Ato o orsodne([email protected]) correspondence for *Author Bristol Bristol, of University UK. Building, 1TD, Sciences BS8 Medical Biochemistry, of School ieSine,Thk nvriy edi9087,Japan. 980-8578, Sendai University, UK. Tohoku 1TD, Sciences, BS8 Life Bristol Bristol, of University Building, Sciences Medical a .McGough J. Ian recycling SNX27–- -to-plasma-membrane in mediated heterogeneity molecular of Identification ARTICLE RESEARCH ß 4940 2014 September 3 Accepted 2014; May 12 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 2 been for has 1 much endosome Although network 2011). the trans-Golgi Helenius, the from and to or (Huotari internalized exported membrane (TGN) plasma are the to network, they recycling degradation for alternatively, into endosomal the endosome or, to the delivery either of for membrane – vesicles limiting the intraluminal fates the from principal sorted two are in have they cargos arriving transmembrane On INTRODUCTION nexin Sorting Retromer, VPS35, VARP, SNX27, WORDS: KEY sorting essential complex. this of understanding molecular our in up directions data new open these and sorting Overall, retromer-mediated pathway. in trafficking complexities hidden this reveal in VARP with concert in acts retromer that establishing for (Tyrp1), tyrosinase 1 enzymes, protein tryrosine-related melanogenic role of and the of Suppression mistrafficking a to transport. led identified VPS35 endosome-to-melanosome interaction in retromer–VARP Further retromer the . of retromer-interacting analysis confirming for thereby functions cargo, functional retromer cargo-specific affect uniformly of not did complex degree retromer-associated WASH the surprising of suppression this, Extending sorting. a cargo- endosome-to-plasma-membrane retromer-mediated and suggested in heterogeneity differential trafficking effects but in cargo, resulted specific retromer-dependent proteins these of of defects Depletion ANKRD27). known ankyrin-repeat also as (VARP, protein including VPS9-ankyrin-repeat antigen and cancer proteins, (SDCCAG3) colon 3 defined interacting seriologically (ANKRD50), new 50 domain identified of has This number component. interactome retromer a the core the define and VPS35, to we of proteomics endosome-to-Golgi Here, quantitative of employed into pathways. sorting have the transport proteins orchestrates endosome-to-plasma-membrane that cargo assembly protein transmembrane a is Retromer ABSTRACT isnr Fukuda Mitsunori eateto eeomna ilg n ersine,Gaut colof School Graduate Neurosciences, and Biology Developmental of Biochemistry, Department of School Laboratories, Signaling Integrated Wellcome Henry The 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,44–93doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 lra Steinberg Florian , 2 n ee .Cullen J. Peter and 1 ate Gallon Matthew , 1, 3 * rtoisFacility, Proteomics 1 yk Yatsu Ayaka , p5adVs7 n eeormr lsial emdtecargo- proteins the termed membrane classically domains] heterotrimer, a a (BAR) and Vps17, – and Bin/Amphiphysin/Rvs Vps5 yeast, (SNX) subcomplexes nexins possess In [sorting 1998). two SNX-BAR that al., the of of et heterodimer Seaman composed remodeling 1997; is al., et to retromer (Seaman from TGN sorting Vps10 the essential the was of recycling that the pathways ‘retromer’ for termed export complex protein various pentameric the the regulated into into are sorted cargos understood. events poorly and of remains sorting selected retrieval these are during how cargos pathway, how degradative about learned o h aeedsm-soitdRb Saa ta. 09,and 2009), al., protein et GTPase-activating (Seaman Rab7 late-endosome-associated potential the accessory for a these TBC1D5, among are that Included factors proteins accessory sorting. for of hub retromer-mediated number recruitment its aid a a of besides is association CSC that, endosomal retromer the the view selection, this emerging cargo into in an insight role is providing heterogeneity Potentially pathways endosome-to- 2014). underlying distinct transport Cullen, sort into and to proteins endosome-to-plasma-membrane (Burd able cargo and is of CSC array TGN common wide the a how such of question fundamental 2013). al., during al., target et et McDonough a Finsel 2013; as 2011; al., and et al., Lipovsky 2011), 2013; et al., (Kingston et interactions Parkinson Zimprich Vilarin host–pathogen 2011; 2008; and al., of et al., number disease Wen et a Alzheimer (Muhammad in including disease emerging is diseases, Moreover, function events. human retromer sorting of the endosomal in deregulation conductor multiple master of a The as orchestration emerged 2012). of therefore Korswagen, array has and wide CSC retromer (Cullen a sort cargos endosome-to- to distinct also act functionally they but al., and recycling transport, et endosome-to-TGN plasma-membrane roles Zhang distinct in 2011; have only complexes al., These al., not et 2013). et al., Temkin et Wassmer 2011; Steinberg 2004; 2011; al., al., et Harterink et 2009; (Carlton SNX27–retromer three of has the SNX3–retromer component and the retromer core SNX-BAR–retromer, the the 2011). of – as complexes role residing sorting al., CSC the the et with metazoans, expanded, higher (Koumandou in conserved Unsurprisingly, highly been eukaryotic has and throughout ancestor eukaryotic common last 2014). Cullen, and (Burd transport transport cargo-enriched endosome-to-TGN for generate enrichment carriers to cargo remodeling and membrane coat selection a with cargo as Vps10 serves co-ordinating therefore including for yeast cargo in complex Retromer recognize 1998). to al., and shown et Vps29 (Seaman been Vps26, has of and composed is Vps35 that (CSC), complex selective ineigsuisi es e oteietfcto fa of identification the to led yeast in studies Pioneering h iesfcto fterlso ermrhsrie the raised has retromer of roles the of diversification The the before evolved that assembly protein ancient an is CSC The 2 oiioOhbayashi Norihiko , 2 aeJ Heesom J. Kate , ˜o-Gu ele l,2011; al., et ¨ell 3 ,

Journal of Cell Science EERHARTICLE RESEARCH i.1. Fig. e etpg o legend. for page next See ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal 4941

Journal of Cell Science ihsal upeso fedgnu P3 uiga hN lentivirus shRNA an (using 3 VPS35 the endogenous targeting of suppression stable with localized quantitative endosomally interactors. a novel using retromer reveals interactome approach VPS35 proteomic the SILAC of Identification 1. Fig. ARTICLE RESEARCH 4942 the in retromer for role new . lysosome-related a this identify VARP-mediated of we biogenesis 2009), in in al., biogenesis and et Finally, melanosome (Tamura tyrosinase during retromer function. (Tyrp1) enzymes 1 melanogenic retromer protein for tryrosine-related the of of requirement transport underlying analysis endosome a reveal further the describing we framework molecular aid – a endosome-to- to provide retromer-mediated CD97 and transport in plasma-membrane receptor TNF-receptor heterogeneity TNFRSF10A) adhesion mechanistic as the the known MCT1, (also and TRAILR1 transporter member super-family monocarboxylate SLC2A1), as known (also the GLUT1 transporter glucose the – cargos SNX27–retromer distinct functionally late four of analysis regulates comparative (Scha closed lysosome thereby a the with in and fusion endosome VAMP7 conformation R-SNARE the inactive traps and fusogenically Rab21 2006) against al., activity et (Zhang exchange guanine-nucleotide possesses as that known Rab38 and (also Rab32 described VARP for effector endosome-associated and an ANKRD27), 2013), no al., multiple et (Hagemann implicated cytokinesis with a protein in endosome-associated protein an ANKRD50, SDCCAG3, containing function; include domain functionally These have ankyrin-repeat recycling. we that membrane endosome-to-plasma- retromer-mediated proteins of context accessory the in validated retromer number new a identified has of including this complex, interactors, WASH established the of to components addition all In isotope VPS35. interactome human stable the labeling of quantify (SILAC)-based to of culture spectrometry cell mass employment high-resolution in and the acids amino describe with labeling here of heterogeneity we and functional the retromer, into Gomez insight additional 2009; provide and al., et on (Derivery filaments 2009). network Billadeau, branched regulating homolog endosomal of by SCAR formation the that, the complex and promotes multi-protein Arp2/3, protein a complex, syndrome (WASH) Wiskott-Aldrich the 10 bars: Scale right. the to magnification Boxed higher (red). at VPS35 shown endogenous are SDCCAG3 against areas or antibody RME-8 an Myc, with against co-stained raised and antibodies with were stained cells and HeLa fixed untransfected cells and VARP–Myc HeLa with VPS35. transfected with transiently colocalize indicated. RME-8 also the and are of SDCCAG3 proteomics VARP, enrichment SILAC (E) fold VPS35 and the peptides in of proteins number indicated The proteins. binding indicated for the analyzed to subsequently and (GFP-IP) were GFP-nanotrap GFP–VPS35 a or to GFP subjected extracts with transduced Cell lentivirally blotting. cells RPE1 western from by derived confirmed were associate. interactions Novel to known (D) complexes represent protein Colours or database. interactions STRING protein–protein SILAC the the using in performed identified was interactome. components proteomics VPS35 interactome the VPS35 in of found analysis The were Network (C) interactors classifications. retromer those known between within of proteins edge majority of the the overlap greater thicker the the the greater node, category; the red that nodes, the in larger classified The proteins peptides. of two number of minimum a with and rtisietfe nteVS5SLCpoemc iha with to proteomics SILAC annotations VPS35 Ontology the in identified assign proteins to VPS35 used the was DAVID in interactome. ‘transport’ and in ‘localization’ involved localization’, proteins of of ‘establishment preponderance a Gene revealed (B) annotations antibodies. Ontology anti-tubulin and anti-VPS35 with immunoblotted ofrhrepoeterl fteCCa nedsmlscaffold endosomal an as CSC the of role the explore further To 9 T) ro otesal eepeso fGPVS5 were GFP–VPS35, of re-expression stable the to prior UTR), A yae rmcnrlRE el n P1cells RPE1 and cells RPE1 control from Lysates (A) fre l,21) Through 2012). al., et ¨fer . .-odenrichment 2.5-fold m m. ta. 02 i ta. 02.Wsenbo nlssof with associated analysis were 1E). (Fig. blot VARP, endosomes retromer-decorated myc-tagged Western immunofluorescence expressed and a thetransiently 2012). and RME-8, 1D), and (Fig. SDCCAG3 confirmed endogenous al., that protein established analysis each GFP et of versus enrichment Jia GFP–VPS35 2012; Harbour immunoprecipitated 2010; al., al., et complex al., Harbour VPS35, et et WASH 2009; (Derivery Billadeau, to CSC the and the Gomez binding with and 2009; WASH direct 2014), of association through al., the mediates which, et FAM21, Freeman Shi component 2009; 2009; previously al., protein al., et a (Popoff et DNAJC13), sorting as retromer-mediated known in implicated (also the RME-8 characterized further of we role addition, In ( peptides). SDCCAG3 quantified five and peptides) quantified ( seven VARP ( were ANKRD50 peptides), quantified proteins 46 enriched, These of analysis. levels functional high and with validation further each for proteins, peptides, quantified numerous three and enrichment top the Of selected S2). we Table these, material (supplementary identified were interactors 2011)]. al., et (Swarbrick a oe n‘salsmn flclzto’ lclzto’and ‘localization’ localization’, of interactors ‘establishment these of in majority roles the had that of revealed more enrichment analysis or two ontology an from quantification of and protein filtering – were peptides criteria after two proteins interactome by VPS35 data 63 the the S1), comprise to Table considered and material quantified 2530 (supplementary SDS-PAGE From proteins digestion. tryptic spectrometry by in-gel mass after (LC-MS/MS) chromatography-tandem separated liquid by combined, quantified 2008). were al., et (Trinkle-Mulcahy Precipitates the method SILAC using GFP-trap precipitated efficient was in RPE1 highly GFP cultured an lysis, was cellular alongside that Following GFP medium medium. expressing SILAC stably heavy cell line resultant in the cell The cultured 1A). (Fig. was of levels the line endogenous to targeting which close at in (sh)RNA-mediated VPS35 line hairpin cell 3 through RPE1 suppressed short stably an To was lentiviral 1998). generated VPS35 endogenous al., first of et expression we (Seaman CSC the end, a retromer VPS35, this obtain the human of to component of of core sought basis interactome mechanistic we high-resolution the quantitative sorting, into insight endosomal unbiased retromer-mediated and new interactome VPS35 gain the To of analysis proteomic Quantitative RESULTS h nooet-lsammrn eyln favreyof variety a examined of and FAM21, recycling and three endosome-to-plasma-membrane RME-8 all of of that the retromer-associated as expression well new the as the suppressed interactors, of individually role we functional proteins, the establish To function of loss SNX27–retromer phenocopies ANKRD50 of suppression RNAi-mediated K a has SNX1 [the with interaction this VPS29 of entirely the of affinity of low is enrichment interaction apparent of SNX-BAR–retromer actin the lack the with The the consistent 2012; 2013). of al., al., of components et et Gomez Hao Harbour SNX-BAR 2012; components 2010; 2009; al., al., et al., et all SNX27 Jia Harbour et 2009; and 2010), (Derivery Billadeau, and al., 2013) complex WASH et al., polymerizing (Harbour et TBC1D5 and (Steinberg VPS26A the VPS29, partners confirmed VPS35-interacting VPS26B, 1C) known (Fig. the analysis of presence Network 1B). (Fig. ‘transport’ 9 T.It hscl ie esal eepesdGFP-tagged re-expressed stably we line, cell this Into UTR. eie hs salse idn ates ubro new of number a partners, binding established these Besides ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal . .-odoe oto.Gene control. over 2.5-fold . . 0-odenriched, 100-fold 0-odenriched, 100-fold d of . . 100-fold 150 m M

Journal of Cell Science EERHARTICLE RESEARCH i.2. Fig. e etpg o legend. for page next See ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal 4943

Journal of Cell Science * DCG,VR rAKD0() oe ra r hw thigher 10 at bars: shown Scale are right. areas the Boxed to RME-8, (B). magnification FAM21, ANKRD50 VPS35, or for VARP deficient cells SDCCAG3, the HeLa of in and LAMP1 accumulation GLUT1 marker lysosomal endogenous lysosomal the of and in staining ANKRD50 increase Immunofluorescent VPS35, an GLUT1. (B,C) to control. indicated leads loading the suppression a against FAM21 as siRNAs shown with is transfected Tubulin were targets. that cells HeLa from VPS35 the endosome-to- transport. in retromer-mediated identified plasma-membrane affects interactors screen novel proteomics the SILAC of Suppression 2. Fig. ARTICLE RESEARCH 4944 (Piotrowski T-lymphocytes in recycling GLUT1 in WASH1 of for regulation requirement the reported previously the with transport Consistent GLUT1 reveals retromer-mediated proteins in accessory heterogeneity retromer other the of Analysis in is complex. process ANKRD50 sorting this SNX27–retromer in for the role with Taken its and role linked mechanistically, cargos TRAILR1. that, a evidence multiple and provide of identify CD97 sorting data endosome-to-plasma-membrane MCT1, endosome-to-plasma- these of the together, in sorting inhibit a function treating phenocopied membrane SNX27–retromer also in to by ANKRD50 of discussed of be loss suppression reversed to bafilomycin later, and detail was Finally, more 3B). with (Fig. GLUT1 degradation lysosomal the cells of ANKRD50-suppressed the level in decrease 2D,E). a into observed whole-cell (Fig. GLUT1 the transporter and internalized pathway, of lysosomal-degradative the missorting the protocol, of a with level biotinylation consistent whole-cell Entirely surface the as in LAMP1-labeled GLUT1, a of decrease a expression through surface of in to decrease determined suppression a localization Moreover, to led GLUT1 2B,C). ANKRD50 (Fig. in compartment increase lysosomal corresponded a which an in surface, cell resulted the to at ANKRD50 expression of GLUT1 the of Like suppression loss to 2B). VPS35, localized (Fig. of mostly recycling suppression was efficient GLUT1 indicating membrane, cells, plasma efficient control highly In was 2A). ANKRD50 (Fig. and RME-8 SDCCAG3, surface. confocal VARP, cell the by at transporter this cells of expression HeLa specific levels whole-cell the of and in analysis biochemical trafficking through and microscopy GLUT1 analyzing proceduresfor established we Hence, the 2013). the al., in et as (Steinberg degradation lysosome enhanced levels, whole-cell and missorting in cell undergoes decrease the transporter corresponding at VPS35 GLUT1 a component of (RNAi)-mediated amount and CSC the surface in interference retromer decrease pronounced the a RNA to SNX27–retromer or leads the 2013). SNX27 by of al., mediated suppression being et latter PDZ-motif- the (Steinberg 2009), (Wieman and al., recycling endocytosis et endosome-to-plasma-membrane of it where steady dependent from rounds membrane At plasma continuous 2013). the undergoes at the al., localized is et on GLUT1 (Steinberg state, GLUT1 focusing transporter initially glucose cargos, SNX27–retromer-dependent the show Data siRNAs. the indicated from the MCT1 with shown. and transfected mean also GLUT1 cells is of HeLa lysates loss of cell the surface total of in representation western GLUT1 Graphical quantitative of (E) by abundance surface determined The the was (D). and MCT1 blotting siRNAs and indicated GLUT1 the of interactors. with abundance retromer transfected certain were of cells suppression HeLa upon decreased are GLUT1 * mean P P , , nHL el,RA-eitdsprsino P3,FAM21, VPS35, of suppression RNAi-mediated cells, HeLa In .5(nardStudent’s (unpaired 0.05 .5(nardStudent’s (unpaired 0.05 6 6 ...[he MT)adsx(LT)idpnetexperiments]; independent (GLUT1) six and (MCT1) [three s.e.m. experiments, independent three in acquired cells (150 s.e.m. t t -test). ts) DE ufc eeso C1and MCT1 of levels Surface (D,E) -test). A etr ltaayi flstsderived lysates of analysis blot Western (A) m .()Tedt hwthe show data The (C) m. n 5 3); needn nlsso h oeo APi LT otn,we sorting, GLUT1 in VARP of role further by a the provide of To 2B–E), analysis analyzed 2C,E). independent (Fig. (Fig. trafficking significant GLUT1 when but procedures of mild disruption a phenotype revealed biochemical quantification subsequent GLUT1 although or robust immunofluorescent GLUT1 VARP a of nor level RME-8 generated a neither include of might Suppression the control. that consistent transcriptional phenotype Furthermore, 3B), complex (Fig. shown). a degradation with lysosomal partially not be blocking only (data by could GLUT1 rescued of ERC level whole-cell the the in not in reduction did or GLUT1 this of 2B–E) accumulation but (Fig. obvious levels, an whole-cell with in correlate reduction a and expression accessory VARP. retromer and endocytic RME-8 SDCCAG3, specific trafficking -labeled to GLUT1 extended of SNX27–retromer-mediated and efficient requirement for SNX1- proteins the in appeared a the heterogeneity majority in (Fig. to (ERC) compartment trapped localized the recycling GLUT1 be 2B,C); internalized (Fig. to of lysosome portion as LAMP1-labeled small suppression, did ANKRD50 a suppression or only FAM21 retromer phenocopy However, completely 2B–E). cell not (Fig. reduced-whole GLUT1 and of expression levels surface FAM21 GLUT1 component decreased complex WASH also the of suppression 2013), al., et sntfntoal eurdfrteSNX27–retromer-mediated the for GLUT1. required of sorting endosome-to-plasma-membrane functionally it not endosomes, upon retromer-decorated to is localized observed is and kinetics retromer associates with degradation VARP although Overall, 4B). GLUT1 the (Fig. suppression phenocopy retromer in to increase VARP failed following clear suppression degradation VARP GLUT1 Again, of suppression. kinetics the quantified ofrhrpoetemlclrhtrgniyi SNX27– in heterogeneity molecular we sorting, the endosome-to-plasma-membrane probe retromer-mediated further To additional of proteins analysis accessory through retromer provided SNX27– is the pathway within retromer heterogeneity endosomal of evidence of Further indicative conditions. are SNX27– VARP-suppressed these data under for cargos degradation these retromer enhanced an of to leading All missorting material S1Aiv). (supplementary lysosomal the Fig. TRAILR1 in internalized exofacial increase an of an revealed accumulation against also of receptor antibody and this levels an of 6Aiii,B,Civ), epitope using (Fig. total assays TRAILR1 assays, and uptake and TRAILR1 cell-surface imaging-based 5Aiii,B,Civ) reduced (Fig. and in CD97 greatly the cytometry rates revealed VARP-suppressed and flow of also degradation GLUT1 analysis employing Further as normal 4B). cells, MCT1, (Fig. displayed assays for parallel specific cells receptor VARP-suppressed was was in MCT1 transferrin This rate transporter of degradation this 4B). enhanced (Fig. of kinetics an abundance to degradation 4A). reduced due (Fig. western the the by immunofluorescence that of established defined and analysis as 2D,E) MCT1, (Fig. Biochemical in of reduction analysis pronounced levels a blot total to subsequent led and previously, 2013) published cell-surface GLUT1, al., as et and, (Steinberg VARP on VPS35 of retromer between Suppression MCT1, effects link VARP. functional cargos and a subtle revealed SNX27–retromer TRAILR1 additional and the CD97 the of to examination contrast their In for required transport is VARP endosome-to-plasma-membrane cargos, SNX27–retromer other For upeso fSCA3ldt eraei LT surface GLUT1 in decrease a to led SDCCAG3 of Suppression ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal A.Itrsigy hsapparent this Interestingly, 3A).

Journal of Cell Science EERHARTICLE RESEARCH NR5 xrsingnrlypeooidals fSNX27– of ANKRD50 of suppression loss analysis, cytometric flow a From retromer. phenocopied generally expression ANKRD50 assays uptake intracellular TRAILR1 S1Av,vi). in lysosomal Fig. the For material using increased increase (supplementary observed 6Aiv,v). an (Fig. an was Similarly, accumulation TRAILR1 with 6Cv,vi). (Fig. 5Aiv,v) of correlated (Fig. accumulation expression levels this surface surface TRAILR1, CD97 the on and effects WASH minor polymerizing produced actin transport. require the and sorting to endosomal and optimal appears for cargos GLUT1 complex FAM21 SNX27–retromer only with study, those present association of the Thus, in examined S1Aiii). supplementary 6B,Ciii; Fig. Fig. trafficking 5Aii,Ciii; TRAILR1 material Fig. and 3C,D; CD97 Fig. MCT1, 2D,E; on (Fig. effects minor additional that only no had notable or depletion FAM21 where through was complex WASH It the conditions suppressed. of disruption ce were MCT HeLa under proteins endogenous from of derived accessory assays lysates staining retromer of Immunofluorescent analysis (D) parallel blot control. Western loading performed (C) a lysates. as 10 total-cell shown bars: in is higher Scale levels Tubulin at cells. GLUT1 shown targets. of HeLa are indicated FAM21-deficient analysis areas the to and Boxed against prior control cells. siRNAs h HeLa with FAM21-deficient 12 in transfected further compartment. uptake a were h transferrin-positive for and 1 DMSO after SNX1- in 10 dsRed–transferrin a bafilomycin bars: and in Scale GLUT1 GLUT1 right. or the of to SNX1, accumulation magnification and intracellular GLUT1 an endogenous to of leads staining suppression FAM21 3. Fig. ial,ada rel icse rvosy upeso of suppression previously, discussed briefly as and Finally, suppression RNAi-mediated SDCCAG3, and RME-8 For m .()HL el eetasetdwt h niae iNsad 8hltr eetetdwt ihrDS r0.1 or DMSO either with treated were later, h 48 and, siRNAs indicated the with transfected were cells HeLa (B) m. m m. i.6vi,wihw ofre o RIR hog uptake 5Cvii; through (Fig. TRAILR1 accumulation S1Avii). for Fig. confirmed material intracellular (supplementary assays we increased which 6Cvii), their Fig. suppression ANRD50 to TRAILR1, and led CD97 both TRAILR1 For of 6Avi). expression surface (Fig. the in decrease similar a alongside an to led ermrbnig qiaett hto ullnt AP was VARP, full-length of that Robust to 7B). (Fig. equivalent binding binding, retromer ANKR retromer isolated for the sufficient of being neither domains with various consistent in were ANKR2 cells overexpressed with HEK293 constructs and deletion experiments ANKR1 VARP mStrawberry-tagged Co-immunoprecipitation regions, in and repeat domain 7A). complex (Fig. VPS9 ankyrin retromer N-terminal the C-terminal an to two comprises VARP VARP of detail. TRAILR1, binding and more CD97 and the MCT1, retromer analyzed of sorting we between endosomal relationship the in functional VARP strong N- the its Given in region a through terminus retromer with interacts VARP ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal , 5 eraei h ufc eeso D7(i.5Avi), (Fig. CD97 of levels surface the in decrease 35% A Immunofluorescent (A) l that lls 4945 1in m M

Journal of Cell Science EERHARTICLE RESEARCH tbehtrtie nwihsprsino nindividual an of suppression 4946 which further in is and CSC heterotrimer the VPS35 7C); was stable (Fig. against and VPS29 effect targeting a off-target siRNA 2A siRNA an Fig. an second of with in result confirmed a the evident with not was also established this 7C; That (Fig. 4B). protein Fig. VPS35 VARP against endogenous that by siRNA decreased observe consistently were to levels intrigued its for were retromer We requires VARP of expression proportion stable a cells, HeLa associated In the their include and might domain which individual each retromer, regions. of to more folding binding a correct the of ANKR1 suggestive in and VPS9 is domains N-terminal, when 1–597 This the the between mutant 7B). or relationship (Fig. complex VARP VARP mutant wild-type 1–461 deletion for VARP the observed that in binding with retromer mutant B). compared of reduced level (Fragment the deletion significantly that 1–461 note was residues to VARP important expressing is was whereas a it mutant binding However, a retromer that with A), to (Fragment detected lost evidence 1–367 residues was provided expressing binding analysis 7B). retromer Fig. deletion in C (Fragment ANKR1 Further 1–597 and residues domain N-terminal VPS9 the the spanning encoding construct a with retained , 0 nclstetdwith treated cells in 50% P3 i o etr h nooeascaino stably of association of endosome suppression the 7D). (Fig. RNAi-mediated perturb VARP myc-tagged 2009), not of transduced al., did ability et Rab38– the VPS35 and (Tamura with Rab32–GTP endosome-associated of consistent GTP various to association bind entirely endosomal to of the and VARP for case association However, the examined VARP. also we endosomal was complex, this WASH the whether the including aiding proteins accessory for scaffold retromer- the on complex and functional of a endosomes. VARP sorting form decorated to on 1C,D), the appear (Fig. colocalization in CSC retromer cargos strong relationship SNX27–retromer functional their requires specific their retromer, VARP and to of the endosomes VARP with proportion together of Taken a stability. binding its for that retromer with indicate association These 2004). of therefore Seaman, expression 2004; al., of data et loss (Arighi and subunits remaining destabilization the overall to leads subunit h salse oeo APi eaooeboeei.Here, biogenesis. melanosome in VARP analyzing observed of to the role turned we established of VARP, the significance and retromer functional between the interaction validate further To during sorting biogenesis cargo VARP-mediated melanosome to linked is Retromer nlgto h vdneta h ermrCCfntosa a as functions CSC retromer the that evidence the of light In ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal Student’s * experiments); independent mean the represent data of The kinetics MCT1. degradation increased suppression in VARP results control. loading a as scanner. shown is Odyssey an on quantitative blotting to western subjected points and time biotinylation indicated after the at with beads lysates streptavidin from captured were Biotinylated proteins evident. fully of became effects suppression h the 24 before at transfection, biotinylated post surface were and and VARP VPS35 against siRNA with were transfected cells HeLa cargo. SNX27–retromer bars: Scale MCT1. for 10 stained VPS35 and or fixed VARP were against siRNA with cells transfected HeLa in signal. decrease a immunofluorescent to MCT1 leads suppression VARP CD97. (A) and MCT1 cargos SNX27–retromer the of endosome- transport affects to-plasma-membrane suppression VARP 4. Fig. m .()Dgaainasy fbiotin-labeled of assays Degradation (B) m. t -test). P , .5(unpaired 0.05 6 ...(ten s.e.m. b -actin

Journal of Cell Science EERHARTICLE RESEARCH yooe n ec hi nacddgaain h ee of level the degradation, enhanced their into enzymes hence these of of and HeLa an missorting loss lysosomes to the in with by to lead Consistent leading situation not function expression). determined blot retromer does the VARP suppression in unlike as retromer decrease western that, apparent cells Tyrp1, melan-a note through in and – reduction cells, 8A,B confirmed tyrosinase dramatic (Fig. a analysis of and to levels led immunofluorescence the the siRNAs, in independent in expression, retromer VPS35 two suppressed of Suppression using we melan-a. line enzymes, cell the melanocyte these explore To of 2009). Tyrp1 al., and et trafficking sorting (Tamura tyrosinase endosome melanosome enzymes maturing the the melanogenic for to the required of are transport activity 2012), and (GEF) but al., factor VARP, exchange et nucleotide of (Ohbayashi guanine activities Rab21 binding its VAMP7 not and Rab32/Rab38 the otn n rfikn fteecuilpoen during proteins crucial these the of in VAMP7 retromer trafficking with and biogenesis. concert melanosome Rab32/Rab38 and in the act VARP sorting that of suggest retromer activities between binding and link functional VARP, the together, and establish Taken further 8C–E). data (Fig. these A the with bafilomycin treatment inhibitor by restored lysosomal partially were Tyrp1 and tyrosinase hrceie nmr eal nadto,w aefurther have have we we – addition, SDCCAG3 In detail. and of more three VARP interactors, in most ANKRD50, new characterized of detecting – number Besides proteomic a which our identified to VPS35. validates we proteomics which approach, of interactors, SILAC retromer interactome quantitative established the employed have define we Here, DISCUSSION ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal n yooe.Saebr:10 bars: Scale lysosomes. and endosomes late LAMP1-decorated with colocalization its and CD97 of endogenous distribution the stained examine and to fixed were the targets against indicated siRNA with transfected cells Student’s (unpaired P3.Dt nBso the show mean B in Data or VPS35. VARP ANKRD50, RME- SDCCAG3, FAM21, 8, for deficient cells in CD97 surface-resident of analysis cytometric interactors. of VPS35 suppression the are by levels perturbed surface CD97 5. Fig. 6 ... * s.e.m.; P , .2;** 0.025; t AB Flow (A,B) ts) C HeLa (C) -test). P , 0.01 m m. 4947

Journal of Cell Science EERHARTICLE RESEARCH 4948 endosome-to-plasma- molecular the SNX27–retromer-dependent in recycling. membrane heterogeneity of underlying the of the details multiple conclusion is major study using a utilizing present cargos, way By distinct 2013). structurally quantitative and al., numerous new et a of (Steinberg role approaches in the methodological dissect interactors finely to be established can tools retromer efficient of as loss upon GLUT1, employed display like TRAILR1 cargos and that phenotypes CD97 2013). robust MCT1, al., the because et so Steinberg endosome-to-plasma- did 2011; of We of al., role et regulator emerging (Temkin recycling major the membrane on a focused as we SNX27–retromer FAM21. analysis, component functional identified WASH the the previously For and the RME-8 of interactors significance retromer functional the clarified o seta o h eyln falSX7rtoe ags Its cargos. SNX27–retromer all of recycling the for essential not this. elucidate and sorting to described, underway be this currently to are remains ANKRD50 of experiments function role SNX27–retromer what loss in Precisely plays cargos. the multiple has across phenocopies data complex its as our strikingly function, but SNX27–retromer functional for protein knockdown essential known was this is it to about that homology known established exception no is repeats with Nothing ankyrin The termini 19 domains. two contains phenotypes. by that protein flanked 220-kDa cargo-specific only a in ANKRD50, resulted or knockdown SNX27– efficient core partial their for essential as fully function, be retromer to appeared none interactors, u nlsshsas salse htteWS ope is complex WASH the that established also has analysis Our established the of any or interactors, new the of Interestingly, ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal anfcto bv h aniae Scale 10 image. bars: main the above higher at magnification shown are areas Boxed and lysosomes. endosomes late LAMP1-decorated colocalization with its and of TRAILR1 distribution endogenous the and examine fixed to were stained targets indicated the against t APadVS5 aai hwthe show B in mean Data VPS35. and ANKRD50, VARP SDCCAG3, RME-8, FAM21, deficient for cells in TRAILR1 surface-resident of VPS35 of interactors. suppression the are by levels perturbed surface TRAILR1 6. Fig. ts) C eaclstasetdwt siRNA with transfected cells HeLa (C) -test). 6 ... * s.e.m.; m m. AB lwctmti analysis cytometric Flow (A,B) P , .2 upie Student’s (unpaired 0.025

Journal of Cell Science EERHARTICLE RESEARCH ewe ermradatasotptwyta eursthe requires that VARP. pathway of activities transport link binding functional VAMP7 a a and and provide Rab32/Rab38 we retromer Tyrp1, VARP-mediated and between in tyrosinase cargo. retromer of for SNX27–retromer role of transport GLUT1 a subset describing is a on in VARP of Importantly, that transport effect suggesting the CD97, minor for and essential MCT1 a of SNX27–retromer- Whereas sorting the only on dependent observed had were sorting. effects strong VARP trafficking, SNX27–retromer-mediated of cargo apparent Perhaps the knockdown in was 2006). VARP of al., analysis specificity GEF our et from a (Zhang on striking as most acts Rab21 effect and endosome-associated 2009) its al., for et through (Tamura biogenesis dynamics a melanosome as endosomal (Scha emerged late VAMP7 has VARP of VARP. regulator interactor retromer The of identified itineraries cargos. the investigation. non-WASH-dependent further in warrants differences certainly these cargos the these of behind reason(s) sorting molecular essential the be to of appears for suppression therefore the VARP by function. affected complex not WASH were (MCT1, that cargos all TRAILR1) those and of all not CD97 affected suppression Thus, VARP interesting that is note TRAILR1. it to Moreover, sorting. and efficient for minor by complex CD97 polymerization WASH only the actin of require has to appear sorting and cargos SNX27–retromer the sorting MCT1 on affect effects not 10 cells. bar: does VPS35-suppressed Scale versus subs suppression right. control and of the mixed comparison were to side-by-side cells magnification le direct of a higher populations a with two a allows transduced the This stably at hours, were antibodies. shown populations 48 anti-Myc is cell After and HeLa area siRNA. anti-VPS35 wi Two VARP. VPS35-specific Tub with of transfected or antibodies. localization stained cells anti-VARP control endosomal and and HeLa either the fixed anti-VPS29 with from for transfection anti-VPS35, Lysates required the to with not levels. and prior is immunoblotted protein VARP–Myc VPS35 lysed were VARP with encoding (D) VSP35 were in immunoblotted control. targeting decrease cells loading subsequently oligos a a transfection, siRNA were as causes post different shown immunoprecipitates components two hours the retromer and 48 VPS29 of and At targeting Knockdown method, siRNA constructs. (C) RFP-trap VARP antibodies. the indicated anti-RFP using the and precipitated VPS35 with were transfected proteins VARP. transiently (mSt)-tagged of were mStrawberry region cells N-terminal HEK293 VARP. the of to region binds Retromer 7. Fig. o eea esn,w oue u td ntenewly the on study our focused we reasons, several For fre l,21) tpasa salse oein role established an plays It 2012). al., et ¨fer A ceai ersnaino h APcntut.()VS5itrcswt h N-terminal the with interacts VPS35 (B) constructs. VARP the of representation Schematic (A) m m. epigt otlt htb euaigtesaiiyand stability the is regulating it by cells, that HeLa the in postulate in suppression to step retromer terminal tempting upon this levels endogenous in of decrease VARP the timing late-endosome– Given antagonizing pathway. the maturation retromer) endosome limiting controlling (i.e. hence complex and cargo-sorting and a VAMP7 of context (Scha of fusion lysosome state fusogenic endosome late 2004) and a early events. al., between constitute transport switching might et in regulation association (Simpson of retromer–VARP point endosomes the to early that binding suggest on in Rab21 VARP while Rab38 of and activating The Rab32 roles late-endosome-associated 2014). of distinct forms Dikic, active and functionally endosome Popovic and late 2012; upon diverse al., switch biogenesis et out to (Popovic autophagosome titrating starvation TBC1D5 towards by itineraries allows that, trafficking -like VPS29, interaction the to through an to binding binds ATG8, VPS35 endosome also to protein TBC1D5 late binding 2009). for the al., Rab7–GTP et to of (Seaman availability targeting the the in retromer modifying implicated of been Rab7 has control proposed Rab TBC1D5 the to of protein VPS29 timing GTPase-activating of the binding with Direct switches. retromer GTPase bridging a provides link protein molecular accessory retromer further a as VARP of identification ial,tedsrbdrl fVR ncnrligthe controlling in VARP of role described the Finally, the biogenesis, melanosome in role specific this Besides ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal fre l,21) so neeti the in interest of is 2012), al., et ¨fer h boxed The equently ntivirus anti- 4949 lnis ulin th

Journal of Cell Science EERHARTICLE RESEARCH 4950 8. Fig. e etpg o legend. for page next See ora fCl cec 21)17 9045 doi:10.1242/jcs.156299 4940–4953 127, (2014) Science Cell of Journal

Journal of Cell Science t ihrsVS5o oto iN eeas ujce o1%SDS-PAGE 10% to subjected with also treated were cells siRNA melan-a control of or lysates siVPS35 Cell either graphically. quantified represented were controls are of and those to relative melanocytes bars: VPS35-suppressed Scale cells. the in distribution 20 antibody. melanosome fixed the anti-tyrosinase show were and images cells antibody Brightfield the anti-Tyrp1 were and with cells siRNA immunostained Melan-a VPS35-specific and Tyrp1. or and control tyrosinase with of transfected levels the key in of reduction trafficking dramatic VARP-mediated in role enzymes. a melanogenic plays Retromer 8. Fig. ARTICLE RESEARCH AP rbi oylnl ba,210,Mctg(mouse Myc-tag polyclonal; polyclonal; (rabbit (sheep 24170), RME-8 UK), Myc-tag Bristol, of Serotec), Abcam, University for AbD in-house made 9E10; polyclonal; clone 13-6890), monoclonal; Invitrogen, (rabbit H68.4, Biolegend, clone VIM3B; LAMP1 monoclonal; clone (mouse monoclonal; TfnR (mouse 336302), CD97 (rabbit 52971), GLUT1 Itg Abcam, OASA01719), UK), DR-4- clone Aviva, Bristol, monoclonal; 02; (mouse of TRAILR1 15309), University gift Abcam, a polyclonal; polyclonal; Halestrap, polyclonal; (rabbit (rabbit Andrew monoclonal; MCT1 (mouse from VPS26 11814460001), GFP Roche, 97545), 87442), 7.1/13.1; (Abcam, strumpellin clones Cruz, VPS35 2009), S1181), Billadeau, Santa Epitomics, and and polyclonal; Gomez WASH1 (rabbit polyclonal; 611482), SNX1 Biosciences, proteins; (rabbit following BD FAM21 the 51/SNX1; against clone were study (monoclonal; the in used Antibodies Antibodies METHODS AND MATERIALS 2014). al., might et retromer (Hesketh VARP, recruit fusion to endosome–lysosome ability control colleagues its and through the Owen that, conclusion, of propose own role our like functional Finally, recycling. endosome-to-plasma-membrane one in that is interaction define VARP–retromer these studies together, biochemical taken independent aside, our two That using for pathway. suppression this of VARP required characterization upon effect observe minor is we relatively is the that with VAMP7, inconsistent evidence conclusion of a alongside GLUT1, recycling analysis endosome-to-plasma-membrane VARP, VARP. imaging-based retromer-mediated of that association an with endosome presented interaction through the the for that Moreover, necessary propose They is VARP bind VAMP7. retromer that VARP simultaneously and can establish VARP and VPS29 that they to and retromer VPS29, Importantly, to 2014). binds between directly al., interaction et the (Hesketh identified retrieval also for this sorted proceeds. been lysosome that the have with cargos fusion speculate retromer before ensure relevant to can all late- mechanism that ‘proof-reading’ premature multi-layered One prevent which a to constitutes through check-point fusion. evolved a as endosome–lysosome have act to 2012), might al., et retromer appear Liu 2010; mechanisms al., et multiple (Balderhaar and the tethering with endosome fusion Ypt7-regulated antagonizing in kinetic retromer a fusion as late-endosome–lysosome VARP with (Scha of VAMP7-mediated role concert of the in regulate inhibitor retromer, might Rab38, endosomes, and on Rab32 VARP of availability 20 bars: Scale procedures B. mean other and the All A show fixation. for data before described B,D, h as 9 out for carried with (BafA1) were treated A1 were bafilomycin caused cells nM Tyrp1 VPS35-knockdown 100 and The tyrosinase suppression. of VPS35 loss by the rescues partially Bafilomycin (C–E) anti- antibodies. and anti-VARP specific anti-Tyrp1, tyrosinase anti-VPS35, with immunoblotting by followed -test). uigterve forppr ai wnadcolleagues and Owen David paper, our of review the During m .Telvlo yoiaeadTr1imnfursetsgasin signals immunofluorescent Tyrp1 and tyrosinase of level The m. fre l,21) lnsd h rpsdfnto fyeast of function proposed the Alongside 2012). al., et ¨fer AB upeso fVS5epeso eut na in results expression VPS35 of Suppression (A,B) 6 ... ** s.e.m.; b b atni hw salaigcontrol. loading a as shown is -actin rbi oolnl ln EO1042Y; clone monoclonal; (rabbit 1 P , .1 S o infcn (Student’s significant not NS, 0.01; m C.For (C). m 9,Tr1(a9 os oolnl at Cruz), polyclonal; Santa monoclonal; (rabbit mouse M- Cruz, (rabbit (Ta99; SDCCAG3 Santa Tyrp1 polyclonal; ANKRD50 (goat 19), ab108219), ab108216), tyrosinase Canada), 15969-1-AP), Abcam, Proteintech, Montreal, Abcam, University, polyclonal; McGill polyclonal; McPherson, (rabbit Peter VARP from gift a nihetMpwsue odslyoelpbtengn ontology gene between and plug-in overlap Visualization Cytoscape display to The Annotation, used 6.7). was for version Map Database (DAVID, Enrichment Discovery the was Integrated proteins using VPS35-interacting of set performed the for annotation gene Functional analysis statistical previously and Computational 2013). described al., an et as on Steinberg spectrometer 2012; analysis 4–12% al., LC-MS/MS mass et Nupage to (Steinberg beads (Thermo) on subjected GFP-trap Velos separated and Orbitrap with were Protease (Invitrogen) Samples precipitated gels Roche min. precast was in NP40, 30 lysed GFP for 0.5% were (Chromotek) and Tris-HCl, GFP Cells labeling Cocktail), mM and labeling. full (50 Inhibitor GFP–VPS35 achieve the 2013). buffer to before al., precipitation passages transduced et six for Steinberg lentivirally 2012; 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Journal of Cell Science st 2). (site GUGGAACGUU-3 olwn lgs(ipnGn;Tym,Jpn:5 Japan): Toyama, Gene; (Nippon oligos following UGAAAGAUGAATT-3 LT n C1ascn lg a loue (5 used also was oligo second a MCT1 and GLUT1 CUAAGCUGAGAAGAATT-3 h pnraigfae ofrigrssac osRAVPS35-3 siRNA to resistance conferring frame, into above. reading T1326C) described T1323A, open mis- C1321T, base G1320A, the silent T1317C, 6 introducing (T1314A, by matches generated was VPS35 siRNA-resistant 5 oligo the using suppressed was SDCCAG3 experiments, CACCCUCUCUGCTT-3 eesprse sn NTREpu ua SMARTpools human ON-TARGETplus 5 oligo the using using suppressed was VARP FAM21 (Dharmacon). and suppressed ANKRD50 RME-8, used. only was were case SMARTpool the which from in 3 construct, oligo siRNA-resistant GFP–VPS35 a expressing ARTICLE RESEARCH 4952 SDCCAG3. study to reagents supplying Science, for Biomedical UK) of Sheffield, (Department of Erdmann University Kai thank specifically We Acknowledgements Student’s unpaired were an experiments performing independent of by number of followed error indicated standard to the calculated, and over normalized mean mean The first condition. the of each were percentage for to blotting assays) or blots) (degradation raw western western (comparative The control quantitative experiments. of percentage independent the of from number as indicated shown data the are of data colocalization mean confocal the and blot western quantified All analysis Statistical against construct viral shRNA The 3 pXLG3. the into subcloned was VPS35 mutagenesis and constructs intensity VPS35 signal the fluorescence of percentage the a as as h 24 quantified at and was 12 streptavidin– after Degradation remaining with signal scanner. of on captured blotting transfection Odyssey western post were quantitative an h by 48 detected were proteins and and 36 (GE-Healthcare) proteins biotinylated agarose (24, biotinylation surface and after h established, were siRNA), 24 lysed and were fully 12 cells Cells 0, were at (Pierce/Thermo). HeLa sulpho-NHS-biotin siRNA proteins, with the before the biotinylated transfection, surface post of h of 24 At effects siRNA. degradation requisite the with the transfected measure To assays 2013). al., Degradation assays et as Antibody-uptake (Steinberg previously performed 2012). described was al., as CD97 performed et were and (Steinberg TRAILR1 previously of described detection cytometric assays Flow antibody-uptake and cytometry Flow dithiothreitol- in to boiling by X-100 proteins buffer. and Triton the sample of 1% containing transmembrane elution pooled the with by from followed PBS were contaminants washed proteins, containing and proteins were lysates NaCl cytoskeletal proteins M remove X-100, 1.2 biotinylated in capture, biotinylated Triton extensively capture After 2% to proteins. were used membrane Cells with was surface transfection. (GE-Healthcare) post PBS were streptavidin–agarose h 72 in cells at levels, instructions lysed manufacturer’s surface according the (Pierce/Thermo) protein to available commercially of a with MCT1 quantification biotinylated and GLUT1 the of levels For surface the of Quantification sidctdb nasterisk. an by indicated is a An tests, experiments. statistical all three For the experiments across Student’s calculated individual unpaired was across deviation averaged standard three and least was at experiments from data independent Colocalization significance. statistical determine t 5 9 0. T fVS5wsprhsdfo im (TRCN0000337019). Sigma from purchased was VPS35 of UTR t 9 ts a hnue oaayesaitclsignificance. statistical analyze to used then was -test st )ad5 and 1) (site P 9 .MueVS5wssprse sn the using suppressed was VPS35 Mouse ). 9 vleof -value o LT muoloecnelocalization immunofluorescence GLUT1 For . 9 n o xmnto fsraelvl of levels surface of examination for and , 9 , CUUGAACUA C-3 -CCUGUAGAAGACCCUGAU .5wscniee infcn and significant considered was 0.05 9 -UACGACGCAC- 9 9 -GGUGUAAAU- -AAGAUGUGU- 9 -AAUU- t ts to -test t 5 0 9 alo,J,Bjy . ee,B . osht .M,Rtefr,A,Mlo,H., Mellor, A., Rutherford, M., V. Oorschot, J., B. Peter, M., Bujny, J., Carlton, i,D,Gmz .S,Blaeu .D n oe,M K. M. Rosen, and D. D. Billadeau, S., T. Gomez, D., Jia, A. Helenius, and J. Huotari, rgi .N,Hrnl,L . gia,R . at .R n oiaio .S. Bro J. C., Ostrowicz, Bonifacino, H., and Arlt, R. J., H. C. Balderhaar, Haft, C., R. Aguilar, M., L. Hartnell, N., C. Arighi, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.156299/-/DC1 online available material Supplementary material Supplementary release. Deposited immediate 24570206]. for number for [grant PMC Grants-in-Aid Japan in of by MEXT the supported Tokyo from also the Research from is Scientific grant N.O. a Foundation. by and Research 24657125]; Biochemical (MEXT) and Technology 24370077 Scientific and numbers for Sports [grant Culture, Grants-in-Aid Japan Education, of by of and supported Ministry 086777 is the M.F. 089928, from Society. Research numbers Royal [grant the Trust 083474]. and Wellcome number 093549]; the [grant Studentships by programme PhD supported Year Biology is 4 Cell P.J.C. Trust Dynamic Wellcome the by through supported awarded are M.G. and I.J.M. Funding the wrote P.J.C. and N.O. M.F. F.S., and A.Y. I.J.M., manuscript. proteomics; experiments. performed the melanocyte M.G. performed the and K.J.H. performed F.S. experiments; I.J.M., biology project; cell the the designed P.J.C. and M.F. F.S., I.J.M, contributions Author interests. competing no declare authors The interests Competing ekt,G . Pe G., G. Hesketh, M., Silhankova, J., I. McGough, J., M. Lorenowicz, F., Port, M., Harterink, J. N. M. Seaman, and G. Hesketh, L., C. Freeman, C. H. Korswagen, and J. P. Cullen, J. P. Cullen, and C. Burd, abu,M . ruee,S .adSaa,M N. M. Seaman, and Y. S. Breusegem, E., and M. Harbour, E. Reid, C., Freeman, R., Antrobus, Y., S. Breusegem, E., M. Harbour, J., Z. Chen, M., Xu, D., Jia, S., T. Gomez, S., Ramanathan, M., J. Doyle, H., Y. Hao, Erdmann, and F. Yu, S., Brier, J., Christmann, N., Ackermann, N., Hagemann, D. D. Billadeau, and S. T. Gomez, A., V. Rahden, van S., Weber, F., C. Harrison, C., Hoffmann, C., Ragaz, I., Finsel, A. Gautreau, and D. Loew, B., Lombard, J., J. 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Betist, endosome. sorting. late yeast the at Sci. fusion Cell and J. recycling receptor retromer-mediated lmnswti h A2 alln h AHatnrgltr ope othe to complex regulatory actin WASH the link tail retromer. FAM21 the within elements nooa AHcmlxi eitdb h xedd‘al fFm1binding Fam21 of Vps35. ‘tail’ protein extended retromer the the by mediated to is complex WASH endosomal dynamics. tubule endosomal 3703-3717. regulate that complexes protein for N. M. Seaman, R. ubiquitination. P. by Potts, trafficking 1051-1064. of protein and and K. regulation polymerization actin M. dependent the Rosen, D., in D. Billadeau, involved is and PTPN13 cytokinesis. phosphatase tyrosine protein S. K. sorting. retromer-dependent to dimer regulates SNX retromer the of function the tubulation. with endosomal complex control WASH the of activity the replication. intracellular promote to trafficking H. Hilbi, and L. 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