VPS13C interacts with Rab10 in a phospho-dependent manner on the lysosomal membrane. (A) Representative immunoblot of immunoprecipitated VPS13C-Myc and co-immunoprecipitated Rab10-GFP (N = 4). (B) Representative immunoblot of immunoprecipitated Rab10-GFP and co-immunoprecipitated VPS13C-Myc and endogenous VPS13C (N = 1). (C) Volcano plot of AP-MS data comparing interactors of phosphomimetic Rab10-T73E versus phosphodeficient Rab10-T73A (significant hits shown in magenta), highlighting previously confirmed interaction partners GDI1/2 show higher affinity for phosphodeficient Rab10-T73A, and newly identified interactor VPS13C show higher affinity for phosphomimetic Rab10-T73E (N = 3) (Data S1). (D and E) Confirmation of phospho-dependent interaction of Rab10-GFP and endogenous VPS13C via co-IP. Representative immunoblot of immunoprecipitated Rab10-GFP variants (WT, T73E, T73A) and co-immunoprecipitated endogenous VPS13C with (E) quantification of the VPS13C co-IP efficiency normalized by Rab10-GFP levels in IP fraction (N = 4). (F and G) Representative live-cell confocal images showing mCherry-Rab10 WT and TE, but not mCherry-Rab10 TA (magenta) colocalized with lysosomes (LAMP1-mGFP, green) and ER (BFP-KDEL, blue) in COS7 cells with zoom-out (F) and zoom-in (G) (scale bar: zoom-out: 10 µm, zoom-in: 1 µm). Yellow arrows indicate co-localization. (H) Quantification of the percentage of LAMP1-positive vesicles that colocalize with Rab10 (N = 3, from n > 10 cells). (I) Quantification of the percentage of cells with Rab10-positive ER (N = 3; from WT: n = 18 cells, T73E: n = 43 cells, T73A: n = 39 cells). (J and K) Representative live-cell confocal images showing colocalization of VPS13C-mClover (green) with Rab10 WT and Rab10 T73E, but not with Rab10 T73A (magenta) in COS7 cells (zoom-out [J]; zoom-in [K]) (scale bar: zoom-out: 10 µm, zoom-in: 1 µm). Yellow arrows indicate co-localization. (L) Pearson correlation coefficient of VPS13C and Rab10 colocalization comparing Rab10 WT, Rab10 T73E, and Rab10 T73A mutants (N = 3, from n = 13 cells). (M) Quantification of the percentage of VPS13C-positive vesicles colocalized with Rab10 WT, Rab10 T73E, and Rab10 T73A (N = 3, from n > 9 cells). Data are represented as mean ± SEM; one-way ANOVA with Tukey multiple comparison test (E, H, I, L, and M); *P < 0.05 (E), ****P < 0.0001 (E, H, I, L, and M). Source data are available for this figure: SourceData F4.