 2 days. Before lysis, cells were washed 2× with PBS. After the final wash was aspirated, the cells were lysed in 1 mL CoIP buffer (1% NP40, 5 mM NaF, 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA in 1× PBS) and placed on 4°C rotator for 20 min. The lysates were then centrifuged at 17,000 × g for 5 min and the supernatant was transferred to a new tube.

To begin CoIPs, lysates were first precleared by incubating with protein A/G beads for 1 h on 4°C rotator to reduce non-specific binding. After incubation, samples were centrifuged at 10,000 × g and the supernatant was collected and transferred to a new tube. Subsequently, 2 μL of rabbit anti-myc or mouse anti-myc ascites antibodies were added to each sample and incubated overnight on 4°C rotator. The next day, protein A/G beads were added to each antibody/lysate mixture and incubated for 2 h on RT rotator. The flow-through was then removed and beads were gently washed 3× with CoIP buffer by pipetting. After the final wash, beads were incubated in elution buffer (50% CoIP buffer, 50% 4× Laemmli sample buffer) at RT for 15 min and then at 100°C for 15 min. Samples were then briefly centrifuged and the supernatant was transferred to a new tube. Samples were kept at −20°C until samples were analyzed for the presence of the interactor by Western blot analyses.

The ADP-Glo Assay (Promega) was used to examine the ability of each DGKθ interactor to stimulate DGKθ activity (Balzano et al., 2011; Nagaraj et al., 2017). This assay kit employs a fluorescent reagent to quantify the amount of ADP generated by DGKθ’s kinase reaction. The reaction mix consists of 10 ng/μL purified DGKθ, 0.5 mM of the water-soluble DAG substrate 1,2-dihexanoyl-sn-glycerol (Dic6), 1 mM ATP, 3 mM MgCl₂, and 500 ng/μL of the potential activator. The reaction mixture was incubated at 37°C for 10 min and stopped by the addition of an equal volume of the ADP-Glo Reagent. This was incubated at RT for an additional 40 min to deplete the remaining ATP. Kinase Detection Reagent was then added and incubated at RT for 40 min, which converts the kinase-generated ADP to ATP which is quantified using a luciferase/luciferin-based reaction. The resulting luminescence is then measured with a Molecular Devices SpectraMax^® i3x Multi-Mode Microplate Reader.

To gain insight into the mechanism of DGKθ’s modulatory role in compensatory endocytosis, we performed APEX2 proximity labeling with APEX2-tagged DGKθ in cortical rat neurons. Mass spectrometry analysis was used to identify potential interactors, with a particular focus on proteins with a known role in synaptic vesicle recycling. We chose to employ APEX2 proximity labeling because its rapid labeling of proteins (∼1 min) within a small radius (∼10 nm) results in specific identification of interactions and has been previously used in multiple similar contexts (Rhee et al., 2013; Hung et al., 2014, 2016; Lam et al., 2015; Loh et al., 2016; Chung et al., 2017; Han et al., 2017; Martell et al., 2017). To identify specific, functionally relevant DGKθ interactors, we depolarized neurons prior to labeling in live cells for some samples.

We first confirmed the expression, localization, and activity of our DGKθ-GFP-APEX2 construct (Figure 1A). Mature cortical neurons expressing DGKθ-GFP-APEX2 were incubated with biotin-phenol (BP) for 30 min, followed by a 1 min incubation with H₂O₂ to catalyze the labeling reaction. This was performed with or without KCl to induce depolarization (see section “Materials and Methods”) (Figure 1B). The DGKθ-GFP-APEX2 construct expresses well in neurons in both depolarized and non-depolarized neurons, as evidenced by both a GFP and DGKθ antibody. The DGKθ antibody recognizes the construct at 150 kDa and endogenous DGKθ at 100 kDa (Figure 1C). We then tested the BP labeling efficiency of the DGKθ-GFP-APEX2 construct in live neurons. The resulting Western blots showed that APEX2 labeling of DGKθ-interacting proteins is dependent on BP and H₂O₂ and there are no significant differences in labeling efficacy between the depolarized and non-depolarized samples. The detection of endogenously biotinylated proteins is seen in the lysates from control reactions, with BP or H₂O₂ omitted (Figure 1D).

To examine the localization of the construct, mature neuron cultures were transfected with DGKθ, DGKθ-GFP-APEX2, or mCherry. Neurons were fixed and stained with antibodies against DGKθ, GFP, vGlut1, or DsRed. As expected, DGKθ-GFP-APEX2 is expressed cytosolically throughout the cell, as evidenced by near-complete colocalization with DGKθ and the mCherry cell-fill (Figure 1E, lower panel). DGKθ-GFP-APEX2 is also expressed synaptically, illustrated by GFP signal within spines and colocalization with vGlut1 expression (Figure 1E, upper panel). Altogether, these results show that the DGKθ-GFP-APEX2 construct expresses well in neurons, co-localizes with overexpressed DGKθ, robustly labels proteins, and that labeling is dependent on H₂O₂.

To capture changes in DGKθ interactions with neuronal activity, we labeled mature neurons expressing DGKθ-GFP-APEX2 with and without depolarization. The selection of potential interactors was based on interaction changes dependent on depolarization. To identify DGKθ interactors, we conducted three mass spectrometry experiments with the same conditions. The detailed data from each mass spectrometry experiment can be found in Supplementary Tables 1–3 and Supplementary Figure 1. This data is also publicly available in the Proteomics Identifications Database (PRIDE) at http://www.ebi.ac.uk/pride/archive/projects/PXD030495.

Because proteins were trypsinized before enrichment with streptavidin, the site of biotinylation is known for each peptide identified. Importantly, multiple biotinylated peptides corresponding to DGKθ-GFP-APEX2 were identified in each mass spectrometry experiment, serving as our positive control (Figure 2A). Each experiment produced similar numbers of total biotinylated peptides, ∼2,200–2,700, corresponding to ∼550–700 proteins (Figure 2B). Importantly, because we employed label-free quantitation, the relative abundance ratio for each biotinylated peptide is calculated as (abundance in KCl samples)/(abundance in control samples). Figure 2C displays the log₂(abundance ratio) for each identified peptide in one of the three experiments, ranging from those peptides only found in KCl samples, those present in both samples in different amounts, and those peptides only found in control samples. Although some peptides may have only been found in one condition, there were usually other peptides from the same protein that were present in the other condition. Importantly, most of our analysis focused on differences in biotinylation and relative abundance between conditions. There was a high degree of similarity between the data sets from each experiment, increasing confidence in potential DGKθ interactors. A total of 51% of all peptides identified were found in all three experiments, with 17% being the largest amount of peptides only identified in 1 experiment (Figure 2D).

Because the goal of this project is to investigate the mechanism of DGKθ’s newly discovered regulatory role in endocytosis, we focused our analyses on proteins known to have a role in the synaptic vesicle cycle, particularly synaptic proteins. Many of those proteins had some biotinylated peptides found only within the depolarized samples, such as dynamin-1, heat shock cognate 71 (Hsc70), and syntaxin-binding protein 1 (Munc18-1) (Figure 2D). Other proteins had peptides with a high abundance ratio and a high number of PSMs (peptide-spectrum matches), such as calcium calmodulin-dependent kinase II alpha (CaMKIIα). Most of the proteins we identified as potential interactors have biotinylated peptides present in samples with and without depolarization. To select candidate proteins of interest, we first isolated proteins that were biotinylated in at least 2 experiments. Then, we selected proteins with the same biotinylated peptide in the same condition in at least 2 experiments. The final candidate proteins selected were all synaptic or directly involved in vesicle cycling. These proteins either had biotinylated peptides with a relatively high number of PSMs or a significant change in the number of PSMs or the site of biotinylation between depolarized and non-depolarized samples (Figure 3A). Our candidate DGKθ interactors consisted of the synaptic vesicle proteins listed in Figure 3B. Also listed are 1 or 2 corresponding peptides (each detected in all 3 experiments) and the number of PSMs for that peptide found in each condition in experiment 1. Supplementary Figure 1 outlines all of the biotinylated peptides and their number of PSMs for each candidate interactor from each experiment. Cofilin-1, CaMKIIα, protein kinase Cα (PKCα), Hsc70, synaptogyrin-1, SNAP-25 and synaptojanin-1 had an obvious, significant change in peptides and their number of PSMs between depolarized and non-depolarized conditions, while proteins like brain acid soluble protein 1 (Basp1) had peptides with unusually high numbers of PSMs. Clathrin heavy chain 1, dynamin-1, syntaxin-7, Syt1, and Munc18-1 had a combination of both characteristics (Figure 3B and Supplementary Figure 1).

Because APEX2 proximity labeling can label transient non-specific interactors, it was essential to further validate the potential interaction between DGKθ and our candidate interactors. To biochemically validate that our candidate interactors are biotinylated, we performed streptavidin pulldowns with DGKθ-GFP-APEX2-labeled lysates. A streptavidin-conjugated antibody confirmed the presence of biotinylated proteins in the input and pulldown samples from both the depolarized and non-depolarized conditions. Dynamin-1, synaptogyrin-1, Munc18-1, clathrin heavy chain 1, Syt1, CaMKIIα, Hsc70, and syntaxin-7 were detected in the eluates from both depolarized and non-depolarized samples (Figure 4A). Basp1, cofilin-1, PKCα, synaptojanin-1 and SNAP-25 were not detected in these eluates, suggesting that they are not biotinylated by DGKθ-GFP-APEX2 (Supplementary Figure 2). These data suggest that most of our candidate interactors, such as canonical synaptic vesicle recycling proteins Syt1 and Munc18-1, are biotinylated by DGKθ-GFP-APEX2, which complements our mass spectrometry results. Therefore, our list of candidate interactors now includes CaMKIIα, clathrin heavy chain 1, dynamin-1, Hsc70, synaptogyrin-1, Syt1, syntaxin-7, and Munc18-1.

To further confirm the interaction between DGKθ and our candidate interactors, we performed coimmunoprecipitation (CoIP) experiments in HEK293FT cells. Due to the difficulty in obtaining sufficient amounts of protein in neurons compared to HEK cell cultures, we performed these experiments in HEK cells for simplicity and increased sensitivity. HEK293FT cells were transfected with myc-DGKθ alone or together with a candidate interactor. First, we confirmed that our myc antibody efficiently pulled down DGKθ in HEK cells. Significant amounts of DGKθ were found in eluates from DGKθ-overexpressed HEK cell lysates, as visualized by a different myc antibody (Figure 4B). We found that CaMKIIα, Syt1, synaptogyrin-1, dynamin-1, syntaxin-7 and Munc18-1 coimmunoprecipitated with DGKθ when both DGKθ and the candidate interactor are overexpressed in HEK cells. Hsc70 coimmunoprecipitated with DGKθ at its own endogenous expression level as well as when overexpressed (Figure 4C). Control pulldowns were conducted using a non-specific mouse IgG and failed to pulldown any of our confirmed DGKθ interactors (Supplementary Figure 3).

We note that clathrin heavy chain 1 was biotinylated but did not coimmunoprecipitate with DGKθ (Figure 4D). This could be because the interaction is specific to neurons or that the proteins are spatially segregated in HEK cells. Further, for both Hsc70 and clathrin heavy chain 1, there was not an obvious difference in expression between endogenous and overexpression of these proteins. We hypothesize that a difference is not visible because the endogenous expression is robust.

Overall, these results confirm our mass spectrometry studies, providing the first evidence that DGKθ interacts with Hsc70, CaMKIIα, Syt1, synaptogyrin-1, dynamin-1, syntaxin-7 and Munc18-1. All of DGKθ’s newly confirmed interactors have a known role in synaptic vesicle recycling, lending further support to the hypothesis that DGKθ is involved in regulating endocytosis. As we expected the DGKθ activators to contain a polybasic region, we also note that Syt1, dynamin-1 and Munc18-1 had the highest percent basicity of the proteins that coimmunoprecipitated with DGKθ (Figure 5A).

Confirmed DGKθ interactors were examined for their ability to stimulate DGKθ’s kinase activity using the ADP-Glo assay (Balzano et al., 2011; Nagaraj et al., 2017). In this assay, kinase-generated ADP is converted to ATP which is then quantified via a luciferase/luciferin-based reaction. This reaction was performed with purified DGKθ alone (10 ng/μL) and with the addition of the following purified, recombinant proteins of each individual DGKθ interactor (500 ng/μL) validated by CoIP as described above: Hsc70, CaMKIIα, Syt1, synaptogyrin-1, dynamin-1, syntaxin-7, and Munc18-1. We found that the addition of 500 ng/μL Syt1 produced a 10-fold increase in DGKθ activity (arbitrary fluorescence units/50 ng DGKθ) over DGKθ alone, the largest increase of all our confirmed interactors (No DGKθ: 2.44 ± 0.09, DGKθ only: 15.27 ± 1.89, DGKθ+Syt1: 149.99 ± 21.91, DGKθ+Stx7: 56.11 ± 19.32, DGKθ+Syngr1: 43.95 ± 6.35, DGKθ+CaMKIIα: 37.83 ± 4.28, DGKθ+Hsc70: 36.29 ± 5.69, DGKθ+Dnm1: 20.66 ± 1.16, DGKθ+Munc18-1: 18.00 ± 1.07; ×10³ arbitrary fluorescence units, n = 6). It is also noteworthy that addition of syntaxin-7 led to a four-fold increase in DGKθ activity, while synaptogyrin-1, CaMKIIα and Hsc70 all led to a 2.5-fold increase in DGKθ activity. Dynamin-1 and Munc18-1 did not cause activation of DGKθ (Figure 5B). From this data, we conclude that Syt1 is the most probable DGKθ interactor and activator, while syntaxin-7 and synaptogyrin-1, proteins also implicated in vesicle recycling, may also play roles in DGKθ activity.