Figure 3.
OSBP proximity proteome reveals apicobasal cargo sorting in the TGN. (A) OSBP cycles between its membrane-bound and cytosolic form. As a consequence of its lipid-transfer activity and its preference toward disordered membranes, OSBP contact sites slide toward PI(4)P-rich regions. When OSBP activity is inhibited by ORPphilins, its lateral dynamics and cycling between the cytoplasm and the contact sites are blocked. (B and C) MDCK cells stably expressing OSBP-TurboID were transfected with βGalT1-BFP to label TGN. Cells were treated with ORPphilins for 1 h and biotin was added to the cells in the last 10 min of the treatment when indicated. Cells were fixed, immunostained, labeled with Streptavidin-Alexa594 to visualize OSBP-TurboID and biotinylation, and then analyzed by confocal microscopy (B). Superplot showing TGN-enrichment of OSBP-TurboID upon OSW-1 and SWG treatments (C). N = number of independent experiments used to calculate means (filled circles). Means were compared by one-way ANOVA with Dunnett’s post hoc test. Bar = 15 μm. (D) Hits that showed a significant increase in abundance values in TurboID-expressing MDCK over wild-type MDCK cells were considered members of the OSBP proximity proteome. Multiple proteins already described as partners or interactors of OSBP were identified. (E and F) Log2-transformed fold change values obtained for SWG/DMSO (E) and OSW-1/DMSO (F) were plotted on volcano plots and proteins belonging to selected GO groups (Cytosolic proteins, ER-, and Golgi-resident proteins) were highlighted. Distribution of total hits and each group among the three statistical categories (no change, significantly more abundant-“up,” significantly less abundant-“down”) were plotted upon SWG (E) and OSW-1 (F) treatments. (G and H) Cargoes with highly basolateral or apical localizations were identified among the OSBP proximity partners and highlighted in the OSBP proximity landscape upon SWG (G) and OSW-1 (H) treatments. Upon ORPphilin treatments, basolateral cargoes become more abundant in the OSBP proximity.

OSBP proximity proteome reveals apicobasal cargo sorting in the TGN. (A) OSBP cycles between its membrane-bound and cytosolic form. As a consequence of its lipid-transfer activity and its preference toward disordered membranes, OSBP contact sites slide toward PI(4)P-rich regions. When OSBP activity is inhibited by ORPphilins, its lateral dynamics and cycling between the cytoplasm and the contact sites are blocked. (B and C) MDCK cells stably expressing OSBP-TurboID were transfected with βGalT1-BFP to label TGN. Cells were treated with ORPphilins for 1 h and biotin was added to the cells in the last 10 min of the treatment when indicated. Cells were fixed, immunostained, labeled with Streptavidin-Alexa594 to visualize OSBP-TurboID and biotinylation, and then analyzed by confocal microscopy (B). Superplot showing TGN-enrichment of OSBP-TurboID upon OSW-1 and SWG treatments (C). N = number of independent experiments used to calculate means (filled circles). Means were compared by one-way ANOVA with Dunnett’s post hoc test. Bar = 15 μm. (D) Hits that showed a significant increase in abundance values in TurboID-expressing MDCK over wild-type MDCK cells were considered members of the OSBP proximity proteome. Multiple proteins already described as partners or interactors of OSBP were identified. (E and F) Log2-transformed fold change values obtained for SWG/DMSO (E) and OSW-1/DMSO (F) were plotted on volcano plots and proteins belonging to selected GO groups (Cytosolic proteins, ER-, and Golgi-resident proteins) were highlighted. Distribution of total hits and each group among the three statistical categories (no change, significantly more abundant-“up,” significantly less abundant-“down”) were plotted upon SWG (E) and OSW-1 (F) treatments. (G and H) Cargoes with highly basolateral or apical localizations were identified among the OSBP proximity partners and highlighted in the OSBP proximity landscape upon SWG (G) and OSW-1 (H) treatments. Upon ORPphilin treatments, basolateral cargoes become more abundant in the OSBP proximity.

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