TBC1D15 interacts with the HOPS complex to inactivate and remove Rab7 from membranes selected by SKIP.Figure 10A/B. In situ SKIP/TBC1D15 complex formation assayed using proximity-based biotin ligation (BioID). A. Neutravidin precipitates (PD) from biotin-treated HEK293T cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with HA-BioID-SKIP or HA-BioID-EV. Representative immunoblots against GFP, HA and VPS18 , TL: total lysate. B. Quantification of biotinylation of GFP-TBC1D15 (top graph) and endogenous VPS18 (eVPS18, bottom graph) by BioID-SKIP, expressed relative to BioID-EV (1.0), n=4 independent experiments. C. Analysis of TBC1D15/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with GFP-TBC1D15 from HEK293T cells using GFP-trap beads. Left panel: schematic of HOPS complex composition, with subunits tested marked in yellow. Right panel: representative immunoblots against GFP and RFP; IP: immunoprecipitation, TL: total lysate. Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex. D. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (G-e1D15, green), transfected with either control siRNA (siC) or oligo pool targeting VPS18 (siVPS18) and ectopically expressing HA-SKIP (red), immunolabeled against endogenous VPS18 (eVPS18, blue) and HA, scale bar: 10µm. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions. E. Colocalization (Mander's overlap) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion, n siC=9, n siVPS18=11 images (2≥ cells per image) analysed from 2 independent experiments. F. Neutravidin precipitates (PD) from biotin-treated HeLa cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with BioID-SKIP or BioID-EV. Representative immunoblots against GFP, HA and VPS18 are shown, TL: total lysate. G. Quantification of GFP-TBC1D15 biotinylation by BioID-SKIP over BioID-EV in response to VPS18 depletion expressed as % of control (siC), n=4 independent experiments performed in HeLa or HEK293T cells. H. Quantification of VPS18 protein abundance from experiments in G, n=4. . Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment. Colocalization (Mander's overlap) between Rab7 and TBC1D15 (WT or R417A mutant) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins, n≥ 5 images (2≥ cells per image) analysed from 2 independent experiments J. Proposed model. Left panel: active (yellow star) membrane-bound Rab7 can partner with its effector RILP to mediate dynein-dependent minus-end-directed transport of late endosomes and/or lysosomes into the perinuclear region, toward the microtubule organizing center (MTOC). Left middle panel: a subpopulation of Rab7-positive vesicles acquires Arl8b, forming a hybrid identity compartment. Arl8b in turn recruits the effector SKIP through the N-terminal RUN domain (red), as well as VPS41 and the core HOPS subunits to endosomes. Right middle panel: SKIP is now in the position to engage Rab7 using its C-terminal segment featuring the KMI motif. This promotes acquisition of VPS39 and reconstitution of the HOPS complex, providing a platform for recruitment of the Rab7 GAP TBC1D15 (yellow) not associated with mitochondrial membranes. Right panel: the resulting Arl8b/SKIP/HOPS/TBC1D15 complex induces inactivation (grey star) and removal of Rab7 from the membrane, while maintaining Arl8b/SKIP identity and liberating the endosome for kinesin-dependent transport toward the microtubule plus-end, into the cell periphery. Regulated disengagement of Rab7 thus enables orderly selection of transport route.