An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation

Abstract

Trafficking of G protein-coupled receptors (GPCRs) through the endosomal-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR). We used the GPCR-APEX2/AUR assay to perform a genome-wide CRISPR interference screen focused on identifying genes regulating expression and trafficking of the δ-opioid receptor (DOR). We identified 492 genes consisting of both known and new regulators of DOR function. We demonstrate that one new regulator, DNAJC13, controls trafficking of multiple GPCRs, including DOR, through the endosomal-lysosomal pathway by regulating the composition of the endosomal proteome and endosomal homeostasis.

Extended Data Figure 1:. DOR is amenable to a C-terminal APEX2 tag.

(a) Example of the flow cytometry gating strategy. Cells were gated by size (cells analyzed: 15510), singlets (cells analyzed: 10114), and then analyzed for fluorescence (cells analyzed: 9744). (b) DOR internalization in HEK293-FLP cells stably expressing UBC:DOR or UBC:DOR-APEX2 analyzed following 30 minutes treatment with the agonist DADLE (10 μM). Agonist induced changes in cell surface DOR between treated and untreated cells was probed with anti-FLAG immunoreactivity and measured with a Beckman Coulter Cytoflex S. N=4 independent experiments, and data were analyzed using a two-tailed paired t-test (p=0.0094). (c) DOR signaling was measured as suppression of cAMP and assayed in HEK293-FLP cells stably expressing UBC:DOR or UBC:DOR-APEX2 and transiently expressing pGLO-20F-IRES-RLUC (pGLO). Cells were simultaneously treated with the agonist isoproterenol (30 nM) to stimulate the endogenous B2AR and the DOR agonist DADLE (10 μM). cAMP levels were probed via the pGLO sensor and sensor luminescence was measured using a Tecan Spark, N=3 independent experiments, and data were analyzed using a two-tailed paired t-test. (d,e) DOR proteolysis in HEK293-FLP cells stably expressing UBC:DOR or UBC:DOR-APEX2 were analyzed by western blot (M1 anti-FLAG; constructs have N-terminal FLAG tag). (d) Cells were treated with agonist DADLE (10 μM) for the indicated time before lysis and western blot. (e) Loss of the major band (arrow shown in (d)) was quantified N=3 independent experiments shown as mean ± SD and analyzed by a two-way ANOVA across the time-course (alpha=0.05). (f) HEK293-FLP cells stably expressing UBC:DOR or UBC:DOR-APEX2 were fixed, permeabilized, and probed for DOR (M1 anti-FLAG; constructs have N-terminal FLAG tag) and DNA (DAPI). Wide-field (30 μm scale bar; DOR red and DAPI blue) and zoomed (10 μm scale bar; DOR greyscale) images are shown from a confocal slice taken on an Andor BC43 microscope. White box represents area in wide-field selected for zoom, N=2 independent experiments, representative image shown.

Extended Data Figure 2:. APEX2-based fluorescence biosensor for GPCR downregulation.

(a) Linear range of APEX2/AUR lysate reaction was probed with specific numbers of HEK293-FLP cells stably expressing UBC:DOR-APEX2 or non-expressing control. Standard range for examining DOR trafficking to the lysosome for this cell line shown in brackets. N=3 independent experiments, linear regression fit to the data and constrained to the origin. (b) Tunable signal-to-background of the APEX2/AUR lysate assay was probed by varying the concentration of AUR in HEK293-FLP cells stably expressing UBC:DOR-APEX2-GFP or a non-expressing control. N=3 independent experiments, linear regression fit to the data and constrained to the origin. (c) Comparison of APEX2/AUR and GFP signal-to-background was analyzed in HEK293-FLP cells stably expressing UBC:DOR-APEX2-GFP or a non-expressing control. Assay specific signal was measured in a Tecan Spark plate reader is plotted. N=3 independent experiments, and data were analyzed using a two-tailed paired t-test (p=0.0396). (d) Single cell analysis of APEX2/AUR intact assay (H202, dark grey) or no reaction conditions (no H202, light grey). Cells were analyzed on a BD FACS Aria II, N=2 independent experiments, representative example shown (cells analyzed: 21818 (No H202) or 22062 (H202)).(e) Single cell analysis of stable deposition of reaction product(s) of the APEX2/AUR assay in HEK293 cells stably expressing CMV:DOR-APEX2. Two populations of cells (unstimulated or stimulated by DADLE for 6 hours) were mixed prior to the APEX2/AUR assay, and analyzed on a BD FACS Aria II immediately following the reaction (red) or after 30 minutes on ice (blue). N=2 independent experiments, representative example shown (cells analyzed: 20690 (0 min) or 20794 (30 min)).

Extended Data Figure 3:. APEX2-based fluorescence biosensor for GPCR downregulation.

(a) Uncropped western blots showing knockdown of β-arrestin-2 or total protein stain, N=3 independent experiments. (b) Quantification of β-arrestin-2 knockdown. N=3 independent experiments were analyzed with a one sample, two-tailed, t test (theoretical mean 100, p=0.0039).(c) Uncropped western blots corresponding to 1G showing APEX2 proteolysis or GAPDH loading control. (d). Comparison of signal loss from between lysate- or intact-APEX2/AUR assay from HEK293 cells stably expressing CMV:DOR stimulated with 10 μM DADLE. N=3 independent experiments shown as mean ± SD, fit to a non-linear regression, and analyzed with a two-way ANOVA across the time-course (alpha=0.05, ns across time course and at all timepoints).

Extended Data Figure 4:. Genome-wide CRISPRi screen for genes affecting DOR expression and trafficking.

(a) Relative surface expression of DOR-APEX2 stably expressed from the CMV or UBC promoter, probed with M1 anti-FLAG, and analyzed with a Beckman Coulter Cytoflex S, N=4 independent experiments. (b) Gene ontology overrepresentation test from the 492 hits from the CRISPRi screen analyzed using PANTHER analysis for “Slim Cellular Component” with a Fisher’s exact test with Bonferroni correction for multiple comparisons, and data plotted based on calculated p-value. (c) Relative RNA expression level of genes at the 25th percentile from 12 human nociceptor cell types examining all detected RNAs or CRISPRi hits. Relative expression level analyzed with a two-tailed paired t-test comparing all 12 cell types (p=3.02E-15). (d) Analysis of relative RNA expression of CRISPRi hits in a published dataset of 12 human nociceptor cell types. CRISPRi hits found shown in black, 25th percentile of RNA expression level of the overall dataset shown with the blue line, and percent of CRISPRi hits above the bottom quartile noted in blue text. (e) Effects of β-arrestin-2 targeting sgRNA on DOR trafficking to the lysosome were analyzed in HEK293-FLP cells stably expressing UBC:DOR-APEX2 and dCas9-BFP-KRAB using the APEX/AUR lysate assay with no DADLE stimulation or 2 h DADLE (10 μM) stimulation, N=4 independent experiments, replicates connected by line. Data were analyzed using a two-tailed paired t-test (p=0.0485).

Extended Data Figure 5:. DNAJC13 regulates trafficking of DOR to the lysosome.

(a) Single cell analysis of APEX2/AUR intact assay following knockdown of DNAJC13 and no DADLE treatment or 2 h stimulation with 10 μM DADLE. N=4 independent experiments connected by lines and analyzed using a two-tailed, paired t-test (p=0.002). (b) Uncropped western blot corresponding to Figure 3B. (c) Quantification of DNAJC13 knockdown as analyzed by western blot. N=3 independent experiments were analyzed using a paired one-way ANOVA (alpha: 0.05): p=0.0004. (d) APEX2/AUR lysate assay following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2 with siRNA pool, or individual siRNAs, targeting DNAJC13 or a control. Cells had no DADLE treatment or 2.5 h stimulation with 10 μM DADLE. N=3 independent experiments were analyzed using a paired one-way ANOVA with Dunnett’s multiple comparisons test (alpha: 0.05). Adjusted p-values vs control: pool (0.003), #9 (0.0015), #10 (0.0310). Data for pooled siRNA, #9, and #10 also shown in Figure 3C. (e) Area under the curve (AUC) analysis of Figure 3D. APEX2/AUR lysate assay time-course of 10 μM DADLE stimulation following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2. Cells were treated with 10 μM for 0 to 6 hours. N=3 independent experiments were analyzed with a two-tailed, paired t-test (0.0130). (f) Total DOR expression analyzed by the APEX2/AUR lysate assay comparing knockdown of DNAJC13 to control. N=3 independent experiments were analyzed with a one sample, two-tailed, t test (theoretical mean 100). (g) Surface DOR expression analyzed flow cytometry comparing knockdown of DNAJC13 to control. N=3 independent experiments were analyzed with a one sample, two-tailed, t test (theoretical mean 100). (h) DOR signaling following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2 with siRNA control or siRNA pool targeting DNAJC13. Cells were simultaneously treated with the agonist isoproterenol (30 nM) to stimulate the endogenous B2AR and the DOR agonist DADLE (10 μM). N=3 independent experiments were analyzed using a two-tailed paired t-test.

Extended Data Figure 6:. DNAJC13 regulates trafficking of DOR to the lysosome.

(a) Validation of rabbit anti-DNAJC13 antibody using two populations of cells independently transfected with control siRNA (HEK293-FLP) or a siRNA pool targeting DNAJC13 (HEK293 FLP UBC:DOR-APEX2). Populations were mixed 48 h after transfection, fixed and permeabilized at 72 h, and then probed with anti-FLAG (DOR), anti-DNAJC13, and DAPI. Image is single confocal slice, scale bar is 5 μm, N=3 independent experiments. (b,c) Localization of DNAJC13 (anti-DNAJC13) and VPS35 (anti-VPS35) in HEK293-FLP cells stably expressing UBC:DOR-APEX2 were stimulated with 10 μM DADLE for 15 min. Image is single confocal slice, scale bar 5 μm in (b) and 1 μm in (c). N=3 independent experiments, “Example 1” comes from white box in Extended Data 6B, and “Example 2” comes from an independent replicate. (d) Example of the adjacency scoring metric (A=adjacent, O=overlap). (e) Localization of DOR (anti-FLAG), DNAJC13 (anti-DNAJC13), and VPS35 (anti-VPS35) were analyzed in HEK293-FLP cells stably expressing UBC:DOR-APEX2 stimulated with 10 μM DADLE for 15 min. Image is single confocal slice corresponding to 4E example 1, scale bar 5 μm. N=3 independent experiments, representative example shown.

Extended Data Figure 7:. Loss of DNAJC13 remodels the endosomal DOR proteome.

(a) Volcano plot of fold change (FC) comparing abundance of proteins following DOR-APEX2 mediated proximity labeling in cells transfected with siRNA control or siRNA pool targeting DNAJC13. N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons. Proteins with an FDR <0.1 are denoted as hits (blue). For visualization, volcano plot is cropped and does not show DNAJC13 (log2FC=−1.33; -log10(FDR)=29.8). (b,c) Total reporter ion intensity from TMT mass spectrometry from DOR-APEX2 proximity labeling in control cells or cells with DNAJC13 knockdown. N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons, control vs DNAJC13 (DNAJC13: FDR= 0.162E-30; ARF1/3: FDR=0.0214). (d) Overexpression of AGAP1 analyzed by western blot. AGAP1 band marked with an arrow, non-specific bands marked with stars, total protein stain was used as the loading control, N=3 independent experiments shown.

Extended Data Figure 8:. Loss of DNAJC13 remodels the endosomal DOR proteome.

(a,b) Total reporter ion intensity from TMT mass spectrometry from DOR-APEX2 proximity labeling in control cells or cells with DNAJC13 knockdown. N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons, control vs DNAJC13 (ARP1: FDR= 0.0003; ARF1/3: FDR=0.0881). (c) Overexpression of dominant negative FAM21 (FAM21-DN) analyzed by western blot. Total protein stain was used as the loading control, N=3 independent experiments shown. (d) Area under the curve (AUC) analysis from APEX2/AUR lysate assay time course from 0–6 hours following 10 μM DADLE stimulation in HEK293 cells stably expressing UBC:DOR-APEX2 and transiently expressing empty vector or FAM21-DN. N=4 independent experiments were connected by lines and analyzed with a two-tailed, paired t-test (FAM21-DN: p=0.0258). (e) Western blots showing knockdown of FAM21 detected in HEK293-FLP cells stably expressing UBC:DOR-APEX2 by western blot 72 h after siRNA transfection. Total protein stain was used as the loading control, N=3 independent experiments, representative example shown. (f) APEX2/AUR lysate assay following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2 with siRNA control or siRNA pools targeting DNAJC13 or FAM21A/C. Cells had no DADLE treatment or 2 h stimulation with 10 μM DADLE. N=3 independent experiments were analyzed using a paired one-way ANOVA (alpha: 0.05) with Dunnett’s multiple comparisons test. Adjusted p-values when compared to NTC: DNAJC13 (0.0352), FAM21 (0.2886).

Extended Data Figure 9:. DNAJC13 knockdown causes expansion of DOR endosomes.

(a-c) Fixed and permeabilized HEK293-FLP cells stably expressing UBC:DOR-APEX2 were probed to detect endosomes (anti-EEA1) containing DOR (anti-APEX). Cells were treated with individual siRNAs targeting DNAJC13 or siRNA control for 72 h prior to stimulated with 10 μM DADLE for 15 min. Scale bar 5 μm. N=3 independent experiments, representative example shown. (d) Volume of DOR- and EEA1-positive endosomes measured following knockdown of DNAJC13. Data corresponding to top 10% (90th percentile) of Figure 6D, data from N=3 independent experiments.

Fig. 1:. APEX2-based fluorescence biosensor for GPCR downregulation

(a) GPCR-APEX2 and Amplex UltraRed (AUR) method (b) Agonist-mediated signal loss in the APEX2/AUR lysate assay. HEK293 cells stably expressing CMV:DOR-APEX2 were stimulated with 10 μM DADLE for the indicated duration before the AUR reaction, data normalized to no agonist treatment, N=3 independent experiments. (c) HEK293-FLP cells stably expressing UBC:DOR-APEX2 were treated simultaneously with DADLE (30 nM) and the antagonist naloxone (indicated concentration) for 6h before the AUR reaction and normalized to maximum fluorescence, N=2 (30 nM, 300 nM, 100 μM) or 3 (all other concentrations) independent experiments. (d) Chloroquine pretreatment of HEK293 cells stably expressing CMV:DOR-APEX2 followed by 6 h stimulation with 10 μM DADLE before the APEX2/AUR lysate assay, data normalized to no agonist treatment. N=3 independent experiments were analyzed with a two-tailed, paired t-test (p=0.0068). (e,f) Single cell analysis of APEX2/AUR intact assay after β-arrestin-2 knockdown and 4 h stimulation with DADLE (10 μM), and representative example shown in (e) and in (f) median fluorescence from N=3 independent replicates analyzed with a two-tailed, paired t-test (p=0.0031). Cells analyzed in (e): 21827 (control/no agonist), 21558 (control/agonist), 22601 (siRNA-β-arrestin-2/no agonist), 22363 (siRNA-β-arrestin-2/agonist). (g) Proteolysis of APEX2-tagged DOR detected by western blot following DADLE treatment (10 μM) of HEK293 cells stably expressing CMV:DOR-APEX2, N=3 independent experiments, representative example shown. (h) Quantification of western blot compared to APEX/AUR lysate assay. HEK293 cells stably expressing CMV:DOR-APEX2 were stimulated with 10 μM DADLE. N=3 independent experiments shown as mean ± SD, fit to a non-linear regression, and analyzed with a two-way ANOVA (alpha=0.05, p=0.0002, variation between assays) and Tukey’s multiple comparisons comparing initial proteolysis (western) vs lysate amplex (2 h: p=0.0018; 3 h: p=0.0053, 4 h: p=0.0210; 6 h: p=0.0108) or total proteolysis (western) vs lysate amplex (3 h:p=0.0083). (i) APEX2/AUR lysate assay (solid line) performed over pH range in a universal buffer. Reaction product (dotted line) denotes data in which the assay was performed at standard pH of 7.4 and then diluted into universal buffer at the noted pH. Data were normalized to the maximum fluorescence (pH 6.5), N=3 independent experiments.

Fig. 2:. Genome-wide CRISPRi screen for genes affecting DOR expression and trafficking.

(a-c) (a) Volcano plot of relative gene enrichment in the high (H) and low (L) signal sorted populations from all seven sub-libraries between FACS sorted bottom and top quartiles following APEX2/AUR intact cell reaction. Relative sgRNA enrichment between the populations was analyzed with a Mann-Whitney U test from N=1 independent experiment. Gene-targeting sgRNAs with an FDR <0.05 are denoted as hits (blue), non-targeting control sgRNAs (orange), and all other gene-targeting sgRNAs (open circles). (b) A subset of hits known to function in secretory or endo-lysosomal pathways are highlighted (upper) and hits selected for follow-up (lower). (c) A more comprehensive list of hits known to function in trafficking are diagramed based on primary known location of function. (d) HEK293-FLP cells stably expressing UBC:DOR-APEX2, dCas9-BFP-KRAB, and the noted sgRNA, were analyzed using the APEX2/AUR lysate assay with no DADLE treatment or 1.5 h stimulation with 10 μM DADLE. N=3 independent experiments for all TSS-targeting sgRNAs and N=6 independent experiments for the NTC. Data were analyzed using an unpaired one-way ANOVA (alpha: 0.05) with Dunnett’s multiple comparisons test. Adjusted p-values when compared to NTC-5433: DNAJC13–05 (0.0008), DNAJC13–71 (0.0032), WDR96+47 (0.0329), WDR96–18 (0.0153), SNX24+20 (0.0027), SNX24+26 (0.0123), CPNE+60 (0.0181).

Figure 3:. DNAJC13 regulates DOR trafficking to the lysosome.

(a) Single cell analysis of APEX2/AUR intact assay following knockdown of DNAJC13 and no DADLE treatment or 2 h stimulation with 10 μM DADLE. The fluorescent reaction product in HEK293-FLP cells stably expressing UBC:DOR-APEX2 was detected with a Beckman Coulter Cytoflex S, representative example shown (cells analyzed: 14935 (control) or 14960 (control+DADLE), 14764 (DNAJC13), 14902 (DNAJC13+DADLE)) and summary data from N=4 independent experiments plotted in Extended Data Fig. 5A. (b) Knockdown of DNAJC13 in HEK293-FLP cells transfected with siRNA control, siRNA pool targeting DNAJC13, or individual siRNAs targeting DNAJC13 was analyzed by western blot. Total protein stain was used as the loading control, N=3 independent experiments, representative example shown, uncropped blots in Extended Data Fig. 5B. (c) APEX2/AUR lysate assay following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2 with siRNA control, siRNA pool targeting DNAJC13, or individual siRNAs targeting DNAJC13. Cells had no DADLE treatment or 2.5 h stimulation with 10 μM DADLE. N=3 independent experiments were analyzed using a paired one-way ANOVA with Dunnett’s multiple comparisons (alpha: 0.05). Adjusted p-values vs control: pool (0.0091), 9 (0.0468), 10 (0.0076). (d) APEX2/AUR lysate assay time-course of 10 μM DADLE stimulation following siRNA transfection with HEK293 cells stably expressing UBC:DOR-APEX2. N=3 independent experiments were fit to a non-linear regression and analyzed with a two-way ANOVA (alpha=0.05, p=0.0233 for variation between siRNA treatments across the time-course, at 3 h p=0.0256 between siRNA treatments, all other timepoints=ns). (e) Calculated t_1/2 for the rate of DOR trafficking to the lysosome in cells transfected with DNAJC13-targeting pool or siRNA control. Non-linear regression fit to data in 3D, N=3 independent experiments, were analyzed with a two-tailed, paired t-test (p=0.0051). (f,g) DOR internalization (f) and recycling (g) 72 h after transfection with siRNA control or siRNA pool targeting DNAJC13. Cell surface DOR was probed with anti-FLAG immunoreactivity and measured with a Beckman Coulter Cytoflex S, N=3 independent experiments, and data were analyzed using a two-tailed paired t-test.

Figure 4:. DNAJC13 localizes to DOR positive endosomes.

(a,b) Localization of DOR (anti-FLAG) and DNAJC13 (anti-DNAJC13) were analyzed in HEK293-FLP cells stably expressing UBC:DOR-APEX2 stimulated with 10 μM DADLE for 15 min. Image is single confocal slice, scale bar 5 μm in (a) and 1 μm (b). N=3 independent experiments, “Example 1” comes from white box in 4A, and “Example 2” comes from an independent replicate. (c). Manual scoring of “adjacency” (i.e., <50% signal overlap of the DNAJC13 with either VPS35 or DOR when both signals appear on the same structure). N=3 independent experiments, total structures counted per condition noted. (d) Pearson’s correlation coefficient calculated for DOR/DNAJC13 or VPS35/DNAJC13 from N=3 independent experiments, line denotes median. (e) Localization of DOR (anti-FLAG), DNAJC13 (anti-DNAJC13), and VPS35 (anti-VPS35) were analyzed in HEK293-FLP cells stably expressing UBC:DOR-APEX2 stimulated with 10 μM DADLE for 15 min. Image is single confocal slice, scale bar 1 μm. N=3 independent experiments, “Example 1” comes from white box in Extended Data 6E, and “Example 2” comes from an independent replicate. (h) Imaris software rendering of the surface of a single endosome from the Z-stack images from (4E) “Example 1”. Scale bar is 300 nm.

Figure 5:. Loss of DNAJC13 remodels the endosomal DOR proteome.

(a) Endosome-targeted proximity labeling using DOR-APEX2. (b) Gene ontology overrepresentation test comparing top 101 proteins (filtered by log₂FC>1 and log₁₀FDR<0.1) most enriched in DOR-APEX2 proximity labeling compared to cytoplasmic APEX2 control (CYTO-APEX2). Top 101 hits were analyzed using PANTHER analysis for “Slim Cellular Component” with a Fisher’s exact test with Bonferroni correction for multiple comparisons, and data plotted based on calculated p-value. Initial proteomic comparison of DOR-APEX2 vs CYTO-APEX2 (see Supplementary Table 3) from N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons. (c) Volcano plot of fold change (FC) comparing abundance of proteins following DOR-APEX2 mediated proximity labeling in cells transfected with siRNA control or siRNA pool targeting DNAJC13. N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons. Proteins with an FDR <0.1 are denoted as hits (blue), hits linked to endosomal WASH/actin are highlighted with orange circles, and hits endosomal Arf/ArfGAP are highlighted with red circles. CCDC85C and DNAJC13 are outside the axis-limits of this plot and not displayed (see Supplementary Table 4) (d,f) Total reporter ion intensity from TMT mass spectrometry from DOR-APEX2 proximity labeling in control cells or cells with DNAJC13 knockdown. N=3 independent experiments were analyzed using edgeR’s exact test with Benjamini-Hochberg correction for multiple comparisons, control vs DNAJC13 (AGAP1: FDR= 0.013; FAM21: FDR=0.005). (e,g) Effects of overexpression of AGAP1 or dominant negative FAM21 analyzed by the APEX2/AUR-lysate assay. Cells had no DADLE treatment or 2 h stimulation with 10 μM DADLE. N=5 replicates connected by lines and analyzed compared to empty vector using a two-tailed, paired t-test (AGAP1: p=0.0138; FAM21-DN: p=0.0039).

Figure 6:. DNAJC13 knockdown causes expansion of DOR endosomes.

(a,c) Fixed and permeabilized HEK293-FLP cells stably expressing UBC:DOR-APEX2 were probed to detect endosomes (anti-EEA1) containing DOR (anti-APEX). Cells were treated with siRNA control or siRNA pool targeting DNAJC13 for 72 h prior to stimulated with 10 μM DADLE for 15 min. Image is single confocal slice, scale bar 5 μm in (a) and 1 μm in (c). N=3 independent experiments, “Example 1” comes from white box in 6A, and “Example 2” comes from an independent replicate. Scale bar for Imaris surface rendering is 500 nm. (b) Diameter of DOR-and EEA1-positive endosomes was measured following knockdown of DNAJC13. N=3 independent experiments, control: 701 structures; siDNAJC13: 498 structures, median shown as dotted line and solid lines for the 25^th and 75^th quartiles. (d) Volume of DOR- and EEA1-positive endosomes measured following knockdown of DNAJC13. N=3 independent experiments, control: 3714 structures; siDNAJC13: 3794 structures, median shown as dotted line and solid lines for the 25^th and 75^th quartiles. (e) M2R trafficking to the lysosome analyzed 72 h after transfection with siRNA control or siRNA pool targeting DNAJC13 with the APEX2/AUR assay. Cells had no carbachol treatment or 4h stimulation with 10 μM carbachol. N=5 independent experiments were connected by lines and analyzed using a two-tailed paired t-test (p=0.0058). (f,g) β2AR or MOR recycling 72 h after transfection with siRNA control or siRNA pool targeting DNAJC13. Cell surface DOR was probed with anti-FLAG immunoreactivity and measured with a Beckman Coulter Cytoflex S. N=4 independent experiments were connected by lines data and analyzed using a two-tailed paired t-test (β2AR: p=0.0337, MOR: p=0.0070). (h) Working model describing the role of DNAJC13 in GPCR trafficking to lysosome and downregulation. Loss of DNAJC13 disrupts endosome homeostasis, a disrupted endosomal proteome with changed levels of key endosomal proteins, and impeded trafficking to the lysosome (downregulation) as well as the cell surface (recycling).