TOM1 is enriched on signaling endosomes positive for EGFR in IpgD-expressing cells. (A) Control MEF cells were fixed and immunostained using the indicated antibodies to assess the localization of TOM1. (B) Pearson's coefficient assessing the colocalization between TOM1 and EEA1 on one hand and TOM1 and GIPC on the other hand was calculated for control MEFs or in MEFs expressing IpgD. ns, nonsignificant differences between the indicated conditions (Student's t-test). Data show the mean±s.e.m. (n = 3, representing 92–121 cells). (C) Control or MEF cells stably expressing IpgD were transfected to express EGFR–GFP, serum-starved for 3 h in the presence of cycloheximide, fixed and stained for endogenous TOM1 (red in merge) and GIPC (blue in merge). Scale bars: 10 µm. (D) Enlarged boxes from the IpgD-expressing cell shown in C. Arrowheads indicate EGFR–GFP internalized in TOM1- and GIPC-positive endosomes. (E) Quantification of TOM1–EGFR colocalization in control or IpgD-expressing cells. Individual endosomes positive for TOM1 and EGFR were counted in control cells or IpgD-expressing cells. Data show the mean±s.e.m. (n = 3, representing 30–60 cells). P<0.05 (Student's t-test).

The VHS domain of TOM1 binds to monophosphorylated phosphatidylinositol with a preference for PI5P. (A) The indicated recombinant proteins were used to probe PIP Strips^TM. After extensive washes, bound proteins were revealed using an anti-GST antibody. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (B) Same as in A but proteins were incubated on PIP Arrays^TM (upper panel). Arrows indicate the lowest concentration of binding to PI5P of each protein. The lower panels show typical curve fitting for the interaction of each domain with the indicated phosphatidylinositol. AU, arbitrary units. (C) Wild-type or KRKK-mutant VHS domains of TOM1 were incubated with phosphatidylcholine∶phosphatidylethanolamine liposomes containing 6% of the indicated monophosphorylated phosphatidylinositol. After flotation on a sucrose cushion, the top fractions were collected and the amount of lipid-bound protein was assessed by western blotting (upper panels). Quantification is shown in the lower panels as the mean±s.e.m. from three independent experiments. ns, nonsignificant (Student's t-test).

TOM1 knockdown reverses delayed EGFR degradation in IpgD-expressing cells. (A,B) HeLa cells were transfected with a control siRNA (siControl) or siRNA against TOM1 (siTOM1) and transiently expressed GFP (Control cells) or GFP–IpgD (IpgD-expressing cells). The cells were then serum-starved and left untreated (−EGF) or stimulated with 200 ng/ml EGF for 1 h (+EGF), in the absence or presence (+bafA1) of 100 nM bafilomycin A1 to inhibit lysosomal degradation. Cells were then fixed and stained for endogenous EGFR (green in merge) and EEA1 (red in merge) and imaged by confocal microscopy. Scale bars: 10 µm. (C) The amount of EGFR per cell was quantified and expressed as a percentage of that present in the control. Results are presented as the mean±s.e.m. (three independent experiments). ^#P<0.0001 versus control cells; ***P<0.0001 between the indicated conditions (Student's t-test). (D) HEK293T cells transfected with EGFR–GFP and IpgD as indicated were silenced for TOM1 expression (siTOM1), serum-starved and stimulated with EGF (100 ng/ml) for 2 h. Cell lysates were probed with the indicated antibodies.

TOM1 knockdown reverses the inhibition of bulk endocytosis in IpgD-expressing cells. (A) Control MEF cells or MEFs stably expressing IpgD transfected with control siRNA (siControl) or siRNA targeting TOM1 (siTOM1) were incubated overnight with 40 µg/ml rat IgG with or without bafilomycin A1 (+/−bafA1, 100 nM). Endocytosed IgG was revealed by immunofluorescence using fluorescent anti-rat antibodies (IgG). Differential interference contrast pictures (DIC) are shown to illustrate the presence of cells. Scale bar: 10 µm. (B) The number of labeled vesicles per cell was counted for each condition and is expressed as a percentage of control. Results are presented as the mean±s.e.m. (three independent experiments). ***P<0.0001 between the indicated conditions (Student's t-test).

Binding to PI5P is necessary for TOM1 to block endocytosis. (A) Fluid-phase endocytosis was assessed as in Fig. 5A by assessing rat IgG uptake by IpgD-expressing cells transfected with a siRNA targeting the 3′-UTR region of TOM1 (siTOM1-UTR). DIC, differential interference contrast. Scale bar: 10 µm. (B) Quantification of rat IgG uptake is expressed as in Fig. 5B. Overexpression of wild-type TOM1 as a GFP fusion protein reverses the phenotype, but this is not observed following overexpression of the KRKK mutant that is unable to bind to PI5P. NT, non-transfected cells. Data show the mean±s.e.m. (n = 3, representing 16–66 cells). ^#P<0.001 versus control; *P<0.05 between indicated conditions; ***P<0.001 between indicated conditions (Student's t-test).