abne -17 olue France. Toulouse, F-31077 Narbonne, Ato o orsodne([email protected]) correspondence for *Author d’He Laboratoire O1i IPefco novdi h euainof Fre regulation the in involved maturation effector endosomal PI5P a is TOM1 ARTICLE RESEARCH ß eevd2 oebr21;Acpe 9Dcme 2014 December 19 Accepted 2014; November 20 Received 2 the along 1 amplified early be even specific and continue a of can from receptors characterization signaling of where variety the endosomes, signaling in by termed paradigm inactivated endosomes, challenged This rapidly signaling. been their be internalized has downregulate newly to to pathway, endosomes thought early classical are late this receptors into In cell-surface maturation degradation contents. the their their mediate involves to of route endosomes, lysosomes with canonical fusion early and The endosomes of years. formation of recent the succession in has a pathway rapidly this of of evolved consists understanding our pathway target and (Huotari compartments, This to discrete degradation and 2011). or coming medium Helenius, transport cargoes extracellular and retrograde the differentiate recycling, or for to surface them platform cell sorting pathway. the regulated a from tightly as and serves complex a It is route endocytic The INTRODUCTION domain VHS endosome, TOM1, endosomal Signaling Phosphoinositides, the EGFR, WORDS: in KEY of steps function new maturation a discrete to system. point regulating and in TOM1, PI5P of recruitment PI5P the maturation is that through endosomal suggest prevents endosomes endosomes strongly signaling in signaling data that enrichment bulk our on fluid-phase together, demonstrate and and Taken PI5P degradation EGFR endocytosis. we interaction in by delay this the Finally, TOM1 for responsible in of motif. recruitment involved binding the TOM1 We PI5P. the of of partner characterize domain binding direct the the new that a the identify show is of we TOM1 Here, sorting protein (EGFR). endosomal adaptor receptor the factor the in growth implicated epidermal by is IpgD, produced intracellular phosphatidylinositol phosphatase multiple that (PI5P), of reported 5-monophosphate regulation previously We and steps. organelles specifically trafficking of of maintenance lipids cascades, identity signaling of the of class organization the major in involved a represent Phosphoinositides ABSTRACT 17 olue France. Toulouse, 31077 mole He Marle NS PS(ntttd hraooi td ilgeSrcuae,25ruede route 205 Structurale), Biologie de et Pharmacologie de (Institut IPBS Universite CNRS; and I2MC U1048, INSERM 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,8587doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ´le ´de ´ uar,Isiu atu,774PrsCdx1,France. 15, Cedex Paris 75724 Pasteur, Institut culaire, n Tronche `ne n Marcellin `ne rcBoal ´ric ´ aooi,309Tuos ee 3 France. 03, Cedex Toulouse 31059 matologie, 1 aaMansour Rana , `re 4 NEMU8,Unite U786, INSERM 2,3 1, * dl Burlet-Schiltz Odile , ´ 3 alSbte,342Tuos,France. Toulouse, 31432 Sabatier, Paul Universite 1 ´ ainGayral Marion , ePathoge de ´ eTuos;US PS,F- IPBS;, UPS; Toulouse; de ´ hglaflexneri Shigella i microbienne nie 5 2,3 H eToulouse, de CHU hlpeJ Sansonetti J. Philippe , 1 sel Saland Estelle , nices nteaon fP5:oeivle h direct the PI3P–PI(3,5)P involves a one involves PI5P: second to of PIKfyve, lead 5-kinase the amount the pathways by potential the phosphatidylinositol of three phosphorylation in context, increase Viaud this the 2013; In an regulation (Shisheva, 2014a). Outside the trafficking al., in 2003). and et implicated domain- dynamics be al., to membrane homeodomain) shown et of been (plant (Gozani has PI5P PHD ING2 nucleus, a the protein as though with acts containing expression PI5P interaction gene in 2006), and nucleus, al., its organization the et chromatin In (Jones of cells. have signaling regulator in findings PI5P stress of new to function response years, the recent clarify to in in helped However, concentrations low membranes. one at present cell about 5- is known phosphatidylinositol which phosphoinositide- (PI5P), phosphoinositides, is monophosphate identified much harboring least Not the that well 2013). of proteins autophagy (Balla, are modules and recruiting compartments, binding trafficking by endocytic vesicular function of on regulators mainly known reside which hog h neto ftevrlnefco pD PI(4,5)P a IpgD, factor virulence the bacterium of the injection with the through infected cells in described o eonzda atrrgltr fti ot D al and Paolo (Di route are this of family regulators phosphoinositide master as the the recognized from Among now organelles. lipids these of factors, identity regulatory the maintain to is needed into is receptors of converted signaling are the where endosomes terminated. endosomes to early signaling common classical these Upon is endocytosis. 2009). maturation, compartment al., clathrin-independent et Miaczynska and APPL1-positive Zoncu clathrin-dependent 2001; 2007; Sorkin, transient Camilli, and De Zastrow This von and 2004; Fiore al., et (Di route endocytic P3)adpopaiyioio ,-ipopae[PI(3,5)P 3,5-bisphosphate 3-monophosphate phosphatidylinositol al., and phosphatidylinositol et and Clague example, organelles 2006; (PI3P) an Camilli, processes of De As trafficking and face and 2009). Paolo signaling Di cytosolic of 2003; variety (Lemmon, the vast recruit a to on able coordinate are They effectors 2012). Kiger, specific and Jean 2006; Camilli, De oatrteedctcptwy(ae ta. 01.Namely, 2011). receptor al., factor growth et epidermal (Ramel induce PI5P pathway and of 2013) endocytic levels al., increased the et control (Puhar alter to opening 2006), to al., hemichannel et of survival Pendaries modulation 2002; Akt the al., the to et of shown (Niebuhr activation been pathway this the has and membrane remodeling phosphatase, with plasma membrane lipid the induce at infection bacterial PI5P of the Upon pool of primary 2002). expression al., or pathogen, et (Niebuhr phosphatase ytblrnfml Vade l,21a n h hr uses third the and the 2014a) of al., 3-phosphatases et the PI(4,5)P (Viaud and family PIKfyve the myotubularin of action concerted ie h yai aueo hscmatet ih regulation tight compartment, this of nature dynamic the Given 1 Gae , 2 sasuc fP5.Ti atrptwywsoriginally was pathway latter This PI5P. of source a as 4 enr Payrastre Bernard , tnChicanne ¨tan 1 enMreXuereb Jean-Marie , 1,5 and 2 P5 ot ihthe with route –PI5P hglaflexneri Shigella 1 , 815 2 2 4- ], ,

Journal of Cell Science pDepeso nue iei h muto IPon PI5P of amount the blockade this in that As speculated we rise 2011). 2011), al., a al., et (Ramel et induced endosomes (Ramel (trans- expression pathways endosome-to-TGN IpgD the retrograde also or network) PI5P recycling Golgi dextran, the signals. like markers altering survival fluid-phase without of maintain the endocytosis bulk the delaying to the endosomes, blocked impair EGFR late and of to ligand endosomes degradation its early of of maturation independently activation (EGFR) ARTICLE RESEARCH 816 of blockade insight the gain undertook in we and enrichment, involved PI5P effectors by mechanisms induced PI5P maturation molecular endosome new the characterize into to order In in endosomes cells signaling IpgD-expressing on enriched is TOM1 RESULTS protein maturation. the endosomal blocks effector, endosomes recruitment PI5P signaling TOM1 PI5P-dependent to new that show a and this TOM1, unravel adaptor for to we responsible approach be Here, could proteomic that phenotype. fishing effectors effector PI5P a PI5P new specific undertook characterize a and of endosomes, enrichment the on by explained be could ufml ebr O11adTML Fg A lower allowed purified 1A, immunoblotted that (Fig. we TOM1 result, two this TOM1L2 peptides confirm the to and two of order In neither TOM1L1 panel). The in members Hong, found 2006). and are subfamily (Seet al., TOM1 box of binding et identification clathrin Katoh a on and found 2004; 2005; 2010), are (Bonifacino, al., steps which et trafficking of Wang in both involved domain, proteins 492-residue TOM1) several and a 1A, (GGA and domain (Fig. GAT (Vps27-Hrs-STAM) is was VHS a domains a it contains characterized TOM1 It panel). different as panel). middle of interest, upper composed particular protein 1A, (Fig. of early control candidate with (chicken)] endosomes compared myb-1 PI5P-endosomes a in of 4.1-fold be [target enriched mass-spectrometric TOM1 quantified. to protein was to the appeared proteins hits, of the subjected enrichment Among relative the were and The analysis markers. endosomes endosomal gradient with sucrose the purified fractions shows discontinuous these S1A Fig. of a material endosomes characterization Supplementary on 1991). early al., endosomes et the (Gorvel late separate the to from a method used described We control (PI5P-endosomes). from previously IpgD endosomes overexpressing early those or purified cells on approach proteomic a ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal nooe nIg-xrsigcells. early IpgD-expressing on in enriched endosomes is TOM1 1. Fig. nooe E)fo .Dt hwthe show Data mean D. early from to (EE) recruitment endosomes TOM1 of Rab5. Quantification and (E) (S473) blotted pAkt were TOM1, and for 3xPHD, cells and BHK IpgD from expressing B in as purified were Early endosomes PI5P-endosomes. from 3xPHD TOM1 of displaces Overexpression as (D) used controls. were loading RhoGDI and Hsp90 IpgD indicated antibodies. of the expression with without immunoblotted or were with cells BHK supernatants of altered Post-nuclear not expression. is IpgD TOM1 by of level expression The (C) overall AKT. phosphorylated pAKT, right. the indicated on antibodies the and using gel immunoblotted SDS-PAGE on loaded proteins were and gradient, a sucrose on discontinuous purified cells were or IpgD cells expressing control BHK Early from (B) endosomes red). in (underlined spectrometric analysis mass two by the identified of peptides specificity and the TOM1L2 showing isoforms, TOM1L1, related and closely TOM1 two for the sequences human sequence of panel, alignment Lower shown. of is Numbering residues binding). chain (CHC heavy region clathrin binding the and and domains VHS GAT the showing domains structural human TOM1 by panel, identified Middle peptides analysis. two LC-MS/MS the shows of column sequence right the cells; control IpgD-expressing versus in cells of endosomes enrichment early the on reflects TOM1 ratio PAI TOM1 for shown; number is access The TOM1. analysis for spectrometric results mass panel, Upper (A) 6 ...( s.e.m. n 5 3).

Journal of Cell Science ee fTM a o lee nteps-ula supernatant post-nuclear the in altered S1B). not Fig. material was (supplementary TOM1 of level eiidta O1agetto a neddet the to due we 2013), indeed al., was et augmentation Bua 2003; TOM1 al., that regulate et al., to verified shown et (Gozani was (Pendaries PI5P expression nuclear induced types the As gene we 2011). cell of al., IpgD as other et Ramel PI5P-endosomes, phosphorylation in 1B, 2006; on Fig. previously the accumulation described in by its have and (monitored shown residue) Akt S473 As of cells. hamster activation baby (BHK) IpgD-expressing or kidney control from endosomes early ARTICLE RESEARCH Fg B,wihi ossetwt erimn fTM to TOM1 localization. of its Interestingly, in recruitment change a (32.6%). a coefficients not with Pearson’s and higher consistent endosomes the is signaling change much which not 2B), does is (Fig. IpgD GIPC of expression and between indeed colocalization is the TOM1 whereas EEA1 colocalization), clearly and of stronger TOM1 2B) (2.7% low the (Fig. between a colocalization coefficient of of on that Pearson’s whereas Quantification showed the localized fraction GIPC. EEA1, using with was minor colocalization with observed TOM1 a was colocalized cells, colocalization only to vesicles MEF Interestingly, used these in further vesicles. 2A, was Fig. peripheral As antibody immunofluorescence. in by siRNA this TOM1 shown of by Therefore, localization the TOM1 signal. determine a of specificity this gave the knockdown demonstrating of antibody S1E), upon Fig. this Brissoni material reduced (supplementary 2012), 2005; greatly Hong, al., staining This and was et immunofluorescence. Seet by Tumbarello staining 2004; vesicular (Katoh punctate 2006; al., TOM1 et al., Seet endogenous et 2004; and al., S1C,F; overexpressed reported et was Fig. for what with material specifically Consistent previously S1D,G). (supplementary (si)RNA Fig. in interfering TOM1 quantification small against a for directed with size expected transfected (MEF) in the cells fibroblast at both band embryonic ( hands, single mouse a our TOM1 detects in In antibody this and 2012). cells, cells al., others HeLa as et we human TOM1L2, for (Tumbarello isoform that inefficient recently related signaling was did closely antibody antibody the blotting an of against to The western turned directed marker we by 2006). therefore, TOM1 studies; specific immunofluorescence al., detect GIPC a to et protein used 2012), the (Varsano with al., endosomes colocalize later et to recently, a to reported More (Tumbarello or 2006). been 2005) al., Hong, has and et TOM1 (Katoh Seet (Brissoni 2004; endosomes compartment al., et endosomal EEA1-positive Seet as 2004; to known al., et (also TOLLIP, endofin to or is with shown been interaction system ZFYVE16) has its endosomal TOM1 through Indeed, the recruited, literature. be in the in TOM1 debated of quite localization precise The endosomes cells signaling IpgD-expressing TOM1-positive in to internalized is EGFR S473 of phosphorylation the 38.33 by the monitored 1D, in decrease (Fig. as a well- Akt, observed also of a We activation 1D,E). probe, (Fig. endosomes recruitment 2003). TOM1 the 3xPHD al., with to interfered et probe the 3xPHD (Gozani the binding of with Expression PI5P PI5P for competitor IpgD-expressing sequestered characterized in endosomes we to cells, lysate recruitment its cell for total responsible a on increase TOM1 an blotting to by not 1C). level and (Fig. expression endosomes its to protein in the of recruitment ial,t eosrt httebnigo O1t IPwas PI5P to TOM1 of binding the that demonstrate to Finally, , 0ka,adti adi pcfclydpee in depleted specifically is band this and kDa), 60 6 .6;mean 3.66%; 6 ....Teoealexpression overall The s.e.m.). ta. 02,adi atclro nooe Rmle al., et (Ramel endosomes on particular in (Niebuhr and cells The in 2002), PI5P motif. of al., fact levels KRKK the high et and yields protein the expression the IpgD on through that module PI5P-binding domain PI5P this VHS of to TOM1 existence PI5P. the experiments of binds that interactor show strong preferentially concluded results independent a not these we is together, TOM1 four Therefore, Taken of domain S2C). GAT and across the Fig. that PI3P preference confirmed material over this As assay, (supplementary PI5P not GAT flotation for phosphoinositides. liposome TOM1 was the preference for the in slight S2B, However, TOM1 a PI4P. Fig. the affinity of showed material the in domain domain supplementary tested in PI5P also GAT We shown towards the 3C). (Fig. and preference of assay KRKK) VHS binding the 3A,B, liposome (Fig. abolished assays as overlay completely lipid monophosphates greatly by phosphatidylinositol mutations to assessed these binding Strikingly, the alanine, mutant. reduced into hypothesis, K59) KRKK this and verify the K58 to R52, yielding order (K48, residues mediate In these 2000). material mutated residues al., we (supplementary et charged (Misra membranes S2A) positively Fig. with of interaction patch authors the potential a and solved, further liposome that been has domain proposed VHS 2009). was with the of structure al., The PI5P assay 3C). (Fig. et to flotation PI5P (Blanc or and PI4P liposome VHS PI5P PI3P, phosphatidylcholine:phosphatidylethanolamine preference a the is to clear containing This in binding of a VHS). no confirmed showed 3B, binding (Fig. and which Preferential detected DdTOM1, PI3P pmol 25 to for as contrast PI5P for little in monophosphates, avidity as greater phosphatidylinositol a other with showed the TOM1 with of compared domain VHS The nooe ol etruhadrc neato ihthe Strips with to PIP interaction resorted using we direct experiments possibility, this overlay a test lipid to through order In PI5P- be to phosphoinositide. recruitment would TOM1 explain endosomes to phosphoinositides possibility to interesting binds TOM1 An of domain VHS The Fg A H) hshtdlnstlbshshtsand bisphosphates human the Phosphatidylinositol of domain VHS). VHS the monophosphates (Blanc that 3A, phosphatidylinositol (Fig. the amoeba found with we the interacted TOM1 2009), in al., protein et VHS-domain-containing only with obtained results opr h eaieaiiyo hsdmi odifferent to domain to this Arrays order PIP of In used charges. avidity we negative monophosphates, relative of phosphatidylinositol the with accumulation interaction an only compare not the on and that specific based was indicating monophosphates recognized, phosphatidylinositol were weakly or recognized very not were trisphosphates phosphatidylinositol r oiiefrbt O1adGP hnIg sexpressed is IpgD these when GIPC that Overall, and endosomes cells. 2E. TOM1 signaling Fig. in both in for retained in positive is shown are zoomed EGFR between that is in show colocalization highlighted EGFR–GFP results the of and and both Quantification IpgD TOM1 for 2D). 2C; Fig. positive (Fig. in membrane structures boxes GIPC EGFR– vesicular plasma and described 2011), to al., TOM1 we the internalized et as was (Ramel at cells, EGFR GFP endogenous mainly IpgD-expressing for In EGFR previously localized in Control). overexpressed GFP 2C, EGFR cells, (Fig. internalized to control with In fused together cells. endosomes IpgD-expressing signaling to enx netgtdtersde novdi hsinteraction. this in involved residues the investigated next We ie hs ehpteie htTM ol erecruited be would TOM1 that hypothesized we this, Given ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal itotlu discoideum Dictyostelium TM ossetwith Consistent . dO1 the DdTOM1, nvitro in 817 TM .

Journal of Cell Science oepesEF–F,srmsavdfr3hi h rsneo ylhxmd,fxdadsandfredgnu O1(e nmre n IC(lein (blue GIPC and merge) in (red TOM1 endogenous for stained and fixed cycloheximide, of presence the in 10 h bars: 3 Scale merge). for serum-starved EGFR–GFP, express to EERHARTICLE RESEARCH 818 IpgD-expressing for in reason specifically the degradation represent cells. might EGFR and delayed structures the these accumulation on an TOM1 PI5P cause that of might degradation postulated endosomes EGFR signaling we on for Therefore, enrichment stimulation. protein EGF does ESCRT-associated by TOM1 an induced that as immunofluorescence indicating act by S3B), not Fig. or TOM1 material S3A) of biochemically (supplementary either effect Fig. found No material of was EGF. (supplementary degradation depletion with EGFR stimulation siRNA-mediated on and depletion ESCRT-associated to cells resorted an To in degradation. TOM1 we as EGFR of this, regulation serve the investigate to for would tempting particular very in therefore TOM1 protein, sorting was It endosomal that 2009). al., an (Brissoni postulate et of Blanc complex 2006; part (ESCRT)-0 al., transport be et for to required proposed complexes been has TOM1 EGFR for protein ESCRT-associated degradation an not is of TOM1 recruitment cells. IpgD-expressing PI5P-dependent in the endosomes with to TOM1 consistent a are mean TOM1 for the 2011), positive show endosomes Data Individual cells. cells. IpgD-expressing IpgD-expressing or or cells control indicated control in the in colocalization between counted TOM1–EGFR differences were of nonsignificant Quantification ns, (E) IpgD. endosomes. positive expressing han MEFs one in on or EEA1 and MEFs TOM1 control between for colocalization calculated the was assessing hand coefficient other Pearson’s (Student’s (B) the conditions TOM1. cells. on of IpgD-expressing GIPC in localization and EGFR the for TOM1 assess positive to endosomes antibodies signaling indicated on the enriched is TOM1 2. Fig. t ts) aaso h mean the show Data -test). m .()Elre oe rmteIg-xrsigcl hw nC rohasidct GRGPitraie nTM-adGIPC- and TOM1- in internalized EGFR–GFP indicate Arrowheads C. in shown cell IpgD-expressing the from boxes Enlarged (D) m. 6 ...( s.e.m. n 5 ,rpeetn 211cls.()Cnrlo E el tbyepesn pDwr transfected were IpgD expressing stably cells MEF or Control (C) cells). 92–121 representing 3, 6 ...( s.e.m. G,tesga o GRwsls,a osqec fits of consequence mean plasma a 4C; as Fig. the decrease 41.20 lost, a in was resulted to to this EGFR Quantitatively, for degradation. restricted lysosomal signal the was siControl, EGF, 4A, EGFR control (Fig. left in the membrane anticipated, cells, reflecting As or cell. the quantified, per unstimulated was and receptor EGF cell remaining serum per of performed, antibody. amount signal with anti-EGFR After was EGFR of an imaging stimulated intensity GFP–IpgD. with confocal stained were or Semi-quantitative and cells transiently fixed GFP then unstimulated, the were express EGF siRNA and starvation, a to following siTOM1), with transfected or 4, EGFR siControl) transfected (Fig. were 4, TOM1 (Fig. of Cells siRNA targeting control cells. degradation a either IpgD-expressing the with in track stimulation immunofluorescence an to trafficking to EGFR in resorted study TOM1 we of cells, role IpgD-expressing potential in the study to order In the survival and cell cells of IpgD-expressing maintenance the in for degradation responsible EGFR is delayed PI5P-endosomes to recruitment TOM1 n 5 ,rpeetn 06 cells). 30–60 representing 3, 6 ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal .7 cmae ihcnrlcls101 cells control with (compared 2.47% 6 A oto E el eefxdadimnsandusing immunostained and fixed were cells MEF Control (A) ....A xetd pDepeso induced expression IpgD expected, As s.e.m.). P , 2 .5(Student’s 0.05 G) hnsiuae with stimulated When EGF). t -test). dEGFR nd 6 4.2%, and d

Journal of Cell Science EERHARTICLE RESEARCH iN,EF erdto a eae sw published we 65.5 with as 41.20 2011), with delayed al., (compared was et EGF (Ramel degradation previously EGFR siRNA, EGFR total of amount (95.31 the cell alter per not did but internalization, EGFR ycnrs,i pDepesn el,i h rsneo control of presence the in cells, IpgD-expressing in contrast, By 6 5.56%). 6 .7 ncnrlEGF-stimulated control in 2.47% 6 .%remaining 2.3% h el ihtelssmlihbtrbflmcnA abrogated A1 bafilomycin inhibitor lysosomal the treating with Finally, the IpgD. cells for by the induced crucial degradation is EGFR accumulation of TOM1 blockade that indicated clearly This pDaddpee o O1soe euto nEF level EGFR in (35 reduction challenge a EGF showed TOM1 after for depleted and IpgD mean 4B,C; Fig. cells, ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal eeue opoePPStrips PIP probe to used were PI5P. for to preference binds a TOM1 with of phosphatidylinositol domain monophosphorylated VHS The 3. Fig. osgiiat(Student’s nonsignificant u rtiswr nuae nPPArrays PIP A on in incubated as were Same proteins (B) but phosphatidylserine. PS, acid; phosphatidic PA, sphingosine-1-phosphate; S1P, phosphatidylcholine; PC, phosphatidylethanolamine; PE, phosphatidylinositol; PI, antibody. lysophosphatidylcholine; anti-GST LPC, acid; an lysophosphatidic using LPA, revealed were proteins bound uniiaini hw ntelwrpnl sthe as panels lower the mean panels). in (upper shown blotting is lipid-bound western Quantification of by amount assessed the was and protein the collected cushion, were sucrose fractions a top on flotation monophosphorylated After indicated phosphatidylinositol. the of 6% containing with incubated were liposomes TOM1 phosphatidylcholine:phosphatidylethanolamine of or domains Wild-type VHS (C) KRKK-mutant indicated units. the arbitrary for with AU, fitting domain phosphatidylinositol. curve each typical of show interaction panels the of lower PI5P to The binding protein. of each concentration lowest the indicate Arrows 6 ...fo he needn xeiet.ns, experiments. independent three from s.e.m. 6 6 ....Srknl,clsoverexpressing cells Strikingly, s.e.m.). %,bc otelvlo h control. the of level the to back 4%), A h niae eobnn proteins recombinant indicated The (A) t -test). TM fe xesv washes, extensive After . TM uprpanel). (upper 819

Journal of Cell Science erdto.Clswr hnfxdadsandfredgnu GR(re nmre n E1(e nmre n mgdb ofclmcocp.Scale microscopy. confocal by imaged and merge) in (red EEA1 and merge) in (green EGFR endogenous for stained and 10 fixed bars: then were Cells degradation. el rnfce ihEF–F n pDa niae eeslne o O1epeso sTM) eu-tre n tmltdwt G 10ng/ml) (100 EGF with stimulated antibodies. and indicated serum-starved the (siTOM1), expression with TOM1 probed for were silenced lysates were Cell indicated as h. IpgD 2 and EGFR–GFP with transfected cells EERHARTICLE RESEARCH novd ekokdi onwt pcfcsRA This siRNA. specific also a was with 820 endofin down whether it investigate knocked To we (Seet EGFR 2001). of involved, degradation the Hong, in role a and play to proposed TOM1 been has for silenced was were delay cells 4D). (Fig. the this expression when Strikingly, abolished degradation. completely a observed EGFR–GFP we IpgD, transiently retarded expressed cells EGF cells results when upon However, degradation These HEK293T stimulation. its 4B). monitor in one Fig. to EGFR–GFP only in biochemically overexpressing clarity, shown confirmed is (for were condition conditions A1 all bafilomycin under degradation EGFR serum-starve then mean were cells The cells). (IpgD-expressing GFP–IpgD or cells) (Control GFP expressed ( transiently untreated cells. and left IpgD-expressing (siTOM1) in TOM1 degradation against EGFR siRNA delayed or reverses knockdown TOM1 4. Fig. mn h ecie neatn ateso O1 endofin TOM1, of partners interacting described the Among 6 ...(he needn experiments). independent (three s.e.m. m .()Teaon fEF e elwsqatfe n xrse sapretg fta rsn ntecnrl eut r rsne sthe as presented are Results control. the in present that of percentage a as expressed and quantified was cell per EGFR of amount The (C) m. 2 G)o tmltdwt 0 gm G o +G) nteasneo rsne(bf1 f10n aioyi 1t nii lysosomal inhibit to A1 bafilomycin nM 100 of (+bafA1) presence or absence the in (+EGF), h 1 for EGF ng/ml 200 with stimulated or EGF) # P , .01vru oto el;*** cells; control versus 0.0001 inl ontemo GR ssoni supplementary survival in the shown perturb As could EGFR. wanted this of we whether downstream degradation, EGFR investigate signals EGFR in to of blockade next the regulation prevents the cells in involved cells. is IpgD-expressing not in and endofin, degradation IpgD- itself, in TOM1 partner degradation that EGFR indicates its detect clearly retarded As not This the cells. could S3D,E). in expressing we endofin Fig. for S3F, role material Fig. any material (supplementary supplementary by level in shown measured protein decrease as 40% the a decrease and at S3C) expression Fig. mRNA material (supplementary 65% qRT-PCR a in resulted P , ie h atta O1kokoni IpgD-expressing in knockdown TOM1 that fact the Given .01btenteidctdcniin (Student’s conditions indicated the between 0.0001 AB eaclswr rnfce ihacnrlsRA(siControl) siRNA control a with transfected were cells HeLa (A,B) ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal t ts) D HEK293T (D) -test). and d for

Journal of Cell Science li-hs ukedctss sdmntae yfluorescently by demonstrated inhibiting as also was endocytosis, expression bulk S473 IpgD fluid-phase that previously endocytosis showed by of We inhibition TOM1-KRKK the the restores of monitored not mutant but reversed TOM1 wild-type of as indeed Overexpression TOM1 activation, down Akt knocking phosphorylation. S4, PI5P-induced Fig. material ARTICLE RESEARCH ln i o aeayefc nIGdgaain sw showed we as degradation, IgG TOM1 on degradation. down effect EGFR knocking any for no Again, have Strikingly, not cells. with left. did these IgG alone phenotype, in IpgD- of left delayed the IgG 40% In a detectable reversed to 5B). demonstrated close completely Fig. with clearly TOM1-depletion IgG, in uptake of quantification IgG degradation cells, 5A, (Fig. expressing cells IpgD, bafilomycin-A1- treated expressing signal with remaining compared not no immunofluorescence cells with by degradation detected MEF to elsewhere sent control was described In IgG internalized 2003). as we al., experiment, effect, et this (Petiot confirm IgG-uptake abrogating to was an order cells In performed in 2011). depletion al., TOM1 et whether (Ramel uptake dextran labeled idn oteVSdmi srqie o O1t block to TOM1 for (an required PI5P level is that domain cells. expression confirm PI5P-enriched VHS results in high These endocytosis the a 6A). to Fig. at in binding to shown even able the is not not phenotype, 5. example experiments, was Fig. the mutant these although in KRKK in ones restore the bulk the that of expression, from overexpression noted in results Strikingly, the be undistinguishable IpgD blockade gave to reversed controls has by the It clearly restoring completely. induced 6, Fig. TOM1 by in endocytosis shown phenotype wild-type As KRKK phenotype. knockdown of the or restore wild-type to overexpression of able in assay overexpression was uptake quantified TOM1 IgG whether and the investigate used of S2D We to efficiency S2E. Fig. Fig. The material as material protein. supplementary immunoblotting, supplementary KRKK by fusion in or assessed GFP shown was wild-type a overexpression the and as siRNA either TOM1 overexpressed of MEF IpgD-expressing mutant then or 3 control recovery and in the a protein cells, targeting the to deplete siRNA to resorted TOM1 used we We cells, experiment. IpgD-expressing in blockade nodrt ofr rca oeo O1i endocytosis in TOM1 of role crucial a confirm to order In ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal A oto E el rMF tbyepesn IpgD expressing stably MEFs or cells MEF Control cells. (A) of IpgD-expressing inhibition in the endocytosis reverses bulk knockdown TOM1 5. Fig. odtos(Student’s conditions *** experiments). of presence 10 the bar: illustrate Scale to cells. shown are (DIC) contrast pictures interference Differential (IgG). antibodies anti-rat fluorescent using immunofluorescence (+/ with overnight 40 incubated were siRNA (siTOM1) or TOM1 (siControl) targeting siRNA control with transfected xrse sapretg fcnrl eut are mean Results the is control. as and of presented condition percentage each a for as counted expressed was cell per vesicles 2 m aA,10n) noyoe g a eeldby revealed was IgG A1 Endocytosed bafilomycin nM). without 100 or bafA1, with IgG rat g/ml P , m .01btenteindicated the between 0.0001 .()Tenme flabeled of number The (B) m. t -test). 6 ...(he independent (three s.e.m. 9 URrgo of region -UTR 821

Journal of Cell Science EERHARTICLE RESEARCH euao fdfeetclua rcse u IPefcosare trafficking effectors 822 cellular PI5P a but subvert processes involving as cellular to recognized different here now of is bacteria PI5P regulator shown benefit. a the its that for of component, as machinery expression ability IpgD such to the processes, specific reflects the is endolysosomal effect the and of perturbs This and expression pathway. EGFR endosomes maturation the of signaling to following blockade TOM1 endosomes in the flexneri increase on Shigella for large levels The protein responsible endocytosis. PI5P adaptor is fluid-phase the and PI5P in degradation of that, by recruitment evidence TOM1 compelling the provide cells, data IpgD-expressing our together, Taken DISCUSSION h nooa auaindfc n aneac fsurvival of maintenance and defect for signals. responsible maturation is endosomal cells the IpgD-expressing in endosomes signaling ae oehr hs eut hwta O1rcutetto recruitment TOM1 that show results these together, Taken fetrIg eut ntercutetof recruitment the in results IpgD effector Srus ta. 99 n a enipiae nscfamily src in implicated been has TOM1 and with identity 1999) trafficking 31% al., membrane shares in et TOM1L1 role (Seroussi 2010). and a al., TOM1L1 play et orthologs, to (Wang two not proposed endosomal still its are are and TOM1L2, functions regulating TOM1 cellular their understood, Although totally effector 1997). al., et PI5P (Burk its a through therefore TOM1, as Rac1, identifies maturation. work GTPase domain, present small Our (Viaud VHS 2014b). invasion the cell the al., activate and et facing dynamics that to cytoskeleton membranes shown actin has able regulating on laboratory is our function that recently, PI5P its nucleus Very sparse. the in are outside cytosol involved (Keune effectors on PI5P be PI5P found for also However, might affinity domain, 2011). show PHD al., that regulation a et proteins chromatin has nuclear ING2 in other 2003). many involved al., protein et PI5P nuclear (Gozani for a described ever ING2, effector was first The described. sparsely still O1wsoiial ecie satre ftevmbgene v-myb the of target a as described originally was TOM1 ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal sTM-T) I,dfeeta nefrnecnrs.Scale 10 contrast. bar: interference differential DIC, (siTOM1-UTR). *** idt IP T o-rnfce el.Dt hwthe show Data cells. to mean unable non-transfected NT, is PI5P. that to mutant observed bind KRKK not the GFP is of a this overexpression but as following phenotype, TOM1 the wild-type reverses of protein Overexpression fusion 5B. Fig. in as cells 3 the IpgD-expressing targeting by siRNA uptake a IgG with rat transfected assessing block by to 5A TOM1 Fig. for necessary is PI5P endocytosis. to Binding 6. Fig. esscnrl * control; versus P , 6 .0 ewe niae odtos(Student’s conditions indicated between 0.001 ...( s.e.m. m .()Qatfcto frtIGutk sexpressed is uptake IgG rat of Quantification (B) m. A li-hs noyoi a sesda in as assessed was endocytosis Fluid-phase (A) n 5 P , ,rpeetn 66 cells). 16–66 representing 3, .5btenidctdconditions; indicated between 0.05 9 URrgo fTOM1 of region -UTR # P , 0.001 t -test).

Journal of Cell Science rti–rti neato oanwt h oeta obn to bind to potential the with domain invading interaction protein–protein the for advantage massive bacteria. a represents components EERHARTICLE RESEARCH nraei ontemsria inl.I h otx fthe of context an the by In accompanied signals. is by survival infection this downstream and in the EGFR, al., increase for of et responsible PI5P- degradation is IpgD, (Brissoni endosomes expressing delayed receptor to cells in recruitment IL-1 that TOM1 of show mediated degradation we might contrast, the highlighted By but in 2006). as EGFR, role cargo, for specific its protein a by others ESCRT towards an by specificity mammals, some as and in present act that, here not demonstrate presented does (Roxrud clearly in TOM1 data complex 2012) al., the ESCRT et that the However, (Tumbarello (Benmerah by 2008). demonstrated internalization degradation al., and et EGFR authors 1998) in al., The involved et protein 2009). adaptor in al., as Dictyostelium well et ancestral as 2011), an (Blanc al., phylogenetic form amoeboflagellate et proteins the Recent (Herman family in lysosomes. complex TOM1 ESCRT-0 that the shows their to for analysis membranes, delivery endosomal of early subsequent sorting 2001; and on and recruitment TOM1 cargoes al., clathrin that in ubiquitylated proposed roles et parallel the been play (Katzmann has could with It Hrs receptors 2010). associated Hurley, several and be Wollert of least degradation mediating at complex the multi-protein ESCRT-0 or essential an an component, machinery, serve ESCRT ESCRT Hrs, might TOM1 the an that in with suggests debated This as facilitate is 2004). interact al., this et although (Seet to to 2006), literature protein al., shown et endosomes adaptor (Brissoni been component an signaling Moreover, has 2012). as al., to TOM1 autophagy et (Tumbarello in VI maturation autophagosome role recently myosin a was TOM1 targeting play 2008). to al., tumor defined, et proposed potential a (Girirajan well to activity linked less response suppressor immune is the in with TOM1, role important TOM1L2, study with of a identity function although The 59% and 2009) 2007) shares al., al., which et et Collin (Liu 2005; endocytosis al., EGFR et (Li activity mitogenic kinase h H oano O1wsoiial ecie sa as described originally was TOM1 of domain VHS The hglaflexneri Shigella dO1itrcswt s1,aclathrin a Esp15, with interacts DdTOM1 , Tom1l2 uhasbeso ftecellular the of subversion a such , eetapdmc ugssan suggests mice gene-trapped itotlu discoideum Dictyostelium rvaeanathema Breviate tec Msae l,20) eso eeta h mammalian the that here show charged We positively 2000). a al., of et presence (Misra the stretch to thanks membranes lipid hw,lk h H oan obn opopoiis anyto mainly phospholipids, the to bind to to PI(3,5)P domain, VHS binding the like GAT TOM1 shown, in GGA1 the conserved In Second, not in are GAT. 2010). R281) al., involved (R260, et PI4P the residues phosphoinositide Wang of 2004; GTPases basic al., small two et these (Shiba with which family binding, interaction Arf1 Arf of in lack of for involved its residues the explains recognition lacks TOM1 it 2005). Golgi-localized, First, the al., the TGN. of of et domain in GAT Akutsu the c 2004; from involved affinity differs ubiquitin- al., it TOM1, et is several In the (Shiba has and protein ubiquitylated to domain motifs al., GAT interacting contribute et The (Misra contact domain phosphoinositides. also might GAT this TOM1 in The surroundings S3). could mutations, participate Fig. suggested to the material been the supplementary enough has 2000; in R57 close the by example, be For that residues to binding. abolished noted the other be to completely contribute to that has not It suggesting and was 2000). the Misra al., by in binding proposed et residues as (Misra these membranes to collaborators of VHS involvement TOM1 phosphoinositide of the for binding important confirm are in We that K59) binding. and domain a K58 VHS R52, describe balance TOM1 (K48, also the residues We the charged PI5P. positively increased, to of the TOM1 patch favor are of would binding which levels direct modified, be preferential PI5P could PI5P where and that PI3P propose between system, We PI5P. our to directly bind in can TOM1 of domain VHS hog rti–rti neato ihtePI3P-binding the with interaction protein–protein the through and rescue our binding in phosphoinositide demonstrated as in to role trafficking, experiments. dominant cellular domain bind a of GAT has regulation to domain the VHS able in show TOM1 over that, any the demonstrate is we clearly show cells, results domain mammalian Although not Our PI5P. GAT 2009). does towards specificity it al., TOM1 phosphoinositides, et human monophosphorylated (Blanc that defined here not were er r-idn rtis(G) aiyo dposi the in adaptors of family a (GGA), proteins Arf-binding -ear, twsiiilysonta O1i erie oendosomes to recruited is TOM1 that shown initially was It 2 oee,tersde novdi h ii binding lipid the in involved residues the However, . Dictyostelium ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal h A oano dO1wsalso was DdTOM1 of domain GAT the , aeendosome. late LE, endosome; cells. early infected EE, in signals of survival the increasing EGFR, and activated cargoes soluble the both induces of this blockade and domain with VHS interaction its direct a through endosomes (SE) signaling the is to TOM1 recruited PI5P), of levels IpgD (high phosphatase lipid the express by infected cells are When degradation. for and targeted network endosomal the through internalized are cargoes or soluble EGFR stimulated levels, PI5P maturation. endosomal of blockade and endosomes recruitment signaling TOM1 to of Model 7. Fig. ncnrlclswt low with cells control In hglaflexneri Shigella or 823

Journal of Cell Science IPoiiae rmtepam ebaepo fPI(4,5)P of pool membrane plasma the from originates PI5P xlddteivleeto noi ntecnrlo EGFR of that control fact we the the given First, in Second, arguments. cells. endofin IpgD-expressing two of in propose degradation involvement we To the possibility, pathway. that excluded degradative this postulate the out control to TOM1 rule to of tempting cells upstream acting IpgD-expressing therefore was in partner was binding (Seet another It degradation or EGFR endofin 2001). in 2004), involved Hong, al., be et and to (Seet endofin known and being 2004) endofin al., et (Katoh tollip proteins ARTICLE RESEARCH 824 with direct by together PI(3,5)P either and or PI3P PI5P 2009). the phosphatidylinositol produces al., from myotubularins that of et and enzyme Rab5 (Zoncu phosphorylation the an the endosomes of i.e PIKfyve, compartment, precursors PI3P-rich upstream the strong although an EEA1-positive al. endosomes, a Moreover, affect et APPL1 PI3P, Bechtel to blockade. signaling the of seems in the loss PI5P documented to a study, not were to leads endosomes contrary PI5P signaling model, in our to EEA1- increase interesting and in is Rab5 It that, 2013). of al., note level et (Bechtel the endosomes Vps34- at early positive compromised fluid- 3-kinase are and uptake endocytosis phosphatidylinositol phase receptor-mediated the both However, to mice, the 2003). from invalidated al., reported et podocytes was (Petiot control transport ESCRT-0 PI3P in bulk the not to clear. with but affect interaction protein, not its Hrs shown can through is of sorting, sorting PI3P receptor step been affect and early whether transport an also However, at both TOM1 pathway. has and endolysosomal PI5P PI3P for degradation endolysosomal role cargo endocytosis. the a soluble by and of suggesting the endosomes receptor and perturbation both signaling that this affecting to for pathway, demonstrate responsible TOM1 is adaptor clearly PI5P the the of We affect to recruitment PI5P pathway. by used Here, mechanisms endolysosomal 2011). molecular al., the et unravel (Ramel have pathway we effect retrograde no or has recycling this TOM1L2 and the on pathway degradative addition, the nor modulates In specifically system. TOM1L1 TOM1. this in of TOM1 neither with upstream interacting functions redundant an that role that unlikely a demonstrates very plays is on it effects partner IpgD pathway, abrogates degradative totally alone the TOM1 of knockdown the noyoi.I hssuy oeta oefrP5 eie from derived PI5P for PI(3,5)P role potential of a dephosphorylation study, this In endocytosis. h eeomn fsvrlptoois uha acr diabetes cancer, to as related such cytoskeleton, shigellosis. closely pathologies, or actin also several is of expression, development It gene the signaling. of and functions, trafficking regulation cellular membrane the fundamental a as be multiple to such appears of PI5P coordinator TOM1 Therefore, RTKs 7). of crucial (Fig. pathway and endosomal cargoes signaling the fluid-phase the in and result, impeding a blocked, enrichment As is maturation endosomes. PI5P of signaling expression to induces which recruitment IpgD in model phosphatase network. the a endosomal in the propose in and We steps maturation trafficking endosomal discrete in of regulation PI5P of role unprecedented early maturation. very endolysosomal at of endosomes) cargoes signaling the (i.e. of stages blocking the explain could difference ta. 99 Tronche 1999; al., et fetwsatiue oPI(3,5)P PIKfyve The to 2003). attributed al., (RTK) et was kinase (Ikonomov tyrosine effect EGFR receptor the on like effect receptors, no has but endocytosis napeiu td,w hwdta IPpoue yIpgD by produced PI5P that showed we study, previous a In ae oehr h eut nti td ugs an suggest study this in results the together, Taken ` ee l,20) otosfluid-phase controls 2004), al., et re 2 a o vkd norsystem, our In evoked. not was 2 ciga ae tg of stage later a at acting 2 aha (Sbrissa pathway hglaflexneri Shigella 2 This . h xrsino noi RAwsnraie gis GAPDH against normalized was mRNA endofin expression. mRNA of expression The oto a g a rmAcm(b76) eodr horseradish Alexa- and Secondary Biosciences Promega, (ab37361). from BD Abcam were #4267). from antibodies D38B1, from (HRP)-coupled was (clone peroxidase (#H00009765-D01, IgG Technology were Signaling rat anti-endofin EGFR Cell Control from against Sciences), or (#555996) Antibodies Life #ALX-804- Abnova). A-5, Enzo (clone anti-Hrs 382-C050, Upstate-Millipore), anti- (06-730, Biotechnology), Cruz RhoGDI Cruz Santa Santa (sc-13119, sc-138, anti-Hsp90 B-14, Biotechnology), (clone anti-GST Sigma-Aldrich), anti- #4060, (A5441, Biosciences), D9E, BD actin (ab99356, (#610457, (clone anti-EEA1 sc- (S473) anti-TOM1 Technology), H-136, Signaling anti-pAkt Cell follows: Biotechnology), (clone Cruz as anti-Akt Santa The were Abcam), 8312, ZFYVE16). (ab96320, study as anti-TOM1L2 this known Abcam), (also in endofin used silence antibodies to used was (Ambion) UCCCAUCCUGCUAACGACUAUGAUU-3 CAGACACCATG-3 as was and 2011). al., France) et Nice, (Visvikis U895, provided previously (INSERM kindly described Lemichez was Emmanuel peGFP-C2-TOM1 Dr The by chemicals Sigma-Aldrich. Other from Technologies. Life were from were antibodies Fluor-coupled n 5 and H oano O1 l osrcswr eiidb double-strand by the verified in were mutations constructs All KRKK TOM1. the sequencing. of introduce domain to VHS performed was mutagenesis eindadsnhszdacrigt h eune(es)5 (sense) sequence the to the targeting according siRNA The synthesized was cells. and and MEF origin) on (mouse designed study cells 3 MEF this mouse in in s19514 TOM1 only used TOM1, gave deplete therefore mouse to and and differences efficient human origin), the the was (human between to sequence Owing cells mRNA study. HeLa in the in throughout able results were TOM1 indistinguishable siRNAs silence both that efficiently showed to characterization Initial s19515. Silencer and – targeting used oligos different were Silencer Two TOM1 the (#4390843). human 1 was No. used Control control Negative negative Select The Ambion. from were siRNAs Materials METHODS AND MATERIALS a nue yrmvlo oyyln o 4h. 24 for and doxycycline of IpgD selected removal of by Expression were method. induced dilution was Transformants limiting the expression. with single-cell-cloned transgene the suppress (500 using medium system generated G418-containing Tet-Off were in the cultured for Inp54p were validated or Clontech cells MEF from IpgD Briefly, expressing methods. standard stably cells lines cell MEF stable Tet-OFF of Generation CG using peGFP-C2- vector pGEX-KG using the PCR into frame by in cloned Bam amplified and template were a as TOM1 TOM1 of forms mutagenesis Truncated site-directed and cloning Molecular 5 and cDNA, prepare To GeneElute Sigma. the from using kit prepared Miniprep 1 were Mammalian cells RNA transfected Total from RNA Total qRT-PCR h olwn rmr:5 primers: following the CATGGGGAACTCCAGGC-3 codn otemnfcue’ ntutos fe 8h hygromycin h, 48 After instructions. Technologies) manufacturer’s al., (Life (150 et the Reagent (Ramel Plus to Inp54p with or according LTX pTRE2-hyg-2myc IpgD Lipofectamine for described using sequence previously 2011) coding the the including with vector transfected were and 5 primers: following the using amplified was domain VHS The below). 5 endofin, recommendations. follows: manufacturer’s as the were following Primers Technologies), (Life m GGATCCATGGACTTTCTCCTGGG-3 a ees-rncie sn uesrp I ees transcriptase reverse III Superscript using reverse-transcribed was g Iand HI m /l a de oteclswt oyyln (2 doxycycline with cells the to added was g/ml) 9 9 -CG -TGAGCAGTGGTTAGAATC-3 9 URo O1(eerda iO1UR a custom was siTOM1-UTR) as (referred TOM1 of -UTR ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal GAATTCCTCATTTCCGTTCATGGCGC-3 Eco Irsrcinsts(nelndi h rmrsequences primer the in (underlined sites restriction RI 9 n 5 and 9 -CG 9 9 -TGTAGTTGAGGTCAATGAAGGG-3 h A oanwsapiidusing amplified was domain GAT the ; GGATCCACTGACCTGGACATGC-3 H eetPeDsge iN s19514 siRNA Pre-Designed Select 9 -AGGTAGGATGGACAGTTA-3 9 AD,5 GAPDH, ; 9 9 n 5 and h iN s18852 siRNA The . 9 -CG 9 9 GAATTCTCA- Site-directed . -ACATCGCT- m /l to g/ml) m l/ml), 9 9 9 H - 9 - 9 .

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Journal of Cell Science Re ttsia analysis Statistical 15 ARTICLE RESEARCH 826 A. P. Scotti, and J. Lang, A., Milochau, M., Laguerre, F., Boal, Helmsta W., Bechtel, R., Kato, T. Y., Balla, Yamaguchi, T., Shiba, Y., Katoh, M., Kawasaki, M., Akutsu, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.166314/-/DC1 online available material Supplementary material Supplementary Re the Scientifique; Recherche la de National Pyre Centre the by and Sante la Me Recherche de la National pour Institut Me from Recherche grants la by de supported was study This manuscript. Funding the through co-wrote H.T. project and the F.B. of analysis. experiments. development and the the design performed in H.T. experimental assisted and data. O.B.-S. J.-M.X. the and G.C., analyzed P.J.S. B.P., E.S., and M.M., experiments M.G., the R.M., designed F.B., and conceived H.T. and F.B. contributions Author interests. financial or competing no declare authors The interests Competing We project. Payrastre this the to endosomes. of relating Ve members to discussions present grateful helpful and are for past Puhar the Andrea all and thank laboratory, to like would We Acknowledgements legends. figure the in indicated Student’s are unpaired significant be the using performed was analysis Statistical rsoi . gsii . rp,M,Mrio,F,Sooa . Lippens, V., Swoboda, F., Martinon, M., Kropf, L., Agostini, B., Brissoni, S. J. Bonifacino, Lord, A., R. Spooner, M., Zeghouf, B., R. Sessions, and P. L., Cosson, Guetzoyan, W., F., E. Boal, Smith, L., Aubry, S., Mattei, J., S. Charette, C., Blanc, B. Antonny, and J. Bigay, Be C., Lamaze, A., Benmerah, h eut r rsne stemean the as presented are results The extruded mol%). with 64:28:2:6 phosphatidylethanolamine:PIP mixed phosphatidylcholine:phosphatidylethanolamine:NBD– were nm, (100 proteins Antonny, liposomes recombinant and purified (Bigay Briefly, previously described 2005). assays as flotation performed Liposome essentially antibody. were anti-mouse-IgG followed antibody HRP-coupled anti-GST an an by using detected were proteins recombinant Bua,D.J.,Martin,G.M.,Binda,O.andGozani,O. ´gional). rmnnei h ikrdmi ftecsen-tigpoenCsp protein cysteine-string the of domain linker the in prominence Rev. Physiol. S. Wakatsuki, domain. Tom1-GAT and 5391. by cargo K. ubiquitinated of Nakayama, recognition K., Kato, rfikn fitrekn1rcpo eursTollip. requires I al. et receptor E. Meylan, interleukin-1 S., Janssens, of N., trafficking Aebi, H., Everett, S., during 9 synaptotagmin Biol. sensor Cell calcium the with exocytosis. interaction regulated human in disassembly Golgi causes J. that D. cells. Stephens, function canine and GBF1 M. and of L. inhibitor Roberts, J., An G. LG186: Clarkson, J., Cherfils, M., J. size. defined complex. 0 of liposomes F. Letourneur, on coats COP of Enzymol. cycle disassembly endocytosis. mediated N. Cerf-Bensussan, homeostasis. al. podocyte et for F. 24 endocytosis Geist, of S., importance Liu, the O., Kretz, C., Schell, hshtdlnstl5popaergltsIG tblt tdiscrete damage. DNA at to response stability in targets ING2 chromatin regulates phosphatidylinositol-5-phosphate 2270. ´ne m 727-743. , /lGTfso rtisi BT fe xesv ahs bound washes, extensive After TBST. in proteins fusion GST g/ml e n uoenfns(EE,FnsEurope Fonds (FEDER, funds European and ´es 21) hshioiie:tn iiswt in mato elregulation. cell on impact giant with lipids tiny Phosphoinositides: (2013). 5 404 23-32. , Traffic AE J. FASEB 95-107. , 93 rnqePn o e nta epwt h uiiainof purification the with help initial her for Pons ´ronique 20) itotlu o1priiae oa neta ESCRT- ancestral an to participates Tom1 Dictyostelium (2009). 20) h G rtis dposo h move. the on adaptors proteins: GGA The (2004). 1019-1137. , dcl;Aec ainl el ehrh;Fondation Recherche; la de Nationale Agence ´dicale; Traffic dcl;TeFec uclrDsrpyAscain(AFM); Association Dystrophy Muscular French The ´dicale; 10 de,M,Blc,J,Hrlbn . ifr . rjc F., Hrnjic, B., Kiefer, B., Hartleben, J., Balica, M., ¨dter, 161-171. , 19) P2Es5itrcini eurdfrreceptor- for required is interaction AP-2/Eps15 (1998). 25 .Cl Biol. Cell J. 11 132-143. , 1537-1551. , ge . cmd .L,Dur-ast .and A. Dautry-Varsat, L., S. Schmid, B., `gue, 20) eltm sasfrteassembly- the for assays Real-time (2005). 140 1055-1062. , 6 ...and s.e.m. 21) p3 eiinyreveals deficiency Vps34 (2013). c.Rep. Sci. 20) tutrlbssfor basis Structural (2005). esd De de ´ens P vle osdrdto considered -values ur Biol. Curr. ESLett. FEBS .A.Sc Nephrol. Soc. Am. J. 3 20) Intracellular (2006). 2137. , 21) charged A (2011). go Midi- ´gion 21) Nuclear (2013). ´veloppement a.Rv Mol. Rev. Nat. a ´et eitsits mediates 579 16 Methods 2265- , (2010). 5385- , t -test. emn .A. M. Lemmon, en,W,Blsa . omr . oe,D n ieh,N. Divecha, and D. Jones, L., Sommer, Y., Bultsma, W., Keune, D. S. Emr, and M. Babst, J., D. Katzmann, en .adKgr .A. A. Kiger, A. and Shisheva, S. and Jean, L. J. Carpentier, M., Foti, D., Sbrissa, B. C., O. J. Ikonomov, Dacks, and A. Helenius, M. and Giezen, J. Huotari, der van G., Walker, K., E. Herman, ao,Y,Iaaua . uasmr,M n aaaa K. Nakayama, and M. Futatsumori, H., Imakagura, Y., Katoh, oai . aua,P,Jns .R,Iao,D,Ca . uoso,A A., A. Lugovskoy, J., Cha, D., Ivanov, R., D. Jones, P., Karuman, O., Gozani, ao,Y,Sia . ishsi . aaia . aas,H n Nakayama, and D., H. Takatsu, Elouarrat, Y., Yanagida, R., H., Mitsuhashi, Y., J. Shiba, Y., Halstead, Katoh, J., W. Keune, Y., Bultsma, R., D. Jones, ovl .P,Care,P,Zra,M n rebr,J. Gruenberg, and M. Zerial, P., B., Chavrier, P., B. J. Gorvel, Szomju, N., C. Vlangos, S., Williams, M., P. Hauck, S., Girirajan, ibh,K,Guit,S,Pdo,T,Pipt,D . at,F,Sbe . Sheetz, J., Sable, F., Gaits, J., D. Philpott, T., Pedron, S., Giuriato, K., Niebuhr, Bouyssie F., Roux-Dalvai, E., Mouton-Barbosa, iPoo .adD ail,P. P. Camilli, De Camilli, and De G. Paolo, and Di P. P. Fiore, Di ua,A,Tronche A., Puhar, J. Gruenberg, and H. Stenmark, J., Faure, A., Petiot, Tronche C., Pendaries, H. J. Hurley, and M. B. Beach, S., Misra, Uttenweiler- A., Shevchenko, A., Giner, S., Christoforidis, W. Hong, M., and Miaczynska, F. L. Seet, E., Loh, S., L. Loo, S., N. Seykora, and Liu, M. Dans, C., Schmults, L., Dzubow, L., Mei, C., Marshall, W., Li, oln . rno . io,V,Be V., Simon, M., Franco, G., Collin, Urbe J., M. Clague, H. K. Klempnauer, and B. Haenig, S., Worpenberg, O., Burk, hshioiiesgaln ntenucleus. the in signalling Phosphoinositide fedctsdrcposo otn fpoahpi yregulating by D procathepsin of sorting or morphogenesis. recycling/degradation body receptors not multivesicular but endocytosed endocytosis phase of fluid controls PIKfyve (2003). 3500. 0. and ESCRT anathema of Breviata evolution amoeboflagellate enigmatic the the in bodies Multivesicular notemlieiua oyptwyrqie h ucino conserved a of function the requires ESCRT-I. pathway complex, complex. sorting body protein Tom1-Tollip endosomal multivesicular the the by into endosomes Commun. onto Res. Biophys. clathrin Biochem. of Recruitment ftecrmtnascae rti N2fntosa nuclear a as functions ING2 protein al. et L. receptor. chromatin-associated S. phosphoinositide Lessnick, J., the S. Field, of H., Zhu, L., C. Baird, rtisot al endosomes. early onto proteins K. PIP4Kbeta. for role vivo in an signaling: N. stress Divecha, Cell of and transducer S. a C. as D’Santos, PtdIns5P J., A. Heck, S., Mohammed, functions. 470. and regulation phosphoinositide al nooefso nvitro. in fusion endosome early response. Elsea, immunologic and abnormal K. 262. McCoy, and Jr, tumors L., and K. White, H. D., S. P. Mosier, S., Solaymani-Kohal, tIs45P2 noPdn() yteSfenr fetrIg eraie host reorganizes IpgD B. effector Payrastre, S.flexneri morphology. the and cell by J. P. PtdIns(5)P into Sansonetti, PtdIns(4,5)P(2) C., quantification. Parsot, P., M. protein label-free and treatment Proteomics B. Monsarrat, library O., Burlet-Schiltz, ligand E., Boschetti, al. L., et Guerrier, G., P. 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Journal of Cell Science hsea A. Shisheva, et .F n og W. Hong, and F. L. Seet, A. Shisheva, and C. O. Ikonomov, D., Sbrissa, H. Stenmark, and E. Stang, M., S., N. Pedersen, C., Dupuis-Coronas, Raiborg, I., Roxrud, J., Mounier, V., Pons, F., Lagarrigue, D., Ramel, ARTICLE RESEARCH Tronche Shin, H., Kobayashi, H., Takatsu, K., Yoshino, T., Shiba, Y., Katoh, Y., C., Shiba, A. Sandberg-Nordqvist, M., Kost-Alimova, D., Kedra, E., W. Seroussi, Hong, and J. B. Hanson, N., Liu, F., L. Seet, W. Hong, and F. L. Seet, n deeds. and proteins ubiquitination. and binding mouse K. ubiquitin endosomal Nakayama, for responsible and and the S. al. Wakatsuki, W., et to 22q13.1 H. S. similar chromosome Imreh, proteins A., human B. STAM. encode Roe, to and and Q., HGS map H. 8C1 Pan, genes Y., chromosome Fu, TOM1 F., J. (1999). Jacobs, I., Fransson, endosomes. TOM1. Chem. Biol. J. mediate of Effect 5-phosphoinositides. to synthesizes kinase, Hrs lipid insulin. Fab1p with yeast of interacts ortholog receptor. Eps15 factor growth of epidermal of isoform degradation localized endosomally signaling. Tronche EGFR F., of termination Gaits-Iacovoni, prevent and J., endocytosis P. B. Sansonetti, Payrastre, G., Chicanne, -hsht ytephosphoinositid cells. the mammalian B. by Payrastre, and 5-phosphate L. J. Mandel, L., r,H,Lpre . edre,C,Casae . iue,L,Pirola, L., Liaubet, C., Chaussade, C., Pendaries, J., Laporte, H., `re, .Cl Sci. Cell J. .Bo.Chem. Biol. J. 21) tIsP esadveso t perne disappearance appearance, its of views and news PtdIns5P: (2013). rh ice.Biophys. Biochem. Arch. .Bo.Chem. Biol. J. 21) hglafenr neto eeae h ii IPt alter to PI5P lipid the generates infection flexneri Shigella (2011). 276 Genomics .Bo.Chem. Biol. J. 42445-42454. , 118 274 575-587. , 20) noi eriscahi oeryedsmsvia endosomes early to clathrin recruits Endofin (2005). 20) noi,a nooa YEdmi protein. domain FYVE endosomal an Endofin, (2001). 21589-21597. , 279 57 380-388. , 4670-4679. , 279 7304-7312. , 20) rdcino phosphatidylinositol of Production (2004). 538 -hshts ytblrnin myotubularin 3-phosphatase e 171-180. , 20) A GAadTm)domain Tom1) and (GGA GAT (2004). 20) noi erisTM to TOM1 recruits Endofin (2004). 19) Ifv,amammalian a PIKfyve, (1999). .Cl Biol. Cell J. .Bo.Chem. Biol. J. c.Signal. Sci. 180 279 1205-1218. , r,H and H. `re, 7105-7111. , 20) An (2008). 4 ra61. , ivks . oe,L,Trio . oe . eone,M,Lore M., Lemonnier, A., Doye, S., Torrino, L., Boyer, O., Visvikis, o ato,M n okn A. Sorkin, and M. Zastrow, von iu,J,Lgriu,F,Rml . lat . hcne . ect,L., Ceccato, G., Chicanne, S., Allart, D., Ramel, F., Lagarrigue, J., Viaud, B. Payrastre, and F. Gaits-Iacovoni, H., Tronchere, F., Boal, J., Viaud, R., J. Testa, T., Ma, X., Lou, H., Gacula, I., Niesman, Q., M. Dong, T., Varsano, J. Kendrick-Jones, A., N. Bright, D., S. Arden, J., B. Waxse, A., D. Tumbarello, oc,R,Prr,R . akn .M,Pruclo . Toomre, M., Pirruccello, M., D. Balkin, M., R. Perera, R., Zoncu, H. J. Hurley, and T. Wollert, W. Hong, and F. L. Seet, S., N. Liu, T., Wang, oipouigCF oi iak olpt rge a1dpnetcl invasion. cell Rac1-dependent trigger to al. Tollip et Traffic hijacks D. toxin Bouvard, CNF1 A., producing Mettouchi, coli G., Flatau, M., Rolando, orleu . urb .M,Prz . arsr,B tal. et B. and Payrastre, binding O., through Pertz, invasion Tiam1. of M., of regulates activation tuner J. 5-phosphate a Xuereb, Phosphatidylinositol and D., Courilleau, lipid stress dynamics. nuclear cytoskeleton and A membranes 5-phosphate: Phosphatidylinositol signaling. and trafficking TrkA regulates Biol. G. and Cell. M. endosomes Farquhar, TrkA and the peripheral 3rd R., with J. Yates, fusion and maturation autophagosome lysosome. Tom1-dependent mediate F. Buss, and auainadsgaigpoete fAP endosomes. APPL of properties P. signaling 1121. Camilli, and De maturation and D. complexes. ESCRT by biogenesis trafficking. membrane in 11 Tom1L2 and Tom1L1 Tom1, domain-containing Biol. Cell Opin. 1119-1128. , 12 579-590. , ora fCl cec 21)18 1–2 doi:10.1242/jcs.166314 815–827 128, (2015) Science Cell of Journal 26 a.Cl Biol. Cell Nat. 8942-8952. , 21) uohg eetr ikmoi It uohgsmsto autophagosomes to VI myosin link receptors Autophagy (2012). 19 436-445. , a.Commun. Nat. 14 21) oeua ehns fmlieiua body multivesicular of mechanism Molecular (2010). 20) hshioiiesic otosthe controls switch phosphoinositide A (2009). 1024-1035. , 20) inln nteedctcpathway. endocytic the on Signaling (2007). Nature 5 4080. , 20) ICi erie yAP to APPL by recruited is GIPC (2006). Bioessays 464 21) h mrigrl fVHS of role emerging The (2010). 864-869. , 21) Escherichia (2011). Cell 136 (2014b). (2014a). 1110- , s P., `s, Traffic Curr. 827 Mol.

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