Journal of Cell Science o:10.1242/jcs.129270 doi: 3462–3474 126, ß Science Cell of Journal 2013 May 2 Accepted rnpr swl tde nmmaincls u ehrn for LE tethering cargo. but exchange cells, to mammalian in fuse studied and well tether is meet, transport can distances long they over transported to be before to LE proteins need (HOPS) cells, first yeast cells to SNARE mammalian Sorting contrast in In 2010). with al., Protein et interacts (Ostrowicz fusion vacuole induce subsequently and that by orchestrated fusion complex is HOmotypic tethering (lysosomal) the vacuolar where yeast in 2010). 2004; al., al., et et Cullinane (Gissen syndrome renal (ARC) arthrogryposis, cholestasis and and dysfunction 2012) al., al., et ceroid et neuronal (Uusi-Rauva al., onset lipofuscinosis juvenile classical (BasuRay et 2008), al., Rocha (CMT) et Spinosa 2007; 2013; disease al., as et Charcot–Marie–Tooth (Sobo such 2009), disease diseases C developmental Type in Niemann–Pick result and machinery nutrients. neurodegenerative fusion recycle and severe and transport lysosomal cargo the endosomal in lysosomes degrade Defects and to endosomes order late in acidic (LE) with fuse phagosomes exchangeand with to fuse starting (Bro and cargo dock steps tether, other. of sequential organelles each towards these Fusion of cytoskeleton Subsequently, the complexes. series along organelles fusion a of transport the in and to occurs proteins due organelles motor fuse of and activities move continuously organelles Intracellular Introduction words: Key p150 the and complex HOPS RAB7– complex: tethering multi-protein the mammalian one binds a by endosomes, concomitantly contact. in co-regulated initiate and fusion early processes to directly endosomal tether combined from they late RILP are before of cargo transport that distances regulation and long show acquire very the tethering We over about to lysosomal transported RILP–ORP1L. known that be fuse as is describe to such need and little we diseases, fusion yet Here, developmental for move syndrome, destined and constantly endosomes ARC neurodegenerative Mammalian and severe that system. disease cause organelles dynamics C lysosomal type dynamic in Niemann–Pick Defects are autophagosome. lysosomes and phagosomes and endosomes Late Summary ( correspondence for *Author 4 3 2 1 Kant der van Rik cholesterol the ORP1L and sensor RILP coupled by are controlled tethering processes and transport endosomal Late 3462 n euae h iigo irtbl iu-n rnpr n uin w ao vnsi nooa biology. endosomal in events major couples two efficiently fusion, complex and RAB7–RILP–ORP1L the transport complex, regulation minus-end multiprotein and microtubule single recruitment of combining a By into timing fusion. complex the endosomal HOPS late regulates and on and motor dependent dynein process the a infection, both virus of Ebola control ORP1L and RILP yenmtr R1 hnfntosa hlseo-esn wthcnrligRILP–HOPS–p150 controlling switch cholesterol-sensing a as functions then ORP1L motor. dynein hj Brummelkamp Thijn iiino uo ilg,TeNtelnsCne nttt,Pemnan11 06XAsedm h Netherlands USA The 94305, Amsterdam, CA 1066CX Stanford, 121, Drive, Plesmanlaan Campus Institute, 299 Cancer Medicine, Netherlands Netherlands of The The Netherlands School Amsterdam, Biology, The University 1066CX Tumor Amsterdam, Stanford 121, of 1066CX Immunology, Plesmanlaan Division 121, and Institute, Plesmanlaan Microbiology Cancer Institute, of Netherlands Cancer Department The Netherlands Biochemistry, The of II, Division Biology Cell of Division 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. uho u nesadn fL uini eie rmstudies from derived is fusion LE of understanding our of Much ce ta. 00.Eryedsms autophagosomes endosomes, Early 2010). al., et ¨cker nooa uin bl iu,HP ope,RB,RL,OPL Dynein ORP1L, RILP, RAB7, complex, HOPS virus, Ebola fusion, Endosomal 1 [email protected] lxne Fish Alexander , 2 a Carette Jan , ) 3 2 uoRocha Nuno , enr Janssen Lennert , 1 n aqe Neefjes Jacques and 1 asJanssen Hans , ta. 07 tnak 09 htseiydrcinlt.The directionality. p150 specify Kuijl the 2011; with that al., families interacts 2009) et RILP myosin Paul effector Stenmark, 2002; RAB7 or 2007; al., et kinesin al., Wu et dynein, 2005; al., the et relevant (Hoepfner from of recruitment proteins by transport motor facilitate family Arf and Rab tethering and transport between understood. cross-talk poorly the are processes as well as fusion hrceie yacmlto fcoetrli h late the in al., diseases et cholesterol Vihervaara storage 2009; al., of 2011). et lysosomal (Rocha accumulation compartments in endosomal by observed is for responsible characterized RAB7–RILP is clustering mechanism this to and vesicle VAP-A recruitment ORP1L protein sensor ER the cholesterol motor Guignot and the 2004; between Dynein interactions al., by et 2004). controlled Harrison al., 2004; phagosome al., et as et intracellular (Marsman well facilitating survival as thereby lysosomes maturation to endosomal al., fusion dominant impair et its to (RILP Vallee of mutant shown 2006; expression truncated al., ectopic N-terminally or et negative RILP Jordens cytolytic of 2011; Silencing and al., al., 2012). et et Jordens melanosomes 2009; Daniele al., et early 2001; Rocha 2007; as al., et (Johansson such granules lysosome- organelles microtubule and lysosomes related controlling endosomes, late thus of transport minus-end complex motor dynein–dynactin nteed-yooa ytm aiu ml Tae fthe of GTPases small various system, endo-lysosomal the In 1 aieKrom Sabine , 4, * Glued 1 aacj Ho Nataschja , neatos eso that show We interactions. eerhArticle Research Glued Glued D 9)hv been have 199) uui fthe of subunit uui fthe of subunit Salmonella 1 , Journal of Cell Science Lage l,20) n sciia o fiin yooi nr of al., et entry (Carette cytosolic endosomes efficient late containing for NPC-1 critical maturation from is EGFR virus autophagosome Ebola and in and 2008), al., 2011) implicated et al., (Liang been et has systems (Chirivino HOPS mammalian routing Human in limited. complex function HOPS remains and the of recruitment to from understanding current composition, complex respect the tethering human, with significant actual to Despite yeast the human of functions. and conservation tethering evolutionary endosomal yeast and late between transport for vesicular different essential Ypt7/RAB7 be around be the interactions may that mammals, not indicating to yeast, to contrast in transport In appears found. motor is dynein homologue RILP no vesicles and two on Bro Ypt7 2009; al., bridges et HOPS HOPS RAB7 (Cabrera the that of yeast by sides suggesting The different complex on complex, HOPS 2005). Vps41 and the al., new Vps39 assembles with et a interacting and Gissen recruits and 2009; Ypt7 – al., homologue et VPS33B al., in et Zhu two and (Huizing found 2001; SPE-39 VPS33A Specifically evolved: are has – protein expansion. subunits VPS33B-interacting exist some (Wickner, Vps proteins vacuolar with Vps33 these VPS33 systems and all where components: Vps39 of mammalian (Vps) complex Vps41, Homologues yeast HOPS Vps16, 2010). protein-sorting the in Vps11, vacuolar by Vps18, six understood orchestrated the is comprising best and tethering Golgi (lysosomal) is ER, support (Bro the tethering GTPases endosomes membrane at RAB early other complexes plasma of and tethering fusion 1999) multisubunit and al., unrelated docking et vesicle (Christoforidis promote in between can involved actual also EEA1 (Bro fusion for fusion are and GTPases proteins tethering RAB prevent SNARE fusion. recruiting membrane (i.e. before compartments) control establish membranes, opposing case quality two factors the bridge Tethering not physically vesicles. is to of this function fusion detail, subsequent of and level tethering some for at understood is vesicles igeRB–IPOPLpoencmlxt promote to cargo. a endosomal by late complex regulated of delivery and protein and integrated transport are efficient RAB7–RILP–ORP1L – fusion and endosomal single late transport of – aspects main life Collectively, two that tethering. suggest and observations transport our both RILP–HOPS–dynein-motor times The and controls infectivity. interactions Ebola ORP1L major for has sensor illustrated as processes cholesterol delivery these cargo Uncoupling on complex. effects fusion protein for tethering one and in and transport complex coupling HOPS thereby the motor, recruited both dynein be binds to simultaneously fails RILP VPS33b and RILP. of to membranes, mutant disease endosomal syndrome late ARC an to complex HOPS mammalian to (Garg recruited unclear. complex is is complex membranes, HOPS HOPS endosomal mammalian the late the through How of lysosomes 2011). subunits al., in et some molecules CD1 with of interactions ARL8b Cullinane GTPase loading small 2004; ARC lipid the granule al., interactor, controls HOPS in as et Another 2010). (Gissen impairments al., such fusion et by membrane failures, characterized and secretion is multiorgan and which complex VPS33b syndrome, subunits cause HOPS additional SPE-39 the in Mutations 2011). ee edmntaeta A7efco IPrcut the recruits RILP effector RAB7 that demonstrate we Here, effector Ypt7 known only the is complex HOPS the yeast, In to recruitment protein motor of control spatial the While ce ta. 00.Lt endosomal Late 2010). al., et ¨cker ce ta. 00.TeRB effector RAB5 The 2010). al., et ¨cker ce ta. 2012). al., et ¨cker aeedsmltteigadtasotaeculd3463 coupled are transport and tethering endosomal Late h A7efco IPrcut h p150 the recruits RILP effector RAB7 of The absence the motor in dynein tethering functional endosomal late induces RILP hi noeoseuvlnsa eemndb e filtration gel successfully by to were determined similar as complexes proteins equivalents mass S1B). endogenous molecular epitope-tagged (S1A, their high MelJuSo the into Fig. line incorporated material and (supplementary cell complex moderate S1C) melanoma HOPS were levels (tagged) the complex Expression eight HOPS in the mammalian subunits co-expressed other the we of subunits, localization study and To RILP by ORP1L membranes by to regulated recruited is HOPS Mammalian Results e el hs aaso htRL nue Ecutrn even clustering LE induces RILP that conditions clusters show large these data two These or Under cell. one per silenced. in remained was was efficiently endosomes was RILP-labelled DHC DIC chain expression DHC. and of when intermediate presence protein kDa in dynein eliminated only 500 marker stable SDS-PAGE, is is surrogate that (DIC) DHC by its As by assess 1B). determined (Fig. to (DHC) difficult chain dynein the erimn fteHP ope olt nooe for in endosomes involved late also to is complex and HOPS motor mutant the tethering. dynein of RILP of the N-terminus recruitment 2007). the that recruits truncated suggest al., data These RILP, 1E). et N-terminally (Fig. LE to (Johansson VPS11 an motor (RILP RILP of dynein of N-terminus the the Expression binds state, RILP binds GTP-bound of a directly C-terminus in RILP-labelled the RAB7 to While locks VPS11 and 1E). of (Fig. recruitment co- endosomes striking late we case, a this endosomes, observed In cytosolic cells. late we GFP–VPS11-expressing whether a the to in test recruitment and RILP To expressed HOPS 1E). 1D) (Fig. (Fig. promotes VPS11 RILP blotting for observed western was mass distribution and molecular by assessed filtration high as equivalents gel into endogenous their to incorporated similar VPS11 complexes successfully GFP-tagged GFP–VPS11. be with cell in 2011) HOPS could (KO) complex al., knockout et HOPS the VPS11 (Carette the and a line by of reconstituted RILP localization we RAB achieved cells, the small mammalian the between test is via To membranes link Ypt7. yeast LE GTPase to recruited a in is which electron suggesting tethering complex between by LE again sites contact tethering. analysis 1C), at (Fig. with observed for were often LE stained antibodies was cells sections RILP gold) MelJuSo microscopy. and (10-nm fixed resolution, GFP–RILP, anti-GFP higher with in by transfected induced might clusters RILP expression the that visualize To and RILP LEs. motor, tethering dynein in involved functional be of absence the in xrse IPi el hr yenmtratvt was activity motor dynein we this, where test p50 To cells overexpressing tethering. by in its abrogated induce RILP also might RILP expressed whether LE wondered on we locks state, RILP present GTP-bound and active 2009) an al., As in et RAB7 2001; activity. (Cabrera involved tethering motor al., is endosomal dynein homologue) late et RAB7 to in yeast Cantalupo due (the centre 1A) Ypt7 2009; GTP-bound (Fig. active al., 2001) microtubule-organizing et al., late et induces (Rocha the Jordens cells cells in at of RILP (MTOC) of clustering Expression 2001). endosomal 2001; al., al., et et (Cantalupo Jordens transport minus-end to membranes microtubule endosomal induce late to complex motor dynein–dynactin D 9)i F–P1 xrsigclsfie orecruit to failed cells expressing GFP–VPS11 in 199) dynamitin Fg A rsilencing or 1A) (Fig. Glued uui fthe of subunit Journal of Cell Science eiin nmmrn erimn Gse ta. 04.Indeed, be 2004). to al., reported et been (Gissen has the recruitment and membrane (L30P), of in syndrome VPS33b deficient of one ARC mutant causes in co-expressed disease VPS33b, one Mutations was subunits, RILP- S1B). VPS16 complex to HOPS to Fig. partner recruited recruited material interaction not was (supplementary were its S1A,B) VPS33a subunits unless Only co- Fig. HOPS RILP endosomes. eight when material late decorated the (supplementary However, 2A; of (Fig. RILP S1A,B). seven LE with to Fig. HOPS expressed of material recruitment induce ectopic supplementary to upon RAB7 suffice observed active not was constitutively did of subunits expression complex for Additional HOPS expression. distribution the cytosolic of A all S1D). Fig. material (supplementary 3464 ora fCl cec 2 (15) 126 Science Cell of Journal ne o hlseo odtos sOPLte id oER to p150 binds removes then RILP which ORP1L to as VAP-A motor dynein conditions, protein the cholesterol also of low ORP1L binding under 2009). prevents al., and et RAB7 (Rocha binds ORP1L sensor cholesterol co-immunoprecipitation the as performed VPS18 2C). VPS11, (Fig. SPE-39 with we as co-isolated well be between RILP, could late RILP interactions to experiments. confirm and RILP further by recruited To HOPS be 2B). to (Fig. failed endosomes mutant VPS33b latter the ta. 09.T eti R1 locnrl RILP-HOPS HOPS controls ORP1L of the also mutant a of or ORP1L ORP1L subunits wild-type if and various RILP with test complex co-expressed To we 2009). interactions, al., et idn ftedni oo oRB–IPi otoldby controlled is RAB7–RILP to motor dynein the of Binding ABE;20n (C). nm 10 250 bars: (A,B,E); Scale control. loading a used as is staining Actin an antibody. with anti-VPS11 blotted western and SDS-PAGE by separated were cells and HAP1 knockout reconstituted the VPS11 WT, the of Lysates mRFP–VPS11. expressed ectopically and endogenous of levels expression RILP with transfected were mRFP–VPS11 expressing cells ( arrow). (gray VPS11 endogenous and arrow) (black con complexes to detect antibody anti-VPS11 with probed blot, and western and SDS-PAGE by separated o Fractions is indicated. mass) their (and of proteins elution control The filtration. gel by separated size were VPS11 GFP-tagged expressing cells MelJuSo of Lysates complexes. mass molecular high into incorporated omi fbxdrgo nteleft the in region boxed of zoom-in Right: particles). gold (15-nm antibodies anti-GFP with stained were Sections GFP–RILP. expressing cells MelJuSo in bodies multivesicular endosomal late from images Representative RILP. and VPS11 ( image. overview 02.( 2012). al., et (Raaijmakers chain intermediate dynein co-depleted for staining chain by heavy dynein for silencing showing p50 mRFP- co-expressing either and HA–RILP n from images Representative CLSM. by anti- and (green) anti-HA with immunolabelled fixed, were HA–RILP and expressing (DHC) chain heavy dynein for ( MTOCs. recruitment. dynein-motor of endosomal independent late tethering induces RILP 1. Fig. n from images Representative CLSM. by anti- and (green) anti-HA with immunolabelled . . 0 r hw.Rgt etr blot western Right: shown. are 100 0 r hw.Arwed indicate Arrowheads shown. are 100 dynamitin c c D n tbln(e)atbde n imaged and antibodies (red) -tubulin tbln(e)atbde n imaged and antibodies (red) -tubulin . 9.Mresosc-oaiainof co-localization shows Merge 199. C E 0 r hw.Rgtpanel: Right shown. are 100 Glued lcrnmcorp ftethered of micrograph Electron ) etpnl:VS1K HAP1 KO VPS11 panels: Left ) B bu)o o eefixed, were not or (blue) et eJS el silenced cells MelJuSo Left: ) ( A eJS el expressing cells MelJuSo ) F–IPo GFP– or GFP–RILP rmRL (Rocha RILP from D )TaggedVPS11is ifrn ie were sizes different f ann GFP-tagged taining m m Journal of Cell Science iikn o hlseo odtos(ORP1L conditions p150 cholesterol releases low mimicking hte A- sivle nrmvn h OScmlxfrom complex test HOPS To the removing 2009). in al., involved et is low (Rocha VAP-A whether RAB7–RILP Under from remove to motor VAP-A dynein 2009). with the interacts expose subsequently al., that to motif conformation FFAT et an changes ORP1L (Rocha conditions, endosomes cholesterol endosomes to RILP-labelled recruitment to complex ORP1L HOPS unlike the of subunits of all binding allowed wtORP1L S1E,F). Fig. material supplementary Glued D R htefcieypeetdHP subunit HOPS prevented effectively that ORD rmteRB–IPcmlx(i.2D; (Fig. complex RAB7–RILP the from D R)that ORD) aeedsmltteigadtasotaeculd3465 coupled are transport and tethering endosomal Late ope uuist IPlble nooe ugsigthat p150 suggesting like – endosomes is RILP labelled HOPS to of binding RILP HOPS recruitment to allowed subunits again ER complex mutant with Fig. ORP1L material interactions ORP1L (supplementary This abolish 2009) S1G). al., the to ORP1L et mutant: domain (Rocha VAP-A to repeated FFAT protein the ORP1L similar we in is mutation an conditions, that cholesterol AT(D478A) with low experiments under RILP u aaidct htteHP ope srcutdt membranes to recruited is complex Together HOPS the VAP-A. that protein indicate data ER our through ORP1L sensor cholesterol r hw.Saebr:10 bars: Scale shown. are from images. images merge Representative the from area the boxed show images Zoom confocal microscopy. by imaged and antibodies anti-HA with immunolabelled were tie ihH-nioisand HA-antibodies with stained were RILP-HA with in combination L30P GFP-VPS33b or VPS33b ( antibody. anti-VPS11 with and probing blotting western by as assessed arrow) (black VPS11 endogenous with compared arrow) (gray GFP–VPS11 of level expression mRFP–ORP1L or mRFP–ORP1L with combination in RILP–HA and VPS33b ( antibodies. anti-GFP and with anti-HA blotting western and SDS- PAGE, by separation before GFP anti- with (IP) were immunoprecipitated RILP–HA and subunits HOPS GFP-tagged expressing of MelJuSo Lysates RILP. with VPS11 ( ( s.e.m. + (CC) coefficient correlation average the Graphs show panels. upper in indicated in the shown sections the over (VPS33b) intensity GFP mRFP green, Red, intensity; panels. are lower cells in for shown profiles Plot shown. are from images Representative images. merge area the boxed from the show images Zoom microscopy. confocal by imaged n from merge images the Representative from images. area boxed the show images Zoom microscopy. by confocal imaged and antibodies anti-Myc with stained were Myc–RAB7Q67L with combination in GFP–VPS11 expressing cells MelJuSo panels: n euae yORP1L. by regulated RILP and by membranes to is recruited HOPS Mammalian 2. Fig. B D C . eJS el xrsigGFP- expressing cells MelJuSo ) eJS el xrsigGFP– expressing cells MelJuSo ) and VPS18 SPE39, of Interaction ) 0 r hw.Rgtpanel: Right shown. are 100 Glued D idn otoldby controlled – binding R ihasingle a with ORD D R a indicated) (as ORD D m n ORDFF- . m. ( n n A 25). . . Left ) 100 100 Journal of Cell Science i.3. Fig. 3466 e etpg o legend. for page next See ora fCl cec 2 (15) 126 Science Cell of Journal Journal of Cell Science hwn h ein fRL eriigdfeetHP ope uuisand subunits complex HOPS different recruiting p150 RILP of regions the showing ae,ioae IPmtn ihc-sltdFA–P1.( FLAG–VPS18. top co-isolated input; with panel, mutant Bottom RILP western antibodies. isolated and anti-Flag panel, SDS-PAGE and by anti-HA separation with before blotting immunoprecipitated antibody were anti-HA mutants with truncation FLAG- (IP) expressing HA–RILP MelJuso and of VPS18 Lysates tagged VPS18. with mutants truncation RILP n uuis ersnaieiae r hw nsplmnaymtra Fig. material HOPS supplementary indicated in * the shown S2B. for are shown images are Representative s.e.m. subunits. + correlation coefficients CC, correlation panels. Mean left GFP in Green, shown ( mutants); sections coefficient. 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RILP to recruited subunits HOPS of Quantification ) uui ftedni oo id ietyt RILP to directly binds motor dynein the of subunit P D , 5 and 150 .1ad*** and 0.01 C neato fRILP of Interaction ) D D 5 n mle eeinmtnswere mutants deletion smaller and 150 9,rsetvl)wr ie n tie with stained and fixed were respectively) 199, m P .Rgtpnl ltpoie o cells for profiles plot panel: Right m. , .0 hncmae ihW RILP; WT with compared when 0.001 ce ta. 02.To 2012). al., et ¨cker D 50, D 100, D 9 aldto failed 199 D ( A D 5 and 150 etpanel: Left ) Map ) aeedsmltteigadtasotaeculd3467 coupled are transport and tethering endosomal Late D 199 ihRL n el eesbeunl tie o h dynein the for stained p150 subsequently were subunit cells motor co-expressed and RAB7- was the VPS41 RILP GFP-tagged through with this, coupled test directly To are RILP. – effector transport and fusion – and cells in RILP to associate can OScmlxwsasse ySR(i.4) Whereas 4E). (Fig. SPR by the assessed of subunits was VPS41 p150 and complex (aa1–580) VPS18 HOPS to binding Their 5.39 5.80 P4;1.67 VPS41; whether address further To were 4D). p150 results (Fig. and Similar VPS18 HOPS 4D). (Fig. with endosomes obtained clustered to VPS41 aeHP uuiswas subunits HOPS same eobnn A7 IPadteCtriu fp150 of C-terminus purified the and using RILP RAB7, and reconstituted recombinant RAB7–RILP were RILP), on complexes site RAB7–RILP–C25 same the for (compete exclusive 01 n OScmlxrcutet(o eil tethering) vesicle al., et (for transport) Jordens recruitment 2007; complex al., minus-end HOPS et Johansson and 2001; (driving 2001) al., et recruitment (Cantalupo dynein-motor idn a eetdfrVS8adVS1t 2 ( C25 to VPS41 and VPS18 for detected was binding p150 orce o S-nycnrladfo h P response constant SPR the association from equilibrium and control the GST-only curves a for corrected n P4 ihalwbtdtcal fiiy(4.12 affinity detectable but low VPS18 a to with associated VPS41 RAB7 GTP-loaded and 4C). (Fig. determined ocnrtoso A7 IP h A7RL ope rthe and different p150 or at chip complex of GTP RAB7–RILP (SPR) the of C-terminus RILP, resonance presence RAB7, the plasmon of in concentrations surface determined SPR a were responses the to GST–VPS41 bound for and 1–580) were (aa complex GST-VPS18 subunits stable recombinant HOPS The soluble S3B). a Fig. material to and (supplementary into experiments S3A) recruitment Fig. assembled for material sufficient (supplementary RAB7/RILP RILP be soluble by the to endosomes for shown late (as was VPS18 endogenous were of protein) their N-terminus isolated with The 4A) the interact 4B). (Fig. can that (Fig. partners and indicating used well-folded HOPS, at are endogenous running proteins to proteins complexes sizes mass complex recombinant molecular similar higher RILP The and/or into RAB7 incorporated to SPR. VPS41) and by (VPS18 subunits assayed HOPS we two direct, also are subunits HOPS A7RL–2 soitdwt ihrbnigaffinity binding higher [ only a RAB7–RILP with than associated RAB7–RILP–C25 oo ope n ehrn yteHP complex. p150 HOPS of consequence the a at is clustering clustering by endosomal this and late tethering MTOC induces the RILP and of dynein Overexpression the complex by transport minus-end motor endosomal late couples required RILP is infectivity HOPS virus and Ebola RILP for by tethering RAB7–RILP. endosomal Late receptor: endosomal late complex single tethering a a on and fusion transport for for effectively protein RILP, motor to a bind integrating simultaneously can subunits HOPS and M ute – odices nafnt o P1 n VPS41 and VPS18 for affinity in increase (5.98 fold 2–3 further h A7RL ope oHP uuisadta p150 that and subunits HOPS to complex RAB7–RILP the uuisadteCtriu fp150 of C-terminus the and subunits 2 hssget httognrlpoessi aeedsmllife endosomal late in processes general two that suggests This 1 6 6 .Tgte u aaidct htbt OScomplex HOPS both that indicate data our Together ). 6 Glued Glued 10 10 10 5 4 5 M C5fie oitrc ihteHP subunits, HOPS the with interact to failed –C25 M uprs ahrta optsfr h neato of interaction the for, competes than rather supports, 2 n 1.13 and 6 2 1 epciey.TeRB–IPcmlxhda had complex RAB7–RILP The respectively). , 1 10 epciey.Bnigafnte fRL othe to RILP of affinities Binding respectively). , 6 Glued M 2 6 Glued Glued 1 idt IPsmlaeul rmutually or simultaneously RILP to bind 10 .Ti ugssta h -emnlpr of part C-terminal the that suggests This ]. 6 IPrcutdbt p150 both recruited RILP . 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(Figs 3468 e etpg o legend. for page next See ora fCl cec 2 (15) 126 Science Cell of Journal Glued u o ihthe with not but hre(o xml,Rt rEt )adtse o recruitment for tested and R) to E p150 of or the E N-terminus of reversed to We the R species. example, in of (for series surface) charge a protein between conserved the and to RILP exposed likely are 4–) otmttosta fetdRL neatoswith interactions RILP affected that mutations Most S4A–H). Glued rHP uuis(upeetr aeilFig. material (supplementary subunits HOPS or Journal of Cell Science A77LRL ope rteRB–IPC5(h -emnldomain C-terminal p150 the (the RILP, of RAB7–RILP–C25 RAB7Q76L, the to or chip SPR complex a RAB7Q76L–RILP on immobilized GST–VPS41 or 1–580) netdcls VVG-bVifciiywsrdcdb afin of half cytosol by the reduced in was replication infectivity (Wong upon rVSV-GP-EboV GFP (rVSV-GP-EboV) cells. produces infected glycoprotein that 2010) virus al., Virus et Ebola Stomatitis the Vesicular bearing replication-competent with infected hw is Shown ujce oSSPG n etr ltigwt h niae nioisto antibodies indicated the were with Fractions blotting western filtration. and gel SDS-PAGE using to separated subjected and size cells subsequently MelJuSo of were lysates lysates with GST– incubated GST–VPS41, were His–RILP Recombinant or complexes. VPS18(1–512) is mass proteins molecular marker higher of into position The experiments. ( SPR shown. the for used proteins ( cretdt hto S ny tdfeetcnetain fRB,RL or RILP RAB7, of panel: concentrations Right different complex. at RAB7–RILP only) the GST of that to (corrected of presence GTP- of the binding in and C25 and chip, GTP- complex SPR RAB7Q76L–RILP an a to RILP, immobilized RAB7Q76L, was ( GST–VPS41 proteins. or recombinant 1–580) the containing complexes detect P epne.Sonis Shown responses. SPR P1 a –8)adVS1 l esrdi h rsneo GTP- of presence the in measured all VPS41, c and to 1–580) RAB7Q67L–RILP–C25 (aa and VPS18 RAB7Q76L–RILP of complex preformed aafo u P xeiet Fg ) nyoemutant, most one p150 of on Only effects recruitment negligible 4). with (Fig. subunits affected experiments HOPS markedly SPR our RILPD125RE126R, from data htHP tblzsp150 stabilizes HOPS that nooa ehrn,w oepesdRL uat and mutants RILP co-expressed we p50 tethering, endosomal 5A). 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Fig. 9R9R–aels oeti lseigL n ncombination in and of LE that clustering p50 in capable RILP mutants with potent and less RILPD125RE126R RILP still are – – complex of E93RE94R is HOPS the Expression recruit RILP to 1). fail (Fig. wild-type vesicles conditions clustering these Under IPmtnsta alt eri h OScmlxsol also should mRFP–RILP complex mRFP– mRFP, HOPS expressing Cells the RILP, cells. of recruit infection to virus complex, Ebola HOPS fail affect the for that receptor the mutants is RILP RILP virus If Ebola 2011). by al., infection et cellular (Carette successful for of essential tethering. as complex endosomal recruitment late for that mutant required RILP is indicating RILP small by mainly clusters, motor HOPS with dynein vesicular cells the unable yielded containing mutants of HOPS RILP recruit subunit Again, was to S4I). DHC experiment Fig. the The material (supplementary silencing 5B). (Fig. by endosomes repeated single and clusters from fGTP- of A (means+s.e.m.). S omsi-tie D-AEgl ihteidctdrecombinant indicated the with gels SDS-PAGE Coomassie-stained ) ovsaietecnrbto fHP erimn olate to recruitment HOPS of contribution the visualize To eetgntcsre dniid6sbnt fteHOPS the of subunits 6 identified screen genetic recent A dynamitin Glued c c n oasml h ope.Telf aesso h responses the show panels left The with complex. pre-incubated the were assemble RILP to and S RAB7Q76L determined. was S . Glued nvitro in c B 0 r hw.( shown. are 100 .( S. nvitro in n P1 rVS1 cl as 10 bars: Scale VPS41. or VPS18 and h eobnn rtisue nSRsuisaeincorporated are studies SPR in used proteins recombinant The ) dynamitin ope ma+....Bnigwsdtrie nthe in determined was Binding (mean+s.e.m.). complex ) D t irp h yenmtr(alee l,2012)]. al., et (Vallee motor dynein the disrupt [to eJS el xrsigH–IPadGPVS8or GFP–VPS18 and HA–RILP expressing cells MelJuSo ) oVS8(a150 rVS1 l esrdi h presence the in measured all VPS41, or 1–580) (aa VPS18 to c idn fteCtria p150 C-terminal the of binding n orce o idn oGTol ope othe to coupled only GST to binding for corrected and S vrxrsinyeddclswt aysmall many with cells yielded overexpression K D E a 9 rmF–IP15E2Rwere mRFP–RILPD125RE126R or 199 K etpanels: Left ) acltdFo h P epne hw nB. in shown responses SPR the From calculated a fRBQ6,RL rRAB7Q76L-RILP or RILP RAB7Q76L, of Glued idn oRL n confirming and RILP to binding K nvitro in a Glued ma+... acltdfo the from calculated (mean+s.e.m.) m erimn,suggesting recruitment, idn fGTVS8(aa GST–VPS18 of binding Glued .Rpeettv images Representative m. rgetC5 the C25, fragment C Glued S–P1 (aa GST–VPS18 ) Glued recruitment oRILP. to aeedsmltteigadtasotaeculd3469 coupled are transport and tethering endosomal Late Glued fiiyfrRL soitdt A7 neouin HOPS evolution, In RAB7. to associated RILP higher even for Using and for RILP affinity for RILP. affinity low affinity higher exhibit effector 10–15-fold VPS41) alone, and demonstrate RAB7 RAB7 (VPS18 we subunits the experiments, HOPS (SPR) that by late resonance but to plasmon RAB7 surface recruited by and recruited regulated unclear. is remains cells processes, mammalian and complex these in 2010). membranes all HOPS secretion endosomal al., in et involved the granule Cullinane is how complex 2004; in HOPS al., the et impairments Although (Gissen fusion by membrane characterized which syndrome, ARC SPE-39 as is and such VPS33b failures multiorgan subunits complex HOPS cause 2011). additional al., et the Garg 2011; in al., Mutations et al., Chirivino et 2011; al., (Carette et CD1d Ganley autophagolysosome of 2011; loading trafficking, peptide EGFR endosomal implicated and been infection, mouse formation has virus complex VPS41-deficient (Aoyama HOPS Ebola and stage the in addition, gastrulation 2010), In the 2012). al., beyond al., et develop et to (Harrington Ruan fail systems embryos 2012; model al., mammalian et in protect disease can VPS41 Parkinson’s in Mutations 1999). lysosomal against al., and et Sevrioukov endosomal 2005; late defective in biogenesis in result homologue) erimn fbt p150 flow both or endosomal of 5C,D) late (Fig. When recruitment S4J). microscopy Fig. by material (supplementary assessed cytometry RILP was control wild-type infection mRFP-tagged or to when only mRFP relative either expressing mRFP-RILP(D125RE126R) cells expressing cells homologue), A7RL stelt nooa eetrfrteHOPS the for that receptor suggest endosomal data p150 late our and the complex Collectively, is 5E). (Fig. RAB7–RILP ORP1L wild-type observed mRFP-tagged or was only mRFP either infectivity expressing rVSV-GP-EboV mRFP–ORP1L in expressing reduction cells 50% in A 5E). (Fig. EboV IPi ie ytecoetrlsno R1 oregulate to ORP1L sensor cholesterol the by maturation. endosomal timed is RILP ooousVS3 n P-9(P1BVPR exist. subunits additional (VPS16B/VIPAR) HOPS two of SPE-39 and mutations genomes, and or fusion Deletion eukaryotic VPS33b to in prior homologues found correct vacuoles been control with have to endosomes membranes late (Bro to of Ypt7 complex homologue effector interaction RAB7 HOPS its the yeast, the via In motor recruits 2001). dynein al., the et (Jordens for as RILP receptor acts RAB7 endosome member, terms, late One the general 2009). regulators (Stenmark, few key processes as a these defined been of in have family described Rab/Ypt GTP-binding the be small of proteins The can fusion. and vesicle transport membrane a including of life The Discussion ela yenmtrbnigt IP(oh ta. 2009) al., or virus RILP–p150 mRFP–ORP1L Ebola et mRFP, affected (Rocha with also mRFP–ORP1L transfected RILP ORP1L were whether to Cells infection. tested binding we motor 2D), (Fig. dynein recruitment as HOPS well of inhibition as similar level alone. a to attenuated RILP eew eotta nmmasHP uuisaenot are subunits HOPS mammals in that report we Here stecoetrlsno R1 csa euao fHP as HOPS of regulator as acts ORP1L sensor cholesterol the As ce ta. 00.Hmlge o l es OSsubunits HOPS yeast all for Homologues 2010). al., et ¨cker D 9,ihbto fifcino VVG-bVremained rVSV-GP-EboV of infection of inhibition 199, a sncenidcdnuooiiyin neurotoxicity -synuclein-induced Glued carnation rspiamelanogaster Drosophila D R ti uataoihRL–OSand RILP–HOPS abolish mutant (this ORD neatos eoeifcinwt rVSV-GP- with infection before interactions) Glued VS3 ooou)or homologue) (VPS33a n htbnigo hs opee to complexes these of binding that and Glued n OSwspeetdwith prevented was HOPS and D R eaiet oto cells control to relative ORD Plpaahrvle al., et (Pulipparacharuvil eporange deep dvps16A .elegans C. (VPS16 (VPS18 and Journal of Cell Science h iu rdcsGPi h yoo hc sdtce n uniidb lwctmty hw stepretg ftasetd(RPpstv)adi and (mRFP-positive) transfected of percentage the is Shown cytometry. flow * by (mean+s.e.m). quantified and cells detected transfected is to which relative cytosol cells the (GFP-positive) in GFP produces virus the IP9R9R AIP15E2Radete oepesn mRFP-p50 co-expressing either and HARILPD125RE126R RILPE93RE94R, * RILP hc sdtce n uniid ih ae:clswr dniid akdadoelyduigCl rflr e,tasetdcls re,cell green, cells; transfected ( Red, constructs. Profiler. mRFP-labelled Cell the using expressing overlayed cells and infected masked and identified, transfected were yellow, cells rVSV-GP-EboV; panel: of Right infection quantified. and detected is which RILP encreaincefcet +... r hw o niae OSsbnt.* subunits. HOPS indicated for shown are (+s.e.m.) coefficients correlation Mean lsesado eilsprcl mas+sd) el with Cells s.d.). + from (means images cell Representative per vesicles CLSM. and/or by clusters imaged and (blue) DAPI yenmtrfrmnseddrce irtbl transport microtubule minus-end-directed for motor dynein erimn a paetysitdfo A7YT oits p150 to the binds RAB7/YPT7 also from RILP shifted RILP. apparently effector has recruitment tethering. endosomal late induces RILP 5. Fig. 3470 P , .5 ( 0.05. D D 9-o RPRLD2R16-xrsigclsscesul netdb VVG-bVa esrdb yooi F xrsin(means+s.e.m.). expression GFP cytosolic by measured as rVSV-GP-EboV by infected successfully cells mRFP-RILPD125RE126R-expressing or 199- 9 rmF–IP15E2Rwr netdwt VVG-bVfr8hus h iu rdcsGPi h otctslatrscesu infection, successful after cytosol host the in GFP produces virus The hours. 8 for rVSV-GP-EboV with infected were mRFP–RILPD125RE126R or 199 E ora fCl cec 2 (15) 126 Science Cell of Journal A- el xrsigmF,mF-R1 rmRFP-ORP1L or mRFP-ORP1L mRFP, expressing cells HAP-1 ) ( A uniiaino OSsbntrcutett idtp IP(T lc)adRL 15E2R(gray). D125RE125R RILP and black) (WT, RILP wild-type to recruitment subunit HOPS of Quantification ) Glued . 0cutr eesta 0 * 10. at set were clusters 10 uui fthe of subunit P n , . .5 cl as 10 bars: Scale 0.05. 0 r hw.Teqatfcto ntergtsosteaeaenme findividual of number average the shows right the on quantification The shown. are 100 dynamitin D P R eeifce ihrS-PEo o or.Atrscesu infection, successful After hours. 8 for rVSV-GP-EboV with infected were ORD , hr r utpepstosi IPrqie o HOPS for required RILP in positions that revealed multiple and mapped RILP have are N-terminus We the 2001). there to al., HOPS et of Cantalupo recruitment 2001; al., et (Jordens 0.05. rntwr ie,imnlble ihat-Aatbde gen and (green) antibodies anti-HA with immunolabelled fixed, were not or P , n m 0.05, . m. 0frec odto.( condition. each for 20 + P , . ( 0.1 C A- el xrsigmF,mF–IP mRFP– mRFP–RILP, mRFP, expressing cells HAP-1 ) D uniiaino RP RPRL- mRFP- mRFP-RILP-, mRFP, of Quantification ) B eJS el xrsigH–IP HA– HA–RILP, expressing cells MelJuSo ) ihsuccessful with s nfected Journal of Cell Science IPi P tde ihprfe rtis hssget that RAB7–RILP–p150 suggests the This of proteins. part integral purified an with is studies HOPS SPR in RILP R8,wssont eri OSsbntVS1t lysosomal to VPS41 GTPase, subunit HOPS another recruit Recently, machinery to shown lines. recruitment was cell ARL8b, of different amount not between by the did present replaced possibly in We 1E), was differences (Fig. levels. levels VPS11 indicating endogenous state endogenous at upon steady expressed when VPS11 at tagged even observed LE subunits this, HOPS on observe exogenous observed MTOC studies these also 2001; In al., were 2003). the et al., explain (Caplan et subunits Poupon could complex near complex HOPS of HOPS interplay overexpression clustering and Such processes. motor endosomal coupled dynein are the transport fusion minus-end between that for and tethering affinity) highest and the has total the (as RpoenVPA A- eoe h yenmtrfrom motor dynein the the removes with VAP-A interact to VAP-A. motif protein FFAT cholesterol to ER an low exposes motor at and dynein conformation concentrations the changes of ORP1L binding RAB7–RILP. allows cholesterol ORP1L high At concentrations, 2009). al., et endosomal (Rocha dynein late ORP1L the the sensor by cholesterol of timed and recruitment 2010) regulated the is al., RAB7–RILP that to et motor shown Cabrera previously 2010; have al., we et and (Ruan before phosphorylation reported by been complex lipid has HOPS or the phosphorylation of as transient Regulation such very conditions. factors is other were membranes by regulated with conditions and/or complex that HOPS state the of observed that indicate steady association might We This cytosol. fusion. under the in correct localized subunits predominantly for complex ensure to HOPS space and 2011). time al., et (Garg steps before suggested different as in trafficking act endosomal may of they be that to suggesting (our remains data), endosomes different unpublished exclusive, to localise mutually RILP and or ARL8b established. RAB7–RILP, in as for coupled, case are processes the two (Rosa-Ferreira these Whether trafficking 2011). Munro, anterograde and regulating linker kinesin-1 thereby the binds also SKIP, Arl8 2011). al., et (Garg membranes erimn loafc yenmtrrcutetadteC- the and recruitment complex motor HOPS p150 abrogate dynein of that terminus affect RILP been (Bro also of has complex Mutations recruitment HOPS as yeast 2012). complex, the al., and HOPS et Ytp7 mammalian for shown the RILP recently non-overlapping in multiple sites reflect binding may These recruitment. h iigo eil ehrn a ob elcodntdin coordinated well be to has tethering vesicle of timing The Glued nrae h fiiyo idn fRAB7– of binding of affinity the increases Glued complex aeedsmltteigadtasotaeculd3471 coupled are transport and tethering endosomal Late ¨cker n ehrn eedn nlt nooa otn (cholesterol) content endosomal late on dependent movement tethering vesicle coordinating cell switch, and ORP1L molecular a ORP1L. the as sensor act HOPS cholesterol to seems the by to controlled via also tethering relocalize endosomal is complex that vesicles show now We and periphery. complexes RAB7–RILP ale ta. 01 ickrk n htae,20)adHIV and 2003) Le 2009; Whittaker, al., et and (Lehmann well 2008; Sieczkarski as al., RILP 2011; et involves propagation Schaar al., der et (van propagation Caillet and and compartments infectivity influenza endosomal proper late Dengue, require for to including reported been cytosol. viruses have also host Various HIV by infection the virus. and fusion into Ebola regulate entry of ORP1L the late and subsequent RILP infection that into and show successful we 2004; fusion Here delivery for by al., essential efficient virus et is Ebola Conversely, lysosomes Harrison 2004). and 2004; endosomes al., al., intracellular et et compromises severely Marsman (Guignot and infection block this bacterial breaks RILP Overexpression of endo-lysosomal (phago)lysosome. degradative the the with via fusion avoids instance, host For the entering pathway. pathogens of infectivity 6). (Fig. diint h ehrn ahnr.Frlt nooe hsis this endosomes late For machinery. additional tethering in the required machineries, transport to long-distance have addition time may and recruit eukaryotes to factors higher evolution, in during that hypothesize organelles We unclear. was these between Bro 2009; (Stenmark, of initiation fusion for complexes tethering multi-subunit on and in transport later role data cells. a mammalian Our have of RILP. also causes pathway to might endosomal that SPE-39 recruited the and L30P) be VPS33b (VPS33b to that fails VPS33b suggest phenotype of ARC and mutant RILP severe by disease recruited be a also We can that 2010). SPE-39 al., and et VPS33b and (Cullinane that patients kidney recycling show syndrome polarized ARC endosomal in of cells observed liver defective abnormalities the and cause polarization, might implicated epithelial been on have in present endosomes GTPase Recyling RAB endosomes. a 2010), recycling al., et (Cullinane RAB11A with by determined. tethering be with to and infection remains cell transport viruses host these endosomal for important late indeed is Whether RILP–HOPS–dynein 2006). al., et sRL oriae eiua ehrn,i ih modulate might it tethering, vesicular coordinates RILP As ayclua rnpr tp eyo yenmtratvt for activity motor dynein on rely steps transport cellular Many interact to reported been previously have SPE-39 and VPS33b uinmdae ytecmlxo P1,RB,RILP, RAB7, HOPS. OPR1L, and of motor complex dynein and the transport by controls mediated cholesterol fusion endosomal late how shows OScmlxa ela p150 the as of well binding as terminates complex This HOPS for VAP-A. domain protein FFAT ER its binding late exposes from and released membranes is endosomal ORP1L the of of panel), domain fusion (right cholesterol-binding and conditions transport low-cholesterol can efficient In complex for lysosomes. HOPS RILP the to and bind motor both dynein the (left ORP1L conditions panel), subsequent high-cholesterol for In HOPS) fusion. (by SNARE-dependent (by tethering proximity and close transport) require minus-end HOPS fusion the for of RAB7–RILP. destined binding to endosomes of motor control dynein–dynactin in and ORP1L complex of Model 6. Fig. Salmonella ce ta. 00 u h connection the but 2010) al., et ¨cker elctsi nooe but endosomes in replicates Glued oRL.Ti mechanism This RILP. to ´ vesque Late Journal of Cell Science Jhnsne l,20;Rcae l,20) el eelsdi .%Tio X-100, Triton 0.5% in lysed were Cells 2009). al., et Rocha described 2007; previously al., as et produced (Johansson were MBP–C25 and His–RILP His–RAB7Q76L, purification Protein anti-CD63 mouse or were: mRFP- anti- used various Cross- mouse with antibodies 1985), analyses respectively. al., Other blot et proteins, proteins. (Vennegoor Western by fusion recombinant using excluded GFP-labelled home His–GFP been at has generated or reactivity were antibodies His–mRFP anti-mRFP purified rabbit and anti-GFP Rabbit Reagents p150 ORP1L, RILP, RAB7, cloning and Constructs Methods containing and Materials glycoprotein compartments. Ebola cargo endosomal late from of by cytosol host way the here into efficient entry viral exemplified an with as cells delivery, provides This and efficiently steps. tethering processes subsequent fusion additional for these vesicles an Coupling maturing incoming providing 6). prepares (Fig. processes, regulation ORP1L these of sensor of layer cholesterol timing The the of lysosomes. controls fusion and for tethering consecutive endosomes for complex late but HOPS of the transport and different minus-end endosomes for late two motor dynein–dynactin in the processes: involved complexes protein other determined. for be to coupled remains intricately well, and as are tethering structures processes intracellular and fusion Whether transport ORP1L. and senor coupling cells transport cholesterol the provides of by also in co-regulation way time, allowing RAB7–RILP) efficient same of an the surface with at the shared which, involving recruitment a by HOPS solved (or been have RILP have might RAB7-effector may HOPS VPS41, problem the in This of recruitment motif complex. membrane binding for interactions membrane new the required with of addition in loss this, and yeast; the interactions HOPS in – RAB7 found interfered existing have with not might effectors effectors additional These RAB7 ORP1L. and additional RILP two by reflected 3472 ito .Fudz(etrfrTasainlSca ersine Emory Neuroscience, length) GTP- (full Social Abnova. 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VPS39 in 2FLAG-C1 sites Faundez. a EcoR1 of V. – sites Xho1-EcoR1 HINDIII the the into into subcloned inserted and S1) Molecular Table of below. described (Department Angeles). is 2008) Los constructs California, al., other Southern of et of Generation (Liang University Immunology, Liang GFP–VPS18 and Chengyu and Microbiology GFP–VPS16 from 2001). al., gifts et Jordens were 2009; al., et (Rocha previously on uat fRL eegnrtdb C sn hs epcieprimer respective these using PCR by generated were RILP of mutants Point material (supplementary primerset1 using PCR by generated was V5-VPS16 ee ehv hw htteRB fetrRL recruits RILP effector RAB7 the that shown have we Here, ora fCl cec 2 (15) 126 Science Cell of Journal c a ucae rmMillipore. from purchased was S nvitro in Glued ha emepeso ytm eepurchased were system) expression germ wheat n p50 and c tbln(im)mueat-5adanti- and anti-V5 mouse (Sigma) -tubulin dynamitin osrcshv endescribed been have constructs DAfe rtaeihbtrccti Rce.Telst a lae by cleared was lysate Co Talon The pre-equilibrated with (Roche). incubated mM was 8 cocktail supernatant the NaCl, and inhibitor mM centrifugation 200 protease 7.5, pH EDTA-free Hepes, mM 20 o lcrnmcocp M0eeto irsoe(E,Enhvn the Eindhoven, (FEI, microscope electron Plan- CM10 HCX used. a was (Leica). with microscopy LCS Netherlands) was used equipped electron software acquisition For Leica) The (Leica). lenses AOBS; objective corrected or 63 TCS-SP2, Apochromat TCS-SP1, (CLSM, 5 MTi,10m al MMgCl mM buffer 5 into NaCl, scraped mM and PBS 150 ice-cold Tris, with mM (GraphPad washed (50 were Prism cells Meljuso Transfected Graphpad applied filtration Gel figures http://www.graphpad.com). final USA, Fitting California, and concentration. 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