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Research Article
TOM1 is a PI5P effector involved in the regulation of endosomal maturation
Frédéric Boal, Rana Mansour, Marion Gayral, Estelle Saland, Gaëtan Chicanne, Jean-Marie Xuereb, Marlène Marcellin, Odile Burlet-Schiltz, Philippe J. Sansonetti, Bernard Payrastre, Hélène Tronchère
Journal of Cell Science 2015 128: 815-827; doi: 10.1242/jcs.166314
Frédéric Boal
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Rana Mansour
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Marion Gayral
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Estelle Saland
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Gaëtan Chicanne
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Jean-Marie Xuereb
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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Marlène Marcellin
2CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France
3Université de Toulouse; UPS; IPBS;, F-31077 Toulouse, France
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Odile Burlet-Schiltz
2CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France
3Université de Toulouse; UPS; IPBS;, F-31077 Toulouse, France
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Philippe J. Sansonetti
4INSERM U786, Unité de Pathogénie microbienne moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France
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Bernard Payrastre
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
5CHU de Toulouse, Laboratoire d'Hématologie, 31059 Toulouse Cedex 03, France
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Hélène Tronchère
1INSERM U1048, I2MC and Université Paul Sabatier, 31432 Toulouse, France
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  • For correspondence: helene.tronchere@inserm.fr
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  • Fig. 1.
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    Fig. 1.

    TOM1 is enriched on early endosomes in IpgD-expressing cells. (A) Upper panel, mass spectrometric analysis results for TOM1. The access number for TOM1 is shown; PAI ratio reflects the enrichment of TOM1 on early endosomes in IpgD-expressing cells versus control cells; right column shows the sequence of the two peptides identified by LC-MS/MS analysis. Middle panel, human TOM1 structural domains showing the VHS and GAT domains and the clathrin heavy chain binding region (CHC binding). Numbering of residues is shown. Lower panel, sequence alignment of human sequences for TOM1 and the two closely related isoforms, TOM1L2 and TOM1L1, showing the specificity of the two peptides identified by mass spectrometric analysis (underlined in red). (B) Early endosomes from BHK control cells or cells expressing IpgD were purified on a discontinuous sucrose gradient, and proteins were loaded on SDS-PAGE gel and immunoblotted using the antibodies indicated on the right. pAKT, phosphorylated AKT. (C) The overall expression level of TOM1 is not altered by IpgD expression. Post-nuclear supernatants of BHK cells with or without expression of IpgD were immunoblotted with the indicated antibodies. Hsp90 and RhoGDI were used as loading controls. (D) Overexpression of 3xPHD displaces TOM1 from PI5P-endosomes. Early endosomes were purified as in B from BHK cells expressing IpgD and 3xPHD, and were blotted for TOM1, pAkt (S473) and Rab5. (E) Quantification of TOM1 recruitment to early endosomes (EE) from D. Data show the mean±s.e.m. (n = 3).

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    Fig. 2.

    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).

  • Fig. 3.
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    Fig. 3.

    The VHS domain of TOM1 binds to monophosphorylated phosphatidylinositol with a preference for PI5P. (A) The indicated recombinant proteins were used to probe PIP StripsTM. 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 ArraysTM (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).

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    Fig. 4.

    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.

  • Fig. 5.
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    Fig. 5.

    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).

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    Fig. 6.

    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).

  • Fig. 7.
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    Fig. 7.

    Model of TOM1 recruitment to signaling endosomes and blockade of endosomal maturation. In control cells with low PI5P levels, stimulated EGFR or soluble cargoes are internalized through the endosomal network and targeted for degradation. When cells are infected by Shigella flexneri or express the lipid phosphatase IpgD (high levels of PI5P), TOM1 is recruited to the signaling endosomes (SE) through a direct interaction with its VHS domain and this induces the blockade of both soluble cargoes and activated EGFR, increasing the survival of signals in infected cells. EE, early endosome; LE, late endosome.

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Keywords

  • EGFR
  • Phosphoinositides
  • Signaling endosome
  • Tom1
  • VHS domain

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Research Article
TOM1 is a PI5P effector involved in the regulation of endosomal maturation
Frédéric Boal, Rana Mansour, Marion Gayral, Estelle Saland, Gaëtan Chicanne, Jean-Marie Xuereb, Marlène Marcellin, Odile Burlet-Schiltz, Philippe J. Sansonetti, Bernard Payrastre, Hélène Tronchère
Journal of Cell Science 2015 128: 815-827; doi: 10.1242/jcs.166314
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Research Article
TOM1 is a PI5P effector involved in the regulation of endosomal maturation
Frédéric Boal, Rana Mansour, Marion Gayral, Estelle Saland, Gaëtan Chicanne, Jean-Marie Xuereb, Marlène Marcellin, Odile Burlet-Schiltz, Philippe J. Sansonetti, Bernard Payrastre, Hélène Tronchère
Journal of Cell Science 2015 128: 815-827; doi: 10.1242/jcs.166314

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