Endogenous TurboID tagging preserves CATCHR complex function and enables compartment-specific proximity labeling.

(A) Schematic representation of the Golgi and endolysosomal CATCHR complexes analyzed in this study, including EARP (VPS52-, VPS53-, and VPS50-TurboID), GARP (VPS52-, VPS53-, and VPS54-TurboID), and COG (COG4-and COG6-TurboID). TurboID-mediated proximity labeling was performed by biotin supplementation followed by streptavidin purification and mass spectrometry analysis. (B) Functional validation of the TurboID cell lines. Endogenous expression of CATCHR-TurboID fusion proteins rescues trafficking and glycosylation defects associated with depletion of the corresponding endogenous proteins. Rescue was assessed by restoration of O-glycosylation, measured by HPA lectin binding, normalization of LAMP2 glycosylation, recovery of GPP130 abundance, and restoration of B4GALT1 levels. Actin serves as a loading control. (C) Rescue of glycosylation defects in VPS53KO, VPS54KO, COG4KO, and COG6KO cells. HPA binding was quantified in KO cells reconstituted with the corresponding TurboID-tagged protein and is presented as the fold change decrease in HPA signal relative to the parental KO cell line. Reduced HPA binding following rescue indicates restoration of normal glycosylation. (D) Expression of VPS50-TurboID restores the stability of the EARP complex, rescuing the abundance of the VPS52 and VPS53 subunits in VPS50 KO cells. (E) Immunofluorescence analysis of TurboID-tagged CATCHRs detected by anti-TurboID antibodies (first column, left to right) and biotinylated proteins detected with fluorescent streptavidin (second column) in RPE1 cells stained with Golgi marker GOLGA4/P230 (third column). VPS50-, VPS52-, and VPS53-TurboID localize to endosomal compartments, VPS54-COG4-, and COG6-TurboID localize to Golgi membranes. Streptavidin staining closely overlaps with each TurboID construct, demonstrating spatially restricted proximity labeling within the expected intracellular compartments.

CATCHR-TurboID constructs selectively enrich proteins from their cognate intracellular compartments and identify known interaction partners.

(A) Spatial enrichment analysis of proteins identified in each proximity proteome. VPS50-and VPS52-TurboID preferentially enrich endolysosomal proteins, whereas COG4-TurboID significantly enriches Golgi proteins. VPS53-and VPS54-TurboID enrich proteins from both Golgi and endolysosomal system (EL) compartments, consistent with the dynamic localization of GARP and EARP. Endoplasmic reticulum (ER) proteins are not significantly enriched in any dataset, indicating high compartmental specificity. Color indicates the log2 fold change (Log2FC) of individual proteins labeled by the CATCHR-TurboID relative to GFP-TurboID. Circle size denotes protein number. (B) Volcano plots showing proteins significantly enriched by each TurboID construct relative to GFP-TurboID controls. EARP proximity proteomes are enriched for VPS50, VPS51, VPS52, VPS53, GRIPAP1, CCDC186, VPS33B, VIPAS39, RAB11FIP5, and RELCH. VPS54-TurboID identifies GARP subunits together with STX16, TBC1D23, GOLGA1, and CCDC186. COG4-and COG6-TurboID preferentially enrich COG subunits, Golgi coiled-coil tethers, SNARE regulators, and the trafficking factor WWOX. The robust enrichment of known CATCHR interaction partners validates the specificity and sensitivity of the TurboID approach.

Distinct trafficking modules are associated with Golgi and endolysosomal CATCHR complexes.

Dot plots summarize the enrichment of selected trafficking factors identified in the proximity proteomes of VPS50-, VPS52-, VPS53-, VPS54-, COG4-, and COG6-TurboID. Dot size represents average protein abundance and color indicates log2 fold enrichment relative to GFP-TurboID controls. (A) Coiled-coil tethers (CCTs). EARP proximity proteomes are enriched for the endosomal tether GRIPAP1 and the evolutionarily conserved tether CCDC186, whereas GARP preferentially labels CCDC186, GOLGA1, and GOLGA4. COG4-and COG6-TurboID strongly enrich multiple Golgi CCTs, including GOLGA1, GOLGA2/GM130, GOLGA5/golgin-84, GOLGB1/giantin, TMF1, TRIP11/GMAP210, GCC1, GCC2/GCC185, and CUX1/CASP, indicating extensive association of the COG complex with Golgi tethering networks. (B) SNAREs and Sec1/Munc18 (SM) proteins. GARP proximity labeling preferentially enriches STX16 and associated SNARE machinery, whereas EARP is enriched for the VPS33B–VIPAS39 CHEVI complex. COG proximity proteomes are enriched for Golgi SNAREs and regulators, including STX5, BET1L, GOSR1, GOSR2, SCFD1, and SNAP29, consistent with a role for COG in regulating intra-Golgi membrane fusion. (C) Additional trafficking factors identified in each proximity proteome. EARP preferentially labels RELCH and RAB11FIP5, RAB11-associated machinery involved in recycling endosome function. GARP proximity proteomes are enriched for TBC1D23 and Golgi-associated trafficking factors. COG4-and COG6-TurboID identify multiple regulators of Golgi organization and vesicular transport, including SEC23IP, RNF121, SCYL1, RAB1A, RAB6, and the candidate trafficking regulator WWOX. Together, these data indicate that each CATCHR complex functions within a specialized trafficking module composed of CCTs, SNAREs, SM proteins, and Rab-associated factors. (D) Immunoblot validation of selected proximity proteomics hits. Streptavidin pull-downs from TurboID-expressing cells confirm the selective enrichment of CCDC186, VPS33B, VPS53, GOLGA4, TBC1D23, COG3, STX5, TRIP11/GMAP210, and CUX1/CASP by the predicted CATCHR complexes, validating the proteomic datasets.

EARP associates with the CHEVI complex and GARP associates with the vesicle tether CCDC186.

(A) IF analysis of VPS53-myc and endogenous VPS33B. VPS53-positive structures partially colocalize with VPS33B in peripheral TGN/endosomal compartments adjacent to the Golgi, supporting the association of EARP with the VPS33B–VIPAS39 (CHEVI) SM complex. Inset shows enlarged view of the boxed region. TGOLN2/TGN46 was used as a marker for TGN. (B) Proximity labeling analysis of wild-type VPS53-TurboID and a VPS53 mutant carrying substitutions within the predicted MUN-like domain. Streptavidin pull-downs reveal robust biotinylation of endogenous VPS33B by wild-type VPS53-TurboID, whereas mutation of the VPS53 MUN-like domain markedly reduces VPS33B labeling without affecting VPS50 labeling. These results indicate that the VPS53 MUN-like domain contributes to the association between EARP and the CHEVI complex. Input and streptavidin-purified fractions are shown. (C) IF analysis of VPS54-myc and endogenous CCDC186. VPS54-positive structures partially overlap with CCDC186 in TGN-associated compartments, supporting the association of the GARP complex with the coiled-coil tether CCDC186. Inset shows enlarged view of the boxed region. (D) Electron microscopy analysis of cells expressing mitochondrially anchored CCDC186 (mito-CCDC186) or control constructs. Relocalization of CCDC186 to mitochondria induces the accumulation of vesicular membranes around mitochondria. Higher-magnification images reveal uniform vesicle connections (arrowheads) between neighboring mitochondria, demonstrating that CCDC186 possesses vesicle tethering activity.

WWOX is a Golgi-associated COG neighbor required for Golgi organization and glycosylation.

(A) High-resolution immunofluorescence (IF) analysis of myc-WWOX (red) localization relative to the trans-Golgi marker TGOLN2/TGN46 (blue) and the cis-Golgi marker GOLGA2/GM130 (green). WWOX localizes predominantly to medial Golgi cisternae, with a localization quotient (LQ) of 0.27 (n = 20 Golgi regions from 3 cells), placing WWOX in the medial Golgi. (B) Quantification of Golgi area in control and WWOX KD cells. Depletion of WWOX results in a significant reduction in Golgi size, indicating altered Golgi organization (Wilcoxon rank sum test with continuity correction, W = 25487, p = 0.00159). (C) Quantification of COPI localization and HPA lectin binding following WWOX depletion. WWOX KD significantly reduces the colocalization of COPB2 with GALNT2-positive Golgi membranes and increases plasma membrane HPA binding, consistent with impaired Golgi homeostasis and defects in O-glycosylation (n = 30 cells). (D) Representative IF images showing increased HPA lectin binding in WWOX KD cells relative to controls. Increased HPA binding indicates accumulation of incompletely glycosylated O-glycans. Together with the COPI redistribution and N-glycosylation defects shown in Supplementary Figure 4, these results demonstrate that WWOX is required for normal Golgi trafficking and glycosylation.

Golgi CATCHR complexes function as organizing hubs for vesicle tethering and fusion.

Integrated model of CATCHR-associated trafficking modules. EARP associates with the CHEVI complex (VPS33B–VIPAS39), RAB11FIP5, RELCH, and the coiled-coil tether (CCT) GRIPAP1. GARP associates with the vesicle tether CCDC186, TBC1D23, STX16, and Golgi CCT. The COG complex associates with multiple golgins, STX5-dependent fusion machinery, and the newly identified trafficking factor WWOX. These findings support a model in which CATCHR complexes function as central organizers of specialized tethering and fusion modules at the Golgi and endolysosomal system.

DNA constructs

Antibodies