eto,Braatas 3 97 Osnabru 49076 13, Barbarastrasse section, ermradtednmnVs oprt ntertivlof Arlt Henning retrieval the vacuoles in from cooperate proteins Vps1 transmembrane the and Retromer ARTICLE RESEARCH ß eevd2 ue21;Acpe eebr2014 December 9 Accepted 2014; June 20 Received fission and fusion respective of 1 reuse that gradients and transport during recycling generate interactions on to sufficient rely low-affinity be and that should Heinrich proposed sorting model, mathematical have a the on Rapoport from Based membranes be organelle. of to transport target has retrograde recycling the because with are availability coordinated are through that their processes ensure proteins sophisticated simply to more membrane necessary transport, occurs For vesicular carriers. in recycling of the as involved from proteins, such rounds release proteins, further the tethering membrane their for of peripheral or of available recycling case coat it the the make In is forming transport. to processes common the A machinery transport into membrane. mediate sorting these target a and the cargo of with organelle on vesicle principle next sort the the of depends fusion to to final them cells machinery transport eukaryotic vesicles, protein within conserved transport Vesicular INTRODUCTION selected of Recycling, vacuole trafficking Vacuole, Endosomal Dynamin, the Vps1, Retromer, clear WORDS: KEY to Ypt7 the proteins. membrane cooperate and can Vps1 retromer that with demonstrate data our organization. membrane together, retromer sorting vacuole Taken that maintain to consecutive suggest essential further are implying data Vps1 Our and Golgi, destination. at final the the accumulates to at then steps later cargo and Recycled vacuole agreement in function. the behind, regulatory leave stays its Ypt7 Vps1 whereas with and cargo, and the Retromer Vps1, with Ypt7. together dynamin-like GTPase the Rab7 retromer, We Vps10 the the recycling. receptor requires transmembrane during vacuoles the that cofactors from of assay required an recycling the using that dissect function demonstrate of its to retrieval explore us membrane, We in plasma allows vacuoles. the implicated to on or is act Golgi can which the to retromer, lysosome-like endosomes membrane. from that plasma the proteins the show or to Golgi now the lipids We to and others recycle proteins and vacuole, deliver ability the successfully combine to to need that organelles dynamic are Endosomes ABSTRACT Ato o orsodne([email protected]) correspondence for *Author Netherlands. Medicine, The Molecular Utrecht, for CX Center 3584 Biology, 100, Cell Heidelberglaan of Department Utrecht, Centre nvriyo Osnabru of University 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,6565doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 1 uvoReggiori Fulvio , ¨ k eateto ilg/hmsr,Biochemistry Biology/Chemistry, of Department ck, ¨ k Germany. ck, 2 n hita Ungermann Christian and 2 nvriyMedical University ieadfntoaiyo auls oee,ecs iisand lipids excess however, vacuoles, the of maintain functionality To and 2005). and Stevens, pathway, size and cytosol–vacuole-transport (Bowers autophagy the general of type called selective constitutive the autophagy the pathways, pathway, biosynthetic (AP)-3 protein and 2004; adaptor endocytic the Seaman, including 2011; vacuole, recycling al., et their 2007), 2009). Harterink for al., al., 2010; complex et Seaman al., retromer et mannose-6-phosphate et the (Miller cation-independent (Harbour on epsinR the depend and receptor with sortilin interacts and endosomal the Vti1 instance, For idea. SNARE this support that have described examples been Several 2005). Rapoport, and (Heinrich machinery 05 oa ta. 08 emne l,20) naddition, In 2009). al., et Seaman GTPase-activating Rab7 2008; the with interact al., can retromer mammalian et Rojas al., Vps35, et Nakada-Tsukui 2012; is 2005; al., to et CRC yeast, Liu in 2010; the al., or, et Rab7 (Balderhaar of GTPase Ypt7 Rab subunit endosomal late central the to The binds which 1998). 2007; al., al., et (Hierro et Vps35 Seaman and phosphatidylinositol-3- complex Vps29 second cargo-recognition Vps26, trimeric of the The composed the (CRC) 2002). by and al., formed et 2012b) is subcomplex (Burda al., domain PX et phosphate-binding membranes Weering deform to (van domains (in N-BAR Vps5 possess which of SNX6 SNX32), SNX5, or metazoans consists (in Vps17 complex and SNX2) (SNX) or SNX1 small metazoans nexin retrieval sorting a for dimeric subcomplexes. two signals and contains The retromer The sorting molecules 1996). Stevens, contains and cargo (Cooper that binds C-terminus large that cytosolic a the with N-terminus protein 1997). al., transmembrane cells, et luminal type-I Seaman 1997). single-pass metazoan 2011; a al., is al., et Vps10 in (Harterink Wntless, et Vps10 transporter yeast, include, in Wnt Seaman and, the retromer 1998; and receptor the al., mannose-6-phosphate of et 2008; Hurley, Seaman Substrates and to 2012; (Bonifacino proteins membrane Seaman, membrane plasma of and Golgi recycling the in involved complex protein is 2012; known. machinery al., not similar is et a vacuoles (Rong whether on 2 However, required also 2013). dynamin al., on and the et depends Schulze includes which adaptor, process structures, AP-2 The lysosomal the vesicular and 2014). of al., proteins formation the et membrane with fusion (Chen recycle after lysosomes autophagosome (ALR) of to reformation reformation the process for lysosomal membranes fission metazoan autophagic In a 2004). of al., includes et process from Dove recycling the 1998; active al., cells, is et there (Bryant vacuole that the starvation. suggested during have active studies mainly be Some to seems process counterbalance this that although to indications (Mu means growth are a surface There the as recycled. microautophagy be employ to vacuoles need proteins sorting 1, * eea rnpr ahasdlvrlpd n rtist the to proteins and lipids deliver pathways transport Several nedsms h ermrhsbe hrceie samajor a as characterized been has retromer the endosomes, On le ta. 00 ate n ae,2000), Mayer, and Sattler 2000; al., et ¨ller 645

Journal of Cell Science lsv n otmdl aermie netddet h akof lack the to due assays. remains untested appropriate remained tubules, have releases models retromer most and and how cargo elusive However, sorts 2012a). membrane, Weering al., the van et deforms Weering 2007; van al., 2012b; et al., which (Hierro structure, et Golgi tubular the with a fuses into eventually proteins membrane sorts retromer structural on Based 2009). and analyses al., et (Seaman TBC1D5 (GAP) protein ARTICLE RESEARCH 646 5 bar: Scale replacement. medium the after YPD in time to refer points Time Methods. and the Materials of the control in the under described Vps10–GFP li as of white medium Expression by YPD vacuoles. (indicated to from shown Vps10–GFP are YPG of vacuoles from Recycling representative (C) of vacuole. plots the intensity from Fluorescence Methods. and Materials 5 the bar: vacuoles. in from described recycled as staining be FM4-64 can Vps10–GFP 1. Fig. versatile a is retromer the the beyond activity This that has . that recycling. demonstrate complex Vps1. recycling endosomal homolog protein thus membrane dynamin from to the data Vps10 and recycle manner Ypt7 Our active can similar requires retromer also a event the have that we in assay, reveal vacuoles new to this able Exploiting been vacuole. the from recycling epcietm on.Bten6 n 2 el eecutdi ahframe. each in counted were 1 Detai cells bar: (E) 127 C. Scale in and mean C. as are 67 in taken Results Between as were D. point. out images and weaker time C and carried the in YPD respective was from shown into experiment assays expressed transfer of The after was Quantification h YPD. Vps10–GFP (G) 5 in vacuoles. added h on was 6.5 load Cycloheximide after Vps10 synthesis. localization protein new Vps10 to of due views not is relocalization Vps10 (D) ee ehv eeoe nasyt olwmmrn protein membrane follow to assay an developed have we Here, m .()Mdlo p1 rfikn rmedsmsadGlit h aul ne vrxrsincniin n h uaiermvlmechanism removal putative the and conditions overexpression under vacuole the to Golgi and endosomes from trafficking Vps10 of Model (B) m. nvitro in eoeigasy,i a enpsuae that postulated been has it assays, remodeling A oaiaino noeosGPtge ermrsbnt.Vcoemmrn a iulzdby visualized was membrane Vacuole subunits. retromer GFP-tagged endogenous of Localization (A) 6 ..o p1–F oscl sdtrie nsmpoetoso 3.2- of projections sum on determined as dots/cell Vps10–GFP of s.d. GALS rmtradtedsrbto fti hmr a nlzda nC. in as analyzed was chimera this of distribution the and promoter uhwae hnCCsbnt Fg A.Akonretromer known however, A 1A). vacuoles; (Fig. on subunits complex CRC SNX than a Vps17, weaker entire retromer some much the detected the also that of We the vacuoles. suggesting subunit to with 1A), associate overlap (Fig. can partial CRC FM4-64 the their marker analyze detect and also also vacuolar subunits to could CRC two us we other prompted the interestingly Vps29, This and Vps26 2012). and of Vps5 al., localization subunits et the (SNX) (Liu nexin on Vps17 sorting endosomal of detected whereas the Recruitment Ypt7, requires active been 1A). on localization (Fig. depends also vacuole 2012) the has al., to Vps35 et CRC, a (Liu Vps35, retromer membrane However, vacuolar the Golgi. of the recycle to subunit to back membranes endosomal receptors on act protein-sorting to known is retromer The transmembrane vacuole-localized proteins for assay recycling A RESULTS ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal m .()Rccigkntc rmvcoe orlt with correlate vacuoles from kinetics Recycling (F) m. GAL1 rmtrwssopdb hfigcells shifting by stopped was promoter m tcstkna the at taken stacks m e) Scale nes). m m. led

Journal of Cell Science p1.Ti a nedtecs.When case. the likely of indeed recycling which was faster in vacuole, This that result the Vps10. would predicted overexpression on therefore the We cargo of lowering machinery. of recycling amount be the high might sequesters this the that to reasoned due other We to processes. compared time transport long intracellular relatively a required localization like membrane. the of vacuolar consequence the likely from a block is recycling 1E). Vps10 its of (Fig. not appearance vacuole Vps10- look dot-like the observed the closer from did Thus, we potent a emanating Vps10, structures took of h, tubular a relocalization we positive 5 this When during cycloheximide, 1D). cells after (Fig. at of expressed dots added into newly addition relocalization to inhibitor due the not translation because was the puncta Importantly, distribution protein, 1A). Vps10 a (Fig. of Vps10 with appearance endogenous 1C,G), Vps10 to (Fig. most similar h, structures profile 7.5 dot-like After in h. in accumulate 5 to after localized membrane started vacuolar Vps10 then the medium, on and glucose dots vacuoles to on shift the remained After initially puncta 1C). fluorescent (Fig. few visible only and were membrane vacuolar the strong to shifted the of control hs twsucerwihfnto ermrperformed retromer function which 2014). unclear al., et was (Chi previously shown it as 1A), a Thus, (Fig. display In endosomes Golgi to to the correspond 1994). found and likely exclusively very metazoan which al., was localization, the Vps10 punctate et retromer, with to (Marcusson similarities contrast receptor structural Y, some carboxypeptidase mannose-6-phosphate receptor including shares sorting hydrolases the which vacuolar Vps10, various protein transmembrane for the is cargo ARTICLE RESEARCH eee eetdgnsadaaye h fiinyo Vps10 of efficiency the analyzed we and process, recycling genes the selected in involved deleted machinery the identify and To Ypt7 retromer, the quantification). on Vps1 for depends 1G vacuoles Fig. from see Recycling 1F, (Fig. h 2.5 by observed was weaker the from glucose- the pulse to a expressed shift functional we when the of Indeed, after 1B). (Fig. points was medium galactose- repress time containing Vps10 different from of to at localization shifted medium analyzed the Subsequently, were glucose-containing expression. cells to further vacuole, medium the containing to localized inducible available the the than in larger much delay was strong Vps10 machinery. of a pool vacuoles, the expected as we on retrieval, Second, much imaging the endosomes. easily from morphologically live-cell smaller more distinguished to be by easily us we can allow function which First, would consequences. retromer cargo of two monitor to increase have phenotype an would that that this Vps10 expected thought We of of pool. advantage Vps10 surplus vacuolar took this the endosome of We the recycling from the 2002). study recycling al., its al., inhibit et et that (Burda limiting to mutants (Cereghino displaced in vacuolar overexpression largely or upon the is 1995) Vps10 membrane from that vacuolar Vps10 shown the is been recycle has to It retromer membrane. able is that retromer vacuolar hypothesized the from thus proteins transmembrane We membrane. recycle vacuole. to required the on entcdta h hf fVs0fo aulrt h dot- the to vacuolar a from Vps10 of shift the that noticed We o u sa,w vrxrse p1–F rmthe from Vps10–GFP overexpressed we assay, our For whether analyze to decided we question, this address To GAL1 VPS10-GFP GALS rmtr neVs0wscompletely was Vps10 Once promoter. GAL1 rmtr h hf rmvcoet puncta to vacuole from shift the promoter, rmiscrmsmllcsunder locus chromosomal its from rmtr p1 a completely was Vps10 promoter, VPS10 a expressed was inl eie(opradSees 96,rsligi the in resulting 1996), Stevens, and (Cooper was Vps10 retrieval putative reside two retromer where Vps10 the signals whether of parts C-terminus from truncated cytosolic address we the release vacuole, of further the after from Vps10 dots To recycling Vps10-positive Vps10 specifically 2F). on the (Fig. on vacuole, vacuole finally, the with and, strongly on tubules colocalized also structures Vps35 vacuole Vps10-positive subunit to This the CRC left 2E). localized Vps10 The (Fig. as surface. readily time membrane over vacuolar persisted 2012b), localization the dot-like al., on et dots The Vps10-positive on Weering formation time. tubule (van in over recycling involved endosomes is subunits which Vps10 together Vps17, retromer Vps10 subunit the retromer GFP-tagged RFP-tagged during traced functional cargo we with this, its retromer achieve of to To localization process. relative the vacuoles analyzed we on recycling, in involved endosomes, on al., et (Chi those machinery for recycling from general requirements the 2014). differ of the part vacuole is Mvp1 that where the suggests like from This mutants, recycling 2H). retromer (Fig. to conditions contrast in is its vps26 from This expressed when promoter. vacuole, normal the to Vps10 of mislocalization eitdb ermr(h ta. 04,srnl impaired strongly of 2014), deletion a al., However, below). et see (Chi 2C, (Fig. retromer recycling dynamin recycling AP-3 by endosomal the yeast in of implicated the mediated been Vps26 mutants of recently or has absence to which 2007), Interestingly, Vps1, al., bind 2C). et are (Fig. Kama and which pathway 2011; Btn3, trafficking al., et and endosome–Golgi in (Kama Btn2 delay in Btn1, the proteins involved independent batten-disease explain cell also was the not during Recycling of vacuoles. do Vps10 the from inheritance vacuolar recycling of Vps10 vacuole of and dilution independent division the is that because demonstrates process inheritance, vacuole recycling The vac7 for guanine-nucleotide- blocked below). needed also the machinery see Mon1 of subunit 2C, deletion (Fig. (GEF) by factor inactivation exchange This or Ypt7 vacuole. to the wild-type of between need the intensity compared and was the might vacuoles from when on retromer evident signal Vps10–GFP removal more that even before became indicates phenotype cargo which h 5–7.5 strain, after concentrate membrane vacuolar this the on Vps10 in dots surprise, to our indicating localized To 2A,C), vacuoles. not on was (Fig. recognized blocked also is was subunit, Vps10 retromer that vacuole any the of absence from the recycling In assay. our using recycling vrxrsin ots hs emd s fahpodyatstrain yeast haploid employing a vacuole of without use the made condition we from native this, cargo test more To whether recycle a overexpression. to wondered under the able rapidly thus be more sequesters We also cargo would above). retromer the (see of machinery recycling overexpression surface. because vacuole of probably the role from a Vps10 with recycling agree in thus data retromer Our entire localization 2H). the (Fig. Vps10 mutants to retromer comparable in vacuole the endogenously to and mislocalization 2G) Vps10 (Fig. efficiently expressed blocked was vacuole the without was 2014), an al., Furthermore, 2C,D). et is (Fig. (Chi and effect recycling 1995) Stevens, endosomal and in (Ekena involved Vps1 with interacts that protein oudrtn h ptoeprlodro h proteins the of order spatiotemporal the understand To h eoa fVs0fo h aul a eaieyslow, relatively was vacuole the from Vps10 of removal The vps26 uat loe fiin eyln rmtevcoe This vacuole. the from recycling efficient allowed mutants D D htso aulrlclzto fVs0udrthese under Vps10 of localization vacuolar show that , osrc.Rccigo vrxrse Vps10 overexpressed of Recycling construct. C ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal D el Fg B.Dlto ftevcoa a protein Rab vacuolar the of Deletion 2B). (Fig. cells D rmthe from C VPS10 mvp1 eeinddntla to lead not did deletion rmtrrsle in resulted promoter vac8 D Mvp1 from C 647 and ,a

Journal of Cell Science EERHARTICLE RESEARCH rvosyson(eehn ta. 95 akrfe al., et 648 strain Markgraf as this mixed 1995; vacuole, we mutant, al., the retromer the et complement to To mislocalization (Cereghino 2009). Vps10–GFP in shown results expressed previously which endogenously deletion, of retromer a carrying 2. Fig. e etpg o legend. for page next See vrtm Fg IJ.Atricbto fbt tan for strains both of incubation After 2I,J). (Fig. chamber microfluidics time a in strains over 3 these type, of with mating mating tagged monitored opposing then Vps26 of functional strain expressed wild-type which haploid another with ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal 6 Cer.We mCherry.

Journal of Cell Science Cer inli h ili eldrcl fe aig cl as 5 bars: Scale mating. after directly of cell appearance diploid first time the the indicate in indicates points arrowhead signal Time white mCherry Methods. The and mating. before Materials or in after details and J in scheme expressing strain vps26 wild-type a with background iecus.Lk ntewl-yestain R n SNX and CRC situation, wild-type the the in during Like localize would course. retromer time where wondered we mutant, IJ aigasyo tanexpressing strain a 1A. of Fig. assay the in in Mating as analyzed (I,J) also analyzed was was Vps10 proteins of the the Localization of of control localization under and locus promoter, chromosomal their Wild-type from (H) expressed A. in as GFP assayed the of Vps10 introduction in by GAL1 resulting removed site, were this C- Vps10 at The of tag blocked. acids is amino Vps10–GFP 89 truncated mCherry.terminal with C-terminally tagged of C-terminally Recycling were (G) the Vps35 Endogenous and on organelle. Vps17 dots this subunits Vps10 from retromer to transport localize retrograde subunits during Retromer vacuole (E,F) vacuole. of the Deletion the from (D) from A. mutants in Vps10– deletion as mutants. all analyzed deletion in analyzed expressed of different Overview was (C) the GFP bar). with right (see obtained Vps10–GFP table results the lookup of the ‘fire’ intensity the signal using The displayed shown. is is signal medium glucose to shift the auls B p1–F scnetae nvcoe uigrlae Wild- release. during vacuoles and on type concentrated is Vps10–GFP (B) vacuoles. the of of Deletion control (A) the under overexpressed was GAL1 Vps10–GFP Ypt7 (A,B,D–G) retromer, the 1. Fig. on depends vacuoles Vps1. from and recycling Vps10 2. Fig. ARTICLE RESEARCH nodt n uue ntevcoa ebaei the in membrane vacuolar the Vps10- on of tubules formation and concentrated still the dots was Vps1 in Vps10–GFP into the that involved that Given suggest structures. be positive data might which These h, activity Vps10. 10 fission and for 7.5 positive Vps1 after also detected Vps1 follow be were some could to vacuole Vps10, the us of on of allow puncta recycling localization the did vacuolar analyzed during it we into When Vps1–mCherry but cargo. relocalize Vps10 course, the completely to time relative not the did after Vps10–GFP active dots partially as vacuolar only is vacuoles version observed on tagged This tag. previously mCherry terminal the 2004). al., are with et data (Peters Vps1 agree These of localization the arrow). from and Vps10 yellow recycling in view, Vps1 vacuole of detailed role scission a (see with consistent tips accumulated sometimes the Vps10–GFP tubules, on these surface On vacuole 3A). the from (Fig. away dot-like mutants resulted protruded finally retromer that and extensions in in time tubular as over in vacuoles persisted rim structures the These on along 2A). not (Fig. accumulated more and 3A) strongly in (Fig. Vps1 structures it of that role the the realized in understand Vps10–GFP followed To we al., detail, as 2C). et vacuoles, (Fig. from (Chi Vps10 here of endosomes recycling shown the at for function required is retromer and 2014), in involved is Vps1 tubules vacuoles retromer-positive on of fission supports Vps1 vacuole. the retromer from the Vps10 after that recycle min demonstrates rapidly Vps10 experiment 15–30 can most This within 2I). of structures (Fig. release dot-like mating in into vacuole resulted the which from arrow), white 2I, (Fig. 3 , 6 0mn el tre omt.Imdaeyatrmtn,Vps26– mating, after Immediately mate. to started cells min, 90 oflo p1drn hspoes etge p1wt C- a with Vps1 tagged we process, this during Vps1 follow To Cer oaie oteVs0GPpstv vacuole Vps10–GFP-positive the to localized mCherry D rmtri t hoooa ou n eyln ftecieawas chimera the of recycling and locus chromosomal its in promoter rmtrbfr hfigclst P sdsrbdi i.1. Fig. in described as YPD to cells shifting before promoter a tie ihCA omntrvcoemrhlg.See morphology. vacuole monitor to CMAC with stained was vps26 p1–F eyln rmvcoe a netgtda in as investigated was vacuoles from recycling Vps10–GFP D VPS26 el eeivsiae si i.1 h - iepitafter time-point 5-h The 1. Fig. in as investigated were cells lcsacmlto n ees fVs0from Vps10 of release and accumulation blocks VPS10-GFP D MVP1 .Tecntutwsepesdfo the from expressed was construct The C. a oefc nrccigo Vps10 of recycling on effect no has VPS10-GFP vps1 and VPS26-3xmCherry vps26 VPS10 uatoe ie and time, over mutant D GAL1 na in and D C-GFP vps26 mvp1 rmtrand promoter VPS10 D D nythe Only . were mutants. vps1 m m. D t1 hna . nareetwt h ih microscopy light the with deleted we agreement directly, in h 7.5 3A,E–H). at (Fig. than results h 10 at eels.I obnto,ordt niaeta p1is recycling. Vps1 vacuolar into and subunits that endosomal retromer both the vacuoles during indicate of cytosol release from the the data tubules for allow our retromer-positive eventually the and separate background combination, both to cytoplasmic In and necessary the lost. structures, and dot-like were staining to membrane entirely vacuolar shifted retromer oaie opnt n h yols Fg A i.3) nthe In 3I). Fig. 1A; endosomes, (Fig. cytoplasm to the vps1 localized and only puncta was Vps26 Vps17 to whereas localized as cytoplasm, the well and membranes vacuolar as the In Vps35 Vps10. of wild-type, overexpression without subunits retromer tagged exclusively very were which tubules, electron-dense surface, Vps10-positive the vacuole in Moreover, observed the the represent including membrane. from likely at medium, emerging profiles vacuole limiting budding glucose protrusions of inwards vacuole more to remnants and the transfer structures, events fusion endosomal after homotypic of h 10 accumulation and 7.5 to comparison In wild-type, microscopy. the electron by was level ultrastructural and view). the detailed points, 3C,D, time (Fig. vacuole later structures the tubular from at the emanating on even Vps10–GFP dot- with structures colocalized vacuolar sometimes dot-like the stayed to the retromer However, h on 3C,D). 5 (Fig. after accumulations localized Vps10 like retromer the of subunits el akn p7hdadfc nVs0rccigfo vacuoles from recycling Vps10 As in clarified. defect been a not had has Ypt7 Ypt7, lacking Ypt7/Rab7 yeast cells of and role Rab7 precise metazoan the both although and recycling with during interacts retromer retromer endosomes the The with Ypt7 towards of Crosstalk directed Golgi. the initially to endosomes is from by therefore subsequently mediated by vacuole retromer We for the 4C). from compensated (Fig. the pathway was recycling markers the assay that Golgi recycling suggest the the with of h colocalization with 5 colocalization after of of colocalization loss that Vps8 gradual however, to the relative Intriguingly, point, localization Vps10–GFP. made Sec7–mCherry were time analyzed observations Similar we this 4B,C). when h Ypt6 (Fig. 5 20% After to marker first up the assay. increased Golgi within the Vps10 the with of colocalization comparison, of no level in In nearly a showed until accumulating of 4A,C). h starts (Fig. colocalization 5 Vps10 after Interestingly, reached decreased when vacuole. Vps10 This assay, with the Vps8 vacuole. the from during of the away 70% h puncta to to 5 the Vps8 30% first that from the some dramatically or drives increased endosomes colocalization cargo late Vps10 perivacuolar vacuolar excess to the of corresponds some either Golgi that Vps8 suggests as This beginning. Sec7 the our with from and for colocalized protein Ypt6 Vps8 marker endosomal setup. used an experimental as we Vps8 cells, and proteins Vps10-positive wild-type marker and in endosomes the on Golgi resides organelle Vps10 the As which directed. from be to recycling would during structures analyzed cargo Vps10 we the of vacuoles, fate the the understand to To then and endosome the Golgi to recycles Vps10 Vacuolar oaayeapsil oeo p1i ermrfnto more function retromer in Vps1 of role possible a analyze To ete nlzdtertivlo p1 rmtevcoeat vacuole the from Vps10 of retrieval the analyzed then We D uat oee,tesbellrlclzto fthe of localization subcellular the however, mutant, ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal vps1 vps1 D D vps1 el ipae aiu hntpsa both at phenotypes various displayed cells uat hs rflswr oeabundant more were profiles These mutant. nclsepesn hmsmlyGFP- chomosomally expressing cells in , 0 fVs0right Vps10 of 30% , 0 was 60% 649

Journal of Cell Science uuiswr -emnlytge ihGPadepesdfo hi hoooa ou.Vcoe eesandwt M-4a ecie nMtrasa Materials in described as FM4-64 with stained were Vacuoles locus. highl H chromosomal and their F) of from panel Deletion 5 expressed in (I) bars: and box (G,H). nm dashed Scale GFP the 200 with Methods. by (E,F), tagged indicated nm C-terminally (region the 500 G were bars: in panels subunits Scale observed while vacuole. cells, V, vacuole entire nucleus; the show N, from F mitochondrion; and emerging E (arrowheads) Vps35–m Methods. tubules and (C,D) Materials the function. the Vps1 in indicated affects the as in partially microscopy concentrates tagging Vps10 C-terminal where sites that 5 the Note bar: to Vps10–GFP. localize in (D) Vps17–mCherry enriched and structures (C) tubular indicates arrowhead yellow auls hnw analyzed we When vacuoles. the that endosomes on absent. indicating functions is its Ypt7 mutant, of if some even retromer perform still a might retromer from different clearly rflsdrn aulrrccig p1wsCtrial agdwt Cer nthe in mCherry with tagged C-terminally was of Vps1 Deletion recycling. (A) vacuolar 1C. during Fig. profiles in as performed was Analysis EERHARTICLE RESEARCH 650 the retromer of loss whereas cells, the wild-type in function in in found puncta is in Vps10–GFP multiple GTPase Vps10–GFP. the localization expressed of the endogenously on role observations of the revisited clarify initially and We try detail. to more decided we 2C), (Fig. vacuoles. from recycling Vps10 in involved is Vps1 3. Fig. uata nwl-yecls(i.5) nyi h obemutant double the in Only 5B). of (Fig. ( the cells amount wild-type in the in colocalization as low quantified mutant similarly dye a we vacuolar observed the we When with FM4-64, colocalization 2012). Vps10 subunit by al., GEF Vps10 on vacuole-localized Ypt7 et the observations (Liu lacking cells Similar Mon1 with made vacuoles. were visible localization staining clearly FM4-64 and by Vps10–GFP in dots detected expressed in endogenously remains as Interestingly, vacuole, 5A,B). (Fig. the to Vps10–GFP vps26 ete undt u ytmweeecs ag speeton present is cargo excess where system our to turned then We vps26 m D .(–)Utatutrlaayi nwl-ye()and (E) wild-type in analysis Ultrastructural (E–H) m. ypt7 D uat h osqecso oso p7aethus are Ypt7 of loss a of consequences The mutant. D i p1 gi ooaiewt M-4a in as FM4-64 with colocalize again Vps10 did ) vps26 ypt7 m (A–D,I). m D D uatrslsi oa eoaiainof relocalization total in results mutant el,ee huhteeclshv small have cells these though even cells, ypt7 D or mon1 D VPS1 el,w observed we cells, eut naboko p1 eyln rmvcoe.()Vs ooaie ihVs0concentration Vps10 with colocalizes Vps1 (B) vacuoles. from recycling Vps10 of block a in results vps1 ACD erea fVs0GPin Vps10–GFP of Retrieval (A,C,D) D tan FH.Clswr rae si i.1 eoebigpoesdfrelectron for processed being before 1C Fig. in as treated were Cells (F–H). strains ypt7 vps1 D D uat hc r ena lcrndneporsos ,edsm;M, endosome; E, protrusions. electron-dense as seen are which mutant, vps1 tutrs u obnddt niaeta p7saso the these structure. tubular on the in stays of part Ypt7 accumulate not that is not indicate and membrane data the donor did combined followed observe Our yellow Ypt7 could we 5F, structures. we (Fig. However, When membrane h, vacuolar 7 the itself. arrowheads). on after dots machinery release mobile in during the Vps10 Vps10 of of but process part dynamics and suggesting recycling the point), not of structures during time activation h the is vacuoles 7.5 in Vps10-positive involved 5E, is on (Fig. Ypt7 Vps10 that of remained of release Ypt7 the sites after Ypt7 However, points, to its 5E). time early (Fig. analyze colocalized At to surface cargo. mCherry Vps10 vacuole strongly with the the Ypt7 to from tagged relative behavior then release We Vps10–GFP 5C,D). of (Fig. block clear a o nyvcoefsinbtas uin(laie l,2013; al., et (Alpadi fusion The also 2004). al., but affect et fission to Peters shown been vacuole was has only and Vps1 biogenesis not vacuole homeostasis. to could membrane linked Vps1 vacuole previously and vacuoles retromer in whether on involved asked functions be next we also assay, our retromer using that homeostasis demonstrated vacuole affect Having Vps1 and retromer The GAL1pr-VPS10-GFP D uat e oe niaeaessona ihrmgiiain Scale magnification. higher at shown areas indicate boxes Red mutant. ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal vps1 ed oacmlto frtoe nedsms Retromer endosomes. on retromer of accumulation to leads vps1 D el fe vrxrsinfo the from overexpression after cells etsri.Teasywscridota nA The A. in as out carried was assay The strain. test vps1 D uathsadfc in defect a has mutant GAL1 Cherry promoter. nvitro in ight nd

Journal of Cell Science EERHARTICLE RESEARCH iial,w odrdwehrti ol aea feton effect an for observed have previously as would the fusion, vacuolar this in functionality whether Vam3 wondered we similarly, vacuoles. of homeostasis retromer membrane the that upon affect postulate Vps1 thus pattern and We localization retromer S1). levels Fig. 6D). their Vam3 material (Fig. (supplementary in change deletion change not dots a did to Vam3-positive Pep12 due to not was in of localized subunit effect redistribution (V)-ATPase this not general Moreover, vacuolar was a the that to Vma2 given due proteins, not vacuolar was dot-like redistribution a in This concentrated to similarly was 6A) Vam3 (Fig. tagged the equal vacuoles in and smooth wild-type localization a 2A; showing (Fig. along from we distribution changed Curiously, assay of mutants. Vam3 localization these that recycling in steady-state observed change the our might whether SNAREs vacuolar wondered in we 3A), vacuoles Fig. on date. plasma to Vps10–GFP has the lysosomes pathway or a with vacuoles such for vesicles but described synaptic 2012), been Fasshauer, not of and fusion (Jahn well membrane vacuole. example, the retromer for the is, in a from fusion after of described proteins with SNAREs some SNARE of consistent reuse of least and Recycling recycling be at the that also in reasoned function would We observations 2004). al., these SNARE the et with (Peters interacts Vam3 Vps1 recombinant and fusion, vacuole eas ermradVs fettedsrbto fVam3 of distribution the affect Vps1 and retromer Because of localization the affect Vps1 and retromer of deletions As vps1 D D uat(ukrie l,21;Ptr ta. 2004). al., et Peters 2014; al., et (Kulkarni mutant el Fg E.OhrSAE ieVi,Nv or Nyv1 Vti1, like SNAREs Other 6E). (Fig. cells vps35 D uat(i.6) ieie GFP- Likewise, 6B). (Fig. mutant vps1 D el Fg 6C). (Fig. cells (DKY ermradVs aeacuilrl ncnrligvacuole controlling that available in support role the provide crucial uses data a or homeostasis. have These membrane Vps1 Vam3 fusion. and liberates rescue retromer either to addition The then factor. SNAREs recycling Vam7 other idea retromer the with of of the complex deletion a with upon in trapped Vps1 consistent are or SNAREs is of all observation fraction of a fusion This that rescue 6F). to (Fig. able SNARE was mutants 2004), on Vam7 al., fusion et HOPS-driven recombinant (Thorngren and the vacuoles SNARE- of promote for to can addition Pep4 defect which or protein, because Pho8 similar proteins vacuole, marker a was the the and fusion of in mislocalization 2004) defect to due this al., not Importantly, 6F). et active (Fig. Peters mutants of retromer 2014; of al., activity the fusion in et analyzed the fusion we of assaying When block 1994). by from al., vacuoles measured et (Haas be phosphatase fusion of then amount peptidase The can phosphatase. vacuolar inactive the a cleaves peptidase for deleted is strain (BJ3505 One strains. tester measured be can Fusion ucin u sa,wihepot necs fisnatural its and vacuole the of the dissect from to excess recycling us membrane allowed retromer in an has involved machinery receptor, into exploits cargo Vps10 insights which the substrate, mechanistic assay, detailed Our function. provide data Our DISCUSSION pho8 olwda nFg Cwith 1C Fig. in to Golgi. as first the followed directed to is then Vps10 and of endosomes retrieval Vacuolar 4. Fig. rtis(e aeil n ehd o eal) eut are Results details). for marker Methods mean endosome and and Materials Golgi (see with proteins Vps10– transport of retrograde colocalization during the GFP highlight of arrowheads Quantification White (C) process. colocalizations. recycling later the at of Vps10 with time-points vacuoles. colocalizes Ypt6 from mCherry-tagged recycling N-terminal during (B) the Vps8 with protein colocalizes marker Vps10 endosomal (A) mCherry. tagged were with proteins C-terminally marker endosome and Golgi overexpression. pep4 ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal D 6 .Uo uino aulsfo hs tan,the strains, these from vacuoles of fusion Upon ). D ...( s.e.m. n h te tanlcsakln phosphatase alkaline lacks strain other the and ) vps1 D n 5 510clstm on) cl as 5 bars: Scale point). cells/time 25–100 rrtoe uat,w ol eeta detect could we mutants, retromer or vps1 nvitro in D sdsrbdpeiul (Kulkarni previously described as GAL1- sn sltdvcoe rmtwo from vacuoles isolated using promoter-induced p1–F eyln was recycling Vps10–GFP m m. 651

Journal of Cell Science rvddeiec htbt p1adteSXBRprotein SNX-BAR 2014). al., the et (Chi and formation tubule Vps1 endosomal in both cooperate Mvp1 that evidence organelle. this provided of homeostasis the in involved are retromer nlsso el nsoni a efre fe rnfrit P.Snl-ln mgso F,mhryadDP hneswr curdeey1 f s 5 co 10 time bars: every acquired Scale A were vacuoles. membrane. channels on vacuolar DAPI Ypt7 and the mCherry to GFP, on relative of moving structures images structures Single-plane Vps10-positive Vps10–GFP-positive YPD. of indicate into Dynamics transfer arrows (F) after Yellow h D. min. in 7 2 performed as of was out E period carried in was shown in Assay cells time. of over analysis Vps10-GFP of that to compared EERHARTICLE RESEARCH 652 fusion be in This could SNAREs SNARE. of vps1 which Vam7 misorganization the soluble defect, with a agrees fusion of defect homotypic addition the a by similarly, have reversed behave both mutants Vps1 they proteins evidence and i.e. provide recycle retromer data from can our for vacuoles Finally, retromer that required tubule membrane. that the vacuolar initially the enter strong demonstrates from not is provide This does but Ypt7 also 5). membrane (Fig. We GTPase the tubule at 3). Rab function (Fig. this retromer the event of probably that fission occurs fission evidence cytoplasm this the the after into mediates release Once only retromer Vps1 2B). and Fig. formed, 3) 1; concentrates (Fig. (Fig. is retromer tubule a tubule the through a release that for its 6G here to Fig. prior demonstrate (see cargo We detail unprecedented model). an with a events of order the u 1c Fig. in as performed was Analysis retrieval. Vps10–GFP in block a to leads (D) Mon1 GEF the or (C) Ypt7 GAL1 of Deletion recycling. Vps10–GFP for required vacuole. the from transport retrograde retromer-mediated for vps26 essential is Ypt7 5. Fig. uniiaino oa loecnecreaini a are u sn h ao otae eut r mean are Results software. JacoP the using out carried was A in correlation fluorescence total of Quantification rvossuyo h nooa ucino retromer of function endosomal the on study previous A D pooe-eitdoeepeso of overexpression -promoter-mediated D uat nvcoemmrns niaigta p1and Vps1 that indicating membranes, vacuole on mutants ypt7 D uatsris aulswr tie ihF46.()Qatfcto fVs0clclzto ihtevcoa ebaemre FM4-64. marker membrane vacuolar the with colocalization Vps10 of Quantification (B) FM4-64. with stained were Vacuoles strains. mutant VPS10 E oaiaino p7drn h eyln rcs.Ntria Cer-agdYt itiuinwas distribution Ypt7 mCherry-tagged N-terminal process. recycling the during Ypt7 of Localization (E) . vps35 D and uat eutdi h aerccigdfc,w upc htthe that suspect we defect, retromer recycling all same and the assays, in our resulted in mutants similar behaved subunits retromer retromer At the to 2012). recruited al., is Vps1 et how (Cocucci 2012), know tubules. yet dynamin al., not endocytosis of do et we role in present, known Rooij implicated the Smaczynska-de to been 2010; similar al., previously vacuoles, et has requirements (Nannapaneni from their Vps1 in recycling differ 2). the also (Fig. retromer-mediated processes to two in the that contrast a suggesting Mvp1 for In evidence of find not events. did role we membranes, transport will retromer endosomal factor strongly on limiting retrograde findings a retromer recycle as further which the complex, to block this limit of Vps1, will pool also accumulation available This of the 3). but (Fig. absence membranes on the tubulation, accumulated In for components. needed only nooe n auls sVs0dcrtdtblsddnot both did on Vps1 tubules in of Vps10-decorated vacuoles function from as fission separate vacuoles, a and with agree endosomes nicely data Our o srtoe ucincodntdo ebae?A all As membranes? on coordinated function retromer is How A oaiaino p1–F nwl-ye and wild-type, in Vps10–GFP of Localization (A) ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal 6 vps1 ...( s.e.m. D m AE;1 (A,E); m n el.Mroe,Vs snot is Vps1 Moreover, cells. 5 513.(,)Atv p7is Ypt7 Active (C,D) 45–133). m (F). m vps26 D , ypt7 D urse sing and ra or

Journal of Cell Science EERHARTICLE RESEARCH emne l,20) nciaino p7hsbe hw to endosomes shown in retromer-positive been 2012), has on 2008; Ypt7 Vps10 al., al., of of et on et Inactivation has accumulation (Rojas 2009). function Liu cells cause work al., mammalian retromer 2010; et in Previous Seaman in al., work coordinate 2007). with subcomplexes et can agreement al., two (Balderhaar Ypt7 the et endosomes that the into (Hierro suggested though separate a cells as even to complex metazoan this unpublished), purify seems could C.U., we complex (Seaman that (H.A., fact retromer the of heteropentamer and identification 1998) This initial al., process. the et recycling with the agree during unit would one as functions complex 27 at min 90 for incubated buffer fusion ATP-containing the in resuspended strain DKY the from vacuoles to added were vacuoles BJ 5 bars: Scale Methods. and Materials the in described as respectively, FM4-64, and CMAC with stained were membrane homeostasis. the vacuole from in expressed involved are was GFP–Vam3 Vps1 and Retromer 6. Fig. muto aulswssprtdb D-AEadaaye ywsenbotn sn nioisaantVm n a8a odn oto.()Represen (F) and control. retromer loading as from Vac8 isolated and Vam3 vacuoles against antibodies with using assays blotting western fusion a by of analyzed wild-type results and in SDS-PAGE levels by Vam3 separated of was Comparison vacuoles (E) of plasmid. amount centromeric a from expressed was RFP–Vam3 GFP. vps35 with tagged C-terminally was Vma2 Endogenous (D) niae,2 indicated, ermrfnto nedsmladvcoa ebae.()Mdlfrtafcigo rnmmrn rtist n rmtevacuole. the from and to proteins transmembrane of trafficking for Model (H) membranes. vacuolar and endosomal on function retromer D aulsfo Jwl-ye(t and (wt) wild-type BJ from Vacuoles . m a7wsaddt h uinrato ga as.Arpeettv euti hw.Eprmnswr eetda es w ie.()Mdlfor Model (G) times. two least at repeated were Experiments shown. is result representative A bars). (gray reaction fusion the to added was Vam7 M PHO5 rmtr oaiainwsaaye nwl-ye(A), wild-type in analyzed was Localization promoter. vps35 D tan eeioae o h aul uinasya ecie nteMtrasadMtos nequal An Methods. and Materials the in described as assay fusion vacuole the for isolated were strains vps1 AC oso h ermro p1rslsi natrdlclzto fVm.Chromosomal Vam3. of localization altered an in results Vps1 or retromer the of Loss (A–C) uat.Dltoswr eeae nthe in generated were Deletions mutants. p7GPta a ciae yteMn–c1GF first GEF, Mon1–Ccz1 endosomal the occurs. the fusion which membranes HOPS-driven by before in recycling model initiating activated Vps10-positive in a engages was with on that agree stays data Ypt7–GTP vacuole, Our the retromer 5). from (Fig. released carrier the Vps10 in the for of whereas essential part involved not is is Ypt7 but On is that recycling, clear. demonstrate yet could Ypt7 not endosome- is we and mechanism vacuoles, that exact recycling the a although between such suggests fusion, vacuole coordination in This vacuole the the organizing 5). (Fig. blocked to not localizes is situation Vps10 recycling endosomal because However, completely, 2012). al., et (Liu ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal vps35 D B and (B) pep4 vps1 D D akrudsri B30) Isolated (BJ3505). strain background C el.Tevcoelmnand lumen vacuole The cells. (C) m ,nra iw 1 view; normal m, ˚ bakbr) Where bars). (black C m ,dtie view. detailed m, tative 653 nd

Journal of Cell Science scodntddrn ermrdie eyln ed further needs turnover on recycling and effect retromer-driven activity an investigation. during Rab without coordinated How was tested 2C). GAPs is (Fig. We Ypt7 efficiency but putative 2009). recycling, recycling the for al., necessary in of is et identified Ypt7 deletion (Seaman been of inactivation has retromer GAP that whether the GAP with specific likely a complex then to will substrate tubules endosomal become on Rab7/Ypt7 remaining Any ARTICLE RESEARCH 654 1998). as al., fragments et Longtine PCR 2004; overlapping al., et of carried (Janke were recombination previously described manipulations homologous Genetic by S1. out Table material supplementary cerevisiae biology molecular Saccharomyces and manipulation genetic Yeast METHODS AND MATERIALS vacuole. in relevance the its transport of understand physiology fully retrograde to the work retromer-mediated membrane this by of revealed of pathway cargoes that devoid the showed identify largely which (Mu were proteins microautophagy, yet tubules electron-microcopy- on a by might microautophagic suggested observations or mechanisms as exclusive vesicles based These mutually 6H). intraluminal be (Fig. not into mechanism system sorting either unidentified vacuolar endomembrane subsequent by extensive the occur avoid and through and could recycling control Downregulation microautophagy, to be process could expansion. It a membrane changes. is of its environmental change part vacuole to to also the and response function in from vacuolar composition maintain proteins to vacuoles. transmembrane necessary of control likely of quality for removal mechanism a The of part be also could that vacuoles fusion. maintaining for in competent involved are is vacuoles, vacuoles. retromer fragmented on that have present indicating mutants apparently retromer the some is and Furthermore, machinery that a retromer fusion show both either the to data to though applies Our in relates defect defect investigation. 2004) a fusion observed of further al., consequence needs a et the to recycling, 2013; rather Peters same al., Whether or 2014; et function Vam7 fusion-specific (Alpadi the 2004). al., fusion SNARE et membrane by al., soluble Kulkarni for Vps1 et the rescued of of requirement (Thorngren be addition 6F) could the (Fig. to i.e. membrane fusion leads the mechanism, both in deletion and in similar their were backgrounds, defects fusion that the and at is Indeed, organization cooperate always expectation consequences. a retromer one similar and reflects Vps1 vacuole, rather If loss but the to Vps1. due here, or homeostasis case retromer membrane of vacuole special the the that a in believe change is not general do Vam3 we evident other of However, be localization. SNAREs necessarily response in not of must change a localization recycling recycling in by in on defect we a difference Although directed Vam3, function 2004). a than al., to detect et a also transport Dove not 1998; further could al., for that for et (Bryant machinery Vti1 vacuole substrates sufficient the provide like likely to is subunit process SNAREs are V-ATPase Vam3 not. the SNARE endosomes are like the other proteins Vma2 whereas that mutants, transmembrane both here in vacuolar organization lateral observed at its in homeostasis We affected membrane vacuole. of organization the general the onto light u nlsso ermradVs ol nvcoe sheds vacuoles on pools Vps1 and retromer of analysis Our eseuaeta h p1-eitdmmrn retrieval membrane Vps10-mediated the that speculate We le ta. 00.I ilb motn nftr to future in important be will It 2000). al., et ¨ller tan sdi hssuyaelse in listed are study this in used strains vps1 vps1 D uat,even mutants, and vps35 D ytei eimcnann lcs rglcoe obokprotein block 10 of To after concentration final galactose. a microscopy to or added light was glucose cycloheximide with by synthesis, containing washed YP always analyzed medium were in cells and diluted synthetic images, collecting and 2% glucose, Before washed times. containing 2% phase, different medium logarithmic with YP to ml medium h 10–20 18–22 in for grown galactose were cells membrane, with mM washed 0.1 and in medium. min incubated SDC 10 were Emr, for cells and (CMAC) vacuoles, (Vida of 7-amino-4-chloromethylcoumarin analysis staining before h luminal 1 For for 1995). dye without medium in membrane incubation 30 (YP) vacuole with The peptone stained (SDC). extract acids was amino yeast medium essential synthetic in with or (YPG) supplemented phase galactose (YPD), logarithmic glucose containing to medium grown were analysis Cells image and microscopy Light ..adCU eindeprmnsadwoetemanuscript. F.R. the by wrote out and carried experiments experiments designed EM C.U. for and except H.A. experiments all conducted H.A. contributions Author interests. financial or competing no declare authors The interests Competing expert for Xanthakis Despina and suggestions Perz assistance. for Angela technical laboratory and Ungermann project, the this and throughout Babst Markus thank We Acknowledgements MgCl mM 5 of KCl, mM lysis 125 described DKY), mild 30 (see as and in gradient by (BJ up strains step set type were followed Ficoll DKY2168 vacuole reactions a Fusion or S1) 1995). in (Haas, centifugation BJ3505 Table and from spheroblasts material isolated supplementary were Vacuoles fusion Vacuole 2008). previously al., as et examination microscopy (Griffith electron described for processed were Cells microscopy 23 Electron at min 90 constant thorough for under exchange Y04C by with incubated medium Onix and CellASIC SDC stained followed Millipore) to applied (Merck types, was was chambers mixture microfluidics mating strain The strains. different One both cells of mixing distinguish above. or yeast to described MATa haploid CMAC as either mutants, phase in yeast logarithmic mutations of respective complementation carrying analyze To assay mating Yeast 0n i-e1 iho ihu nAPrgnrto ytmfr9 min 90 10 for system with regeneration ATP supplemented an 26 without 6.8 at or with pH His-Sec18 ng Pipes-KOH 10 mM 1 sorbitol, fsmlslcigAPwssbrce rmoiia values. original from subtracted was ATP lacking to samples phosphate of of absorbance nm Background p-nitrophenyl nitro-phenol. 400 alkaline-phosphatase-generated at of absorbance the the addition measure to by membranes detergent-solubilized developed were Reactions betv n olNPH2cmr Pooerx usn AZ). 100 Tucson, (Applied a (Photometrix, with camera software 1–4 equipped of HQ2 stacks microscope SoftWoRx CoolSNAP similar and a and objective on system or Precision) illumination InsightSSI 100 a with equipped loecnesgasbtentocanl.Atraiey nobject-based an Alternatively, channels. two between and signals (Bolte fluorescence JACoP using out carried Cordelie either was colocalization a of Analysis of ImageJ. plane using representative processed One further were Images (SoftwoRx). deconvolution r eetdadoelpi uniiduigai-os mgJPlugin ImageJ in-house a unpublished). C.U., using Piehler, quantified Jacob Kurre, is threshold Rainer overlap certain (H.A., a and above detected sources single-point are where used, was approach oaayeterlaeo vrxrse p1–F rmtevacuolar the from Vps10–GFP overexpressed of release the analyze To mgswr curdo nOypsI-1ivre microscope inverted IX-71 Olympus an on acquired were Images ˚ .2 C. ` e,20)t eemn ero’ orlto ofiin ftotal of coefficient correlation Pearson’s determine to 2006) res, ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal m frcmiatHsVm a de hr indicated. where added was His-Vam7 recombinant of M m ih30n pcn eeue o constrained-iterative for used were spacing 300-nm with m 6 m M-4fr3 i,floe ywsigand washing by followed min, 30 for FM4-64 M A14 betv,a CO aea(C) an (PCO), camera sCMOS an objective, 1.49 NA z saki hw nesohrienoted. otherwise unless shown is -stack ˚ ro oiaeacquisition. image to prior C m ih3 with l a eegonto grown were m 6 2 m feach of g ,20mM CoA, M A1.4 NA m g/ml. z -

Journal of Cell Science iro . oa,A . oa,R,Mrh,N,Efni,G,Kjv,A V., A. Kajava, G., D. Effantin, Fasshauer, and N., R. Murthy, Jahn, R., Rojas, L., A. A. Rojas, A., T. Hierro, Rapoport, and R. Heinrich, M., Silhankova, J., I. McGough, J., M. Lorenowicz, F., Port, M., Harterink, and E. Reid, C., Freeman, R., Antrobus, A., Y. S. Breusegem, E., M. Harbour, W. Wickner, and B. Conradt, A., Haas, oiaio .S n uly .H. J. Hurley, and S. J. Bonifacino, ak,C,Mgea .M,Rtfle,N,Txs . ee,S,Meaa H., Maekawa, S., Reber, C., Taxis, N., Rathfelder, M., M. Magiera, C., Janke, A. Haas, n h ehrad raiainfrHat eerhadDevelopment 016.130.606]. and number Research [grant Health VICI for (ZonMW) Organisation Netherlands DN82-303]; numbers number The [grant [grant and cooperation Research) DFG-NWO Scientific a 822.02.014]; for and Program Organisation 821.02.017 Open Deutsche (Netherlands ALW the NWO by of the supported fellowship of is a F.R. by (DAAD). support Auslandsdienst received Hans-Mu Akademische the H.A. by C.U.). and (to (DFG); P11); foundation Forschungsgemeinschaft (project Deutsche 944 the (SFB) by Sonderforschungbereich supported was work This Funding ARTICLE RESEARCH hn . o,Y,Mo . u,D,Go . h,J,Lu . h,C,Yn,M., Yang, C., Zhu, X., Liu, J., Shi, G., Gao, D., Sun, D., Mao, Y., Zou, R., Chen, D. S. Emr, and G. E. Marcusson, L., J. Cereghino, D. S. Emr, and S. Sarkar, M., S. Padilla, P., Burda, H. T. Stevens, and S. L. Weisman, C., R. Piper, J., N. Bryant, H. T. Stevens, and K. Bowers, Cordelie and S. Bolte, Bro C., Ostrowicz, H., Arlt, K., J. H. Balderhaar, Ayscough, H., K. Sippel, S., Srinivasan, S., Namjoshi, A., Kulkarni, K., Alpadi, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132720/-/DC1 online available material Supplementary material Supplementary rfih . ai . eMazie De M., Mari, J., Griffith, H. T. Stevens, and K. C., Ekena, D. Hughes, C., M. King, W., J. Yu, K., R. McEwen, C., R. Piper, K., S. Dove, H. T. Stevens, and T. A. Kirchhausen, A. G. and Cooper, C. S. Burd, Boulant, F., and Aguet, G. E., Odorizzi, Cocucci, J., Wang, M., West, J., Liu, J., R. Chi, tvn .C,Bnfcn,J .adHre,J H. J. Hurley, and vesicles. synaptic S. J. complex. cargo-recognition Bonifacino, retromer the of C., architecture A. Steven, systems. transport vesicular in Wnt compartments for required is and Wntless receptor Middelkoop, P., sorting H. Wnt secretion. G. the R. of al. Heesbeen, transport et van retrograde T., K. R. Basler, J. C., Weering, T. van C., M. Betist, dynamics. tubule endosomal regulate 123 that complexes protein for hub J. N. M. Seaman, inheritance. vacuole of reactions oeoBrhr,A,Degs . cwb . cibl .e al. et E. Schiebel, E., Schwob, G., Doenges, A., Moreno-Borchart, vitro. yeast of labelling immunogold the and cerevisiae. analysis Saccharomyces ultrastructural the for procedure n uohg uigsrmguaiestarvation. serum/glutamine during autophagy and al. et W. VPS Ye, of localization. subset and a function, and stability, Vps10p receptor receptor Cell regulate sorting protein products vacuolar gene the of domain PtdIns Vps34 yeast the by regulated 3-kinase. is transport retrograde endosome-to-Golgi prevacuolar/endosomal the via occurs TGN the to vacuole compartment. yeast the of out traffic cerevisiae. Saccharomyces 438-454. yeast the in vacuole 427-436. late yeast microscopy. light the in analysis at colocalization fusion and recycling receptor endosome. C. retromer-mediated Ungermann, to and linked M. Babst, R., Brandt, membrane intracellular in formation al. trans-SNARE fusion. et M. control Evangelista, interactions A., Mayer, SNARE A., Schmidt, M., Zieger, K., neat ihVS n srqie o aulrpoensorting. protein vacuolar for required is 15 and VPS1 with interacts al. et H. R. Michell, effectors. T., 3,5-bisphosphate 1922-1933. F. phosphatidylinositol of Cooke, family B., a defines A. Holmes, J., Thuring, multiple for receptor sorting the hydrolases. as vacuolar function yeast its in compartments prevacuolar pit. and clathrin-coated a is of life carriers the in transport seconds Vps1. retrograde protein dynamin-related endosomal the by SNX-BAR-coated promoted of Fission 1671-1678. , 3703-3717. , 6 ehd elSci. Cell Methods 1089-1102. , a.Commun. Nat. 19) uniaieasyt esr ooyi aul uinin fusion vacuole homotypic measure to assay quantitative A (1995). .Cl Sci. Cell J. a.Cl Biol. Cell Nat. .Cl Sci. Cell J. 21) h eea mn cdcnrlptwyrgltsmTOR regulates pathway control acid amino general The (2014). .Cl Biol. Cell J. Nature 21) h ag-eetv ermrcmlxi recruiting a is complex retromer cargo-selective The (2010). 115 4 rs .P. F. `res, 21) N3dpnetrtoe aha mediates pathway retromer SNX3-dependent A (2011). 123 1704-1708. , 17 13 3889-3900. , 490 283-294. , 142 21) oeua ahnsgvrigeoyoi of exocytosis governing machines Molecular (2012). 4085-4094. , .Cl Biol. Cell J. 914-923. , 19) h acaoye eeiieMP gene MVP1 cerevisiae Saccharomyces The (1995). Traffic r,A n egoi F. Reggiori, and A. `re, 20) rti rnpr rmtelt og othe to Golgi late the from transport Protein (2005). 201-207. , 651-663. , 19) p1pcce ewe h late-Golgi the between cycles Vps10p (1996). .Cl Biol. Cell J. 20) Retromer. (2008). 9 1060-1072. , 20) uddtu nosubcellular into tour guided A (2006). 19) -rti iad nii nvitro in inhibit ligands G-protein (1994). 20) eeaino nonidentical of Generation (2005). 133 Cell .Microsc. J. 529-541. , 21) h a TaeYt is Ypt7 GTPase Rab The (2010). 126 .Cl Biol. Cell J. 150 .Cl Biol. Cell J. ici.Bohs Acta Biophys. Biochim. 87-97. , kr . Su C., cker, ¨ 20) ermrfnto in function Retromer (2002). .Cl Biol. Cell J. 19) h yolsi tail cytoplasmic The (1995). 495-507. , ur pn elBiol. Cell Opin. Curr. 20) cryosectioning A (2008). 224 Nature 21) h is five first The (2012). 213-232. , 168 20) Functional (2007). 19) Retrograde (1998). 204 21) Dynamin- (2013). 271-280. , 449 dran F., ndermann, ¨ 206 793-806. , o.Cl.Biol. Cell. Mol. 20) Svp1p (2004). MOJ. EMBO 1063-1067. , 173-182. , ¨hlenhoff .Cl Sci. Cell J. o.Biol. Mol. 20) A (2004). (2014). 1744 20 23 , , , acso,E . oadvk,B . eehn,J . hrkain .and E. Gharakhanian, L., J. Cereghino, F., B. Horazdovsky, G., E. Marcusson, Nakada-Tsukui,K.,Saito-Nakano,Y.,Ali,V.andNozaki,T.Mu ogie .S,MKni,A,II eaii .J,Sa,N . ah A., Wach, G., N. Shah, J., D. Demarini, III, A., McKenzie, S., M. G. C. Longtine, Burd, and D. D. Billadeau, K., B. Sackey, S., T. Gomez, T.-T., Liu, E. J. Gerst, and M. Robinson, R., Kama, ilr .E,Clis .M,MCy .J,Rbno,M .adOe,D J. D. Owen, and S. M. Robinson, J., A. McCoy, M., B. Collins, E., S. Miller, Griffith, N., Epp, K., Peplowska, M., Mari, H., Arlt, F., Ahnert, F., D. Markgraf, emn .N,MCfey .M n m,S D. S. Emr, and M. J. McCaffery, N., D. M. S. Seaman, Emr, and L. J. Cereghino, G., E. Marcusson, N., M. Seaman, J. N. M. Seaman, J. N. M. Seaman, A. C. Casey, S., Chi, W., E. Krueger, B., Schroeder, G., S. Weller, J., R. H., Schulze, A. Ren, Y., Mayer, Li, Z., and Cao, T. Y., Tian, Sattler, L., H., Zhang, A. W., Du, Rojas, L., Ma, Y., M., Prabhu, Liu, Y., Rong, A., G. Mardones, T., Vlijmen, van R., Bu Rojas, L., T. Baars, C., Peters, L., Reustle, C., Highfill, B., Schroeder, S., Jain, D., Wang, S., Nannapaneni, a ern,J .T,Vrae .adCle,P J. P. Cullen, and P. Verkade, T., R. J. K., Weering, V. van Bhatia, P., D. Kloer, J., C. Traer, B., R. Sessions, T., R. J. Weering, J. van A. Merz, and I., W. Wickner, W. A., R. Booth, Fratti, M., K. R., Collins, N., Mishra, Thorngren, G., E. Allwood, I., I. Rooij, Smaczynska-de N. Bright, and E. Read, D., Tattersall, E., M. Harbour, J., N. M. Seaman, ukri . lai . iuag,T n ees C. Peters, and T. Sirupangi, K., Alpadi, A., Kulkarni, E. J. Gerst, and C. Ungermann, V., Kanneganti, R., Kama, ia .A n m,S D. S. Emr, and A. T. Vida, lr . ate,T,Flo T., Sattler, O., ller, ¨ noe yteVS0gene. VPS10 the by encoded D. S. Emr, 20) uohgctbs aulrivgntosivle nltrlmembrane budding. lateral vesicle in inverse involved and invaginations sorting vacuolar tubes: Autophagic (2000). estl n cnmclPRbsdgn eeinadmdfcto in modification and deletion gene PCR-based cerevisiae. economical R. Saccharomyces J. and Pringle, and versatile P. Philippsen, A., endosome Brachat, during export cargo retromer-mediated of maturation. regulation GTPase Rab fluorescent new genes: yeast of cassettes. tagging substitution promoter and PCR-based markers more for proteins, toolbox versatile ftevrlnefco ytiepoes nteetrcpooonparasite protozoan enteric the transport in the protease in involved cysteine is factor that effector virulence Rab7 the novel a of is complex retromerlike in recognition cargo of late mode of new a clustering is vesicles. induce interaction clathrin-coated SNARE-adaptor to A Vps21 (2007). homolog Rab5 C. compartments. the endosomal Ungermann, with and F. cooperates Reggiori, J., ope seta o nooet-og ergaetasoti yeast. in transport retrograde Biol. endosome-to-Golgi for essential complex products. gene Vps10p, VPS35 receptor, and sorting VPS30, protein VPS29, the vacuolar Biol. of the function of the retrieval requires Golgi to Endosome beyond. and retromer. requires Golgi hepatocytes. in tubules autolysosomal of A. vesiculation M. McNiven, and formation. vesicle and invagination vacuole reformation. lysosome al. autophagic regulate et C. Zhang, Bonifacino, Rab7. and and P. Rab5 Sluijs, of der action van G., Raposo, S. J., J. A. Heck, S., Mohammed, proteins. fusion actin and the fission membrane of disorganization the via and endosomes and internalization vesicles the cytoskeleton. endocytic perturb al. of et mutations R. motility Vps1:vps1 McDowell, S., protein Moulder, dynamin-like A., Maysent, D., Pittsley, histolytica. Entamoeba ai o N-A-eitdasml fdsic nooa otn tubules. sorting endosomal distinct of J. J. P. assembly EMBO Cullen, SNX-BAR-mediated and H. for J. Hurley, basis R., S. Carlsson, D., Stamou, during together function Rvs167 amphiphysin R. and K. Ayscough, Vps1 endocytosis. and dynamin W. M. Yeast Goldberg, S., Aghamohammadzadeh, TBC1D5. Rab-GAP the by inhibited and catalysed Rab7 122 is GTPase subcomplex small retromer the cargo-selective by the of recruitment Membrane ooo rmtstetasto rmhmfso ocnetmxn in mixing content to hemifusion from transition fusion. membrane the intracellular promotes homolog transport retrograde endosome-Golgi assembly. controls SNARE Btn1 via orthologue disease Batten protein endosome-Golgi yeast. late in mediates sorting protein, disease-related Batten potential ebaednmc n noyoi nyeast. in endocytosis and dynamics membrane maturation. endosome with 107. co-ordinated is tubulation endosome for Sec17/18p and ATP bypassing docking, rapid drives fusion. vacuole SNARE soluble A 2371-2382. , 142 137 20) euaino ermrrcutett nooe ysequential by endosomes to recruitment retromer of Regulation (2008). 79-92. , 665-681. , ora fCl cec 21)18 4–5 doi:10.1242/jcs.132720 645–655 128, (2015) Science Cell of Journal 31 19) h otn eetrfryatvcoa abxppiaeYis Y carboxypeptidase vacuolar yeast for receptor sorting The (1994). o.Bo.Cell Biol. Mol. 4466-4480. , .Cl Sci. Cell J. Traffic u.J elBiol. Cell J. Eur. MOJ. EMBO o.Cl.Biol. Cell. Mol. 20) ag-eetv nooa otn o erea othe to retrieval for sorting endosomal Cargo-selective (2004). 21) h ermrcmlx-edsmlpoenrecycling protein endosomal - complex retromer The (2012). 21) ltrnadphosphatidylinositol-4,5-bisphosphate and Clathrin (2012). 13 21) ii rpe radw eursdnmn2for 2 dynamin requires breakdown droplet Lipid (2013). emyr . cwr,H,Pate,H n ae,A. Mayer, and H. Plattner, H., Schwarz, M., tenmeyer, ¨ 317-328. , .Cl Biol. Cell J. o.Bo.Cell Biol. Mol. .Cl Biol. Cell J. Nature 125 23 20) elfe eosiuino microautophagic of reconstitution Cell-free (2000). lr .adMyr A. Mayer, and S. hler, ¨ 23 .Cl Biol. Cell J. o.Bo.Cell Biol. Mol. 19) e ia ti o iulzn vacuolar visualizing for stain vital new A (1995). Yeast 2765-2776. , 4693-4702. , 2505-2515. , Cell Traffic 89 27 450 499-508. , 605-621. , 14 77 195 570-574. , 165 .Cl Biol. Cell J. 953-961. , 579-586. , 15 16 203-215. , 183 Cell 111-122. , 558-571. , 5294-5303. , 20) h OVTsbntVps8 subunit CORVET The (2009). 20 20) t2 ok rhlgand ortholog Hook1 a Btn2, (2007). 513-526. , a.Cl Biol. Cell Nat. 119 .Cl Biol. Cell J. 5276-5289. , 667-678. , .Cl Biol. Cell J. 19) diinlmdlsfor modules Additional (1998). 151 21b.SNX-BAR-mediated (2012b). 19) ebaecoat membrane A (1998). 519-528. , 20) uulcnrlof control Mutual (2004). .Cl Biol. Cell J. 151 14 21) dynamin A (2014). Yeast 529-538. , 21a.Molecular (2012a). 128 924-934. , 21) h yeast The (2010). 21) h yeast The (2011). 779-792. , Traffic 203 21 .Cl Sci. Cell J. 20) A (2005). 947-962. , 315-326. , (2012). (1997). (2012). (2009). (2004). 13 .Cell J. .Cell J. 655 94- ,

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