Fig 2: Identification of endoplasmic reticulum (ER)-localized lipid hydrolase, ABHD16A, by proximal proteomics.(A) Cartoon diagram and domain organization of V5-TurboID-Mfn1 and V5-TurboID-Mfn1E209A (human Mfn1 and indicated amino acid numbers). V5-tagged TurboID was added to the N-terminus of Mfn1. Red E209A indicates the catalytically dead (CD) mutation created in the GTPase domain. TMD: transmembrane domain. (B) Representative images and insets of a HeLa cell expressing GFP-Mfn1 (green), mito-BFP (blue), and immunostained with antibody against the V5 tag (magenta, to detect Mfn1 constructs). Yellow arrows indicate GFP-Mfn1 puncta along mitochondria. (C) Cartoon diagram of the strategy used in HeLa cells to biotinylate ER-mitochondria membrane contact site (MCS) proteins with V5-TurboID-Mfn1 vs. V5-TurboID-Mfn1E209A. OMM: outer mitochondrial membrane (blue); IMM: inner mitochondrial membrane (light blue); Drp1: Dynamin-related protein 1 (fission machinery, pink); Mfn1: Mitofusin 1 (fusion machinery, orange); B: biotin. (D) Immunoblot analysis (anti-V5) shows relative expression of V5-TurboID-Mfn1 and V5-TurboID-Mfn1E209A in HeLa cells. (E) Immunoblot analyses of fractions collected by differential centrifugation including: 7000× g pellet containing mitochondria (TOM20, mitochondria), 20,000× g pellet containing light membrane and ER (Calnexin, ER), and supernatant containing cytosol (GAPDH, cytosol). (F) The 20,000× g pellet was solubilized and probed with streptavidin HRP to reveal biotinylation profiles for each sample prior to mass spectrometry. Asterisk denotes a band size indicative of construct self-biotinylation. (G) Average LFQ intensities of proteins biotinylated and enriched in Mfn1 wild type (WT) vs. Mfn1E209A sample in the 20,000× g pellet. Dashed line indicates equivalent enrichment in the WT vs. E209A mutant sample. Data from LFQ intensity are the average of two technical replicates. CANX: Calnexin; RTN4: Reticulon-4; MFN1: Mitofusin-1; OSBPL8: Oxysterol binding protein like 8; ABHD16A: Alpha/beta hydrolase domain containing phospholipase 16 A. (H) Cartoon diagram of human ABHD16A with motif or domain annotations. (I) Representative inset of a U-2 OS cell expressing low levels of ABHD16A-mNG (green), mCh-Sec61β (ER, blue), and mito-BFP (mitochondria, magenta). Arrows indicate ABHD16A localization to ER (blue) and mitochondria (magenta). Scale bar = 5 µm or 2 µm for insets. See Figure 1—source data 1, Figure 1—source data 2, Figure 1—source data 3, Figure 1—source data 4, Figure 1—source data 5, Figure 1—source data 6, Figure 1—source data 7, Figure 1—source data 8, Figure 1—source data 9, Figure 1—source data 10. Figure 1—source data 1.Related To Figure 1D. Figure 1—source data 2.Related to Figure 1E. Figure 1—source data 3.Related to Figure 1F. Figure 1—source data 4.Related to Figure 1G. Figure 1—source data 5.Related to Figure 1D. Figure 1—source data 6.Related to Figure 1D. Figure 1—source data 7.Related to Figure 1E. Figure 1—source data 8.Related to Figure 1E. Figure 1—source data 9.Related to Figure 1F. Figure 1—source data 10.Related to Figure 1F.

Fig 3: Sec22b MCS tethering regulates phagosomal maturation.a Phagolysosome fusion analysis (PLF index) in phagocytic shCTR and shSec22b MEFs loaded with FRET acceptor AF594-HA and exposed to FRET donor-coupled AF488-IgG-beads for 90 min. a Images show representative blue-green pseudocolor of the maximum projection of FRET signal stacks (480/630 ex/em, 15 planes/0.8 µm spacing) in shCTR and shSec22b (n = 6/6 biologically independent samples, 1071/1060 phagosomes shCTR/shSec22b; see also Supplementary Fig. S5a, bar = 10 µm). b Phagosomal pH in shCTR and shSec22b phagocyotic MEFs 45, 60 and 90 min after exposure of cells to IgG-FITC-RBC. (n = 6/6 biologically independent samples, 4163/3049 phagosomes) c PLF index in phagocytic shCTR and shSec22b MEFs transfected with GFP-KDEL, shR-EGFP-Sec22b or shR-EGFP-Sec22b-P33. (n = 10;5/6/6 biologically independent samples, 4757;1363/1426/974 phagosomes for shCTR+KDEL; shSec22b+KDEL /+Sec22b/+P33. See also Supplementary Fig. S5b–d). d PLF index in phagocytic shSec22b MEFs transfected with GFP-KDEL, shR-EGFP-Sec22b, EGFP-MAPPER, EGFP-ORP8 or EGFP-ORP8-Mut. (n = 11/6/15/7/7 biologically independent samples, 4311/2896/476/2080/566 phagosomes KDEL /Sec22b/ MAPPER/ ORP8/ ORP8-Mut. See also Supplementary Fig. S5e). e Quantification of mean phagosomal antigen intensity (anti-EPEA) expressed as a percentage of the mean intensity of non-ingested beads (% Ag remaining) after 90 min incubation of siCTR and siSec22b (50 nM) transfected phagocytic MEFs with EPEA-peptide coupled, IgG opsonized beads. (n = 4/4 biologically independent samples, 81/99 phagosomes siCTR/siSec22b). f JAWS DCs transfected with EGFP-Sec22b and mCh-STIM1. Arrows: periphagosomal puncta containing Sec22b and STIM1. Bar = 3 µm. g Quantification of phagosomal antigen degradation as in (e) in JAWS cells transfected with siCTR or siSec22b (50 nM) (n = 4/4 biologically independent samples, 727/958 phagosomes siCTR/siSec22b. See also Supplementary Fig. S5f–g) h Quantification of phagosomal antigen degradation as in (e) in JAWS cells co-transfected with siSec22b and soluble secreted GFP (ssGFP), GFP-KDEL, EGFP-ORP8 or EGFP-ORP8-mut. (n = 4/4/4/4 biologically independent samples, 753/625/680/1034 phagosomes in siSec22b+ ssGFP/GFP-KDEL/EGFP-ORP8/EGFP-ORP8-mut; see also Supplementary Fig. S5h). Graphs show means + SEM.

Fig 4: Sec22b interaction with ORP8 regulates phagosomal PI(4)P.a Western blot of whole-cell lysates (input) and GFP-trap agarose beads immunoprecipitates (IP) of HeLa cells transfected with FLAG-Sec22b and either EGFP-ORP8, EGFP-ORP5 or EGFP control, using anti-GFP and anti-FLAG. b Phagocytic MEFs co-transfected with mCh-Sec22b (magenta) and either EGFP-ORP8 (green, top panels) or EGFP-ORP5 (green, bottom panels). c Quantification of phagosomal PI(4)P in WT MEFs transfected with GFP-2xP4M and either siCTR (100 nM) or siORP5/8 (50 nM each); n = 9/11 biologically independent samples, 72/79 phagosomes in siCTR/siORP5/8; see also Supplementary Fig. S3b–d. d Quantification of phagosomal PI(4)P in WT phagocytic MEFs transfected with mCh-2xP4M and siCTR+GFP-KDEL, or siSec22b+GFP-KDEL, +EGFP-MAPPER, +EGFP-ORP8 or +EGFP-ORP5. (n = 10, 13/4/6/4 biologically independent samples, 61, 81/29/28/26 phagosomes siCTR+KDEL, siSec22b+KDEL/ MAPPER/ ORP8/ ORP5). e Phagocytic shSec22b MEFs transfected with EGFP-ORP8 or EGFP-ORP8-H514A-H515A (ORP8-Mut). f Quantification of initial peak levels as well as phagosomal PI(4)P in shSec22b phagocytic MEFs transfected with mCh-2xP4M and GFP-KDEL, EGFP-ORP8 or EGFP-ORP8-Mut. (n = 7/11/10 biologically independent samples, 111/123/185 phagosomes KDEL/ORP8/ORP8-Mut). IgG beads were added for 30 min. White bars = 3 µm. Bar graphs are means +SEM.