LETTER. Molecular recognition of a single sphingolipid species by a protein s transmembrane domain

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1 oi:.38/nture742 Moleulr reognition of single sphingolipi speies y protein s trnsmemrne omin F.-Xier Contrers 1, Anres M. Ernst 1, Per Herknt 1 {, Ptrik Björkholm 2,3, Erik Linhl 2,4, Bşk Gönen 1, Christin Tisher 5, Arne Elofsson 2,3, Gunnr von Heijne 2,3, Christoph Thiele 6, Riner Pepperkok 5, Felix Wieln 1 & Britt Brügger 1 Funtioning n proessing of memrne proteins ritilly epen on the wy their trnsmemrne segments re emee in the memrne 1. Sphingolipis re struturl omponents of memrnes n n lso t s intrellulr seon messengers. Not muh is known of sphingolipis ining to trnsmemrne omins (TMDs) of proteins within the hyrophoi ilyer, n how this oul ffet protein funtion. Here we show iret n highly speifi intertion of exlusively one sphingomyelin speies, SM 18, with the TMD of the COPI mhinery protein (ref. 2). Strikingly, the intertion epens on oth the hegroup n the kone of the sphingolipi, n on signture sequene (VXXTLXXIY) within the TMD. Moleulr ynmis simultions show lose intertion of SM 18 with the TMD. We suggest role of SM 18 in regulting the equilirium etween n intive monomeri n n tive oligomeri stte of the protein 3,4, whih in turn regultes COPI-epenent trnsport. Bioinformti nlyses preit tht the signture sequene represents onserve sphingolipi-ining vity in vriety of mmmlin memrne proteins. Thus, in ition to funtion s seon messengers, sphingolipis n t s oftors to regulte the funtion of trnsmemrne proteins. Our isovery of n unpreeente speifiity of intertion of TMD with n iniviul sphingolipi speies s to our unerstning of why iologil memrnes re ssemle from suh lrge vriety of ifferent lipis. Lipiomis of Golgi-erive COPI vesiles unrvelle prtil segregtion of holesterol n most sphingomyelin speies exept for one (N-steroyl sphingomyelin) from the vesile frtion 5.To unerstn the moleulr mehnisms of SM 18 enrihment in vesiles, we investigte in vivo ining of lipis to n p23, memrne proteins involve in COPI vesile iogenesis (Supplementry Fig. 1) 6. Lipi rosslinking 7,8 revele strong sphingolipi lelling of (Fig. 1,, see lso Supplementry Fig. 2), with fivefol higher reovery of riotivity per mole of protein ompre to p23. To nlyse if this intertion of with sphingomyelin is speifi for SM 18, we estlishe liposoml ssy to stuy TMD lipi intertions (see Supplementry Fig. 3). Mltose-ining protein (MBP) fusions of the TMDs of p23 n were nlyse in liposoml Förster resonne energy trnsfer (FRET) system for their intertion with pentenoyl-sm 18 (SM 18:5, s fluoresently lelle nlogue of enogeneous SM 18:O (ref. 9), see Supplementry Informtion), pentenoyl-ermie 18, or pentenoyl-phosphtiylholine (PC) 18 (Fig. 1). Distint FRET ws oserve etween the TMD of n SM 18:5, ut not with the TMD of p23. A mutnt lking the single Trp resiue within the TMD ((TMD)W4A) i not trigger FRET. With ermie 18:5 no FRET ws oserve for ny TMD. With PC 18:5 wek FRET ws oserve with the (TMD), n slightly stronger signl ws otine with p23(tmd). Together these results show mrke speifiity of for sphingomyelin, with oth the hyrophoi moiety n the hyrophili holine phosphte hegroup neee for the intertion. We next nlyse the extent of FRET otine etween the TMDs of n p23 n pentenoyl-sphingomyelins overing the rnge of the mjor enogenous moleulr speies from C14 to C24. A remrkle speifiity ws oserve for the intertion of with SM 18 in liposomes ompose of i-oleoyl-phosphtiylholine/phosphtiylethnolmine/ pentenoyl-sphingomyelin (Fig. 1, left pnel). In liposomes more losely refleting the lipi omposition of the mmmlin Golgi pprtus we gin oserve striking speifiity for SM 18, with wek ut signifint signls for SM n SM 22 (Fig. 1, right pnel). In ontrst, the TMD of p23 i not give rise to omprle FRET with ny of the sphingomyelin speies. To efine the struturl prerequisites for SM 18 ining, we performe n Al sn ross the TMD of strting with position 8 within the TMD, leving three mino is efore n fter W4 unhnge, to minimise iret influene on the iophysil properties of the FRET onor. Two groups of mutnts were oserve, with strong inhiition of FRET with Al sustitutions in positions 8,, 11, 12,, 19 n (group 1), n less inhiition in positions 13, 16, 17, 18 n 21 (group 2) (Supplementry Fig. 4). Although group 1 mutnts showe strong inhiition of FRET, they mintine high egree of speifiity for SM 18:5 (for exmple, V11A, Supplementry Fig. 5). In ontrst, group 2 mutnts V13, T16, L17 n Y21 showe strongly ompromise speies speifiity (Supplementry Fig. 5 f). Mutnt G18A ws not further investigte, euse G18W h not shown n ltertion of SM 18 ining (Supplementry Fig. 3), initing tht G18 is not iretly involve in the lipi intertion. From the ove t we eute tht roxy-terminl motif me of mino i resiues V13, T16, L17 n Y21 of the TMD (V181, T184, L185 n Y189 in the full-length protein) represents struturl eterminnt for the speifi ining of SM 18 (see lso Supplementry Disussion). An energy-minimise projetion of the TMD yiele the struture epite in Fig. 2 (left). In this moel, groove is forme from the Tyr resiue in position 21 to the Vl resiue in position 13. Within the sphingomyelin-ining motif, the -rnhe resiue Ile is foun in position of the TMD. -rnhe resiues were shown to ontriute to TMD TMD intertions,11 y onveying higher rigiity n therey llowing for stronger Lonon ispersion fores. In the moel, this resiue is prt of the vity n thus is likely to ontriute to the ining motif. If this groove inee ommotes the kone of SM 18, the insertion of ulky hyrophoi resiue in position 17 shoul prevent lipi ining. To fill this vity with miniml ltertions of the helil struture 12 we introue Phe resiue in position 17 of the TMD. FRET nlysis showe tht ining of SM 18 ws ompletely inhiite (Fig. 2). To hllenge the existene of C-terminl ining motif, we trnsferre the C-terminl hlf of the TMD to the minoterminl hlf of the non-sphingomyelin-ining TMD of p23. The 1 Heielerg University Biohemistry Center, Im Neuenheimer Fel 328, 691 Heielerg, Germny. 2 Center for Biomemrne Reserh, Deprtment of Biohemistry n Biophysis, Stokholm University, SE-6 91 Stokholm, Sween. 3 Stokholm Bioinformtis Center, Siene for Life Lortory Stokholm University, Box 31, SE Soln, Sween. 4 Theoretil & Computtionl Biophysis, Royl Institute of Tehnology, AlNov University Centre, SE-6 91 Stokholm, Sween. 5 ALMF, EMBL, Meyerhofstrsse 1, Heielerg, Germny. 6 LIMES Life n Meil Sienes Institute, Crl-Troll-Strsse 31, 531 Bonn, Germny. {Present ress: Cell Biology n Biophysis Unit EMBL, Meyerhofstrsse 1, Heielerg, Germny. These uthors ontriute eqully to this work. 26 JANUARY 12 VOL 481 NATURE 5 12 Mmilln Pulishers Limite. All rights reserve

2 RESEARCH LETTER I(F)/I(F) I(F) mx of SM 18:5 with TMD (in %) Input SN IP (%) IP (33%) IP (%) p23 Input SN IP (%) IP (33%) IP (%) Riotivity per mole of protein SM Cer PC Wvelength (nm) Wvelength (nm) Wvelength (nm) I(F)/I(F) SM 14:5 SM 16:5 SM 18:5 SM :5 SM 22:5 SM 24:5 SM 14:5 SM 16:5 SM 18:5 SM :5 SM 22:5 SM 24: I(F)/I(F) p23 Figure 1 speifilly interts with SM 18., CHO ells were grown in the presene of mci of [ 3 H]-photo-sphingosine. Cells were ultrvioletirrite, lyse n sujete to immunopreipittion using ntioies ginst p23 or. Riotivity reovere from input, superntnt (SN) n immunopreipittion (IP) ws visulize y utoriogrphy (upper pnels). Lower pnels, western lot nlysis., Quntifition of immunopreipitte riotivity., In vitro FRET nlysis of MBP TMD fusion proteins n pentenoyl-lipis. Proteoliposomes ontine either SM 18:5, pentenoyl-ermie 18:5 (Cer) or PC 18:5. Re urve, p23(tmd); lk urve, (TMD); lue urve, (TMD)W4A., Proteoliposomes were prepre in the presene of (TMD) (lk rs) or p23(tmd) (grey rs) n 1 mol% of pentenoyl-sphingomyelins, mimiking liquiisorere phse (left pnel) or mmmlin Golgi memrne (right pnel). Bkgrounsutrte fluoresene t re normlize to SM 18:5, n re the men 6 s.. of three experiments. (TMD) p23(tmd) (TMD)L17F (TMD)/p23(TMD) himer YFP (TMD) YFP (TMD)L17F Normlise to I(F) mx of SM 18:5 with (TMD) (%) (TMD) p23(tmd) (TMD) L17F (TMD)/ p23(tmd) himer 2.5% input 5% SN MM 2.5% input 5% SN 2 1 nti-yfp Figure 2 Chrteriztion of the sphingomyelin-ining poket., Energy-minimise struture of (TMD), p23(tmd), (TMD)L17F, n /p23 himeri TMD. Resiues of involve in SM 18 reognition re epite in re, the L17F muttion is highlighte in green., Proteoliposomes ontining (TMD), p23(tmd), (TMD)L17F or the (TMD)/p23(TMD) himer were prepre in the presene of 1 mol% of pentenoyl-sm (18:5) (i-oleoyl-phosphtiylholine/ phosphtiylethnolmine/pentenoyl-sphingomyelin, molr rtio 89::1). FRET mesurements were performe s esrie ove. Dt re the men 6 s.. of three inepenent experiments., In vivo ining of [ 3 H]- sphingolipis to YFP n YFP L17F. Upper pnel, utoriogrphy; lower pnel, western lot. MM, moleulr mss mrker., Moleulr ynmis simultions: snpshot of SM 18 (TMD) intertion. The interting lipi n resiues isplying the signture re highlighte. Blue, (TMD); re, sphingomyelin-ining poket, yellow; SM 18: he group; green, SM 18: kone n N-ylte ftty i. 526 NATURE VOL JANUARY 12 Mmilln Pulishers Limite. All rights reserve 12

3 RESEARCH himeri TMD yiele % of the level of FRET ompre to the TMD of (Fig. 2). Interestingly, the himer shows promisuity with regr to the sphingomyelin moleulr speies, ining signifint mounts of SM n SM 22 in ition to SM 18 (Supplementry Fig. 5), pointing t ontriution of the N-terminl resiues to the orienttion of the sphingomyelin-ining poket. The loss of FRET y mutnt (TMD)L17F suggeste loss of SM 18 ining. To test loss of ining in vivo, full-length wil-type n L17F were expresse s yellow fluoresent protein (YFP) fusion proteins (Supplementry Fig. 6). Sphingolipi lelling showe tht L17F hs lost the pility to in sphingomyelin in vivo (Fig. 2). Sphingomyelin is known to e synthesize in the luminl leflet; however, in vitro experiments suggest tht sphingolipis n flip etween the two leflets stimulte y free ermie tht trnslotes from the ytoplsmi to the luminl leflet of memrnes 13 (see lso Supplementry Fig. 6). In ition, in vivo nlyses using the sphingomyelin-ining toxin equintoxin II point to n ourrene of sphingomyelin in the ytoplsmi leflet of the Golgi memrne 14. To unerstn ining seletivity t the moleulr level, we ompre the struturl fetures of sphingomyelin moleulr speies (see Supplementry Fig. 7). Moleulr ynmis simultions n monolyer experiments 17 suggest tht only the sphingomyelin speies with suitle ynmi volume woul fit into the vity of the TMD forme y V13, T16 n L17, exluing lipis with lrger ynmi volume (SM 14 n 16, see Supplementry Fig. 7). Sphingomyelin speies with smller ynmi volumes, however, woul nee to e exlue from the hyrophoi vity y ifferent mehnism (see Supplementry Disussion). We next investigte the ining signture through series of five moleulr simultions of the TMD of emee in POPC ilyer inluing sphingomyelins with ftty is of C14 to C22. By extening ll tomisti simultions to 1 ms we oserve spontneous iffusion of sphingomyelins to the TMD n lose intertion with the propose ining site primrily for SM 18, n to minor extent lso SM 16 n SM (Supplementry Fig. 8). The polr he group of sphingomyelin wrps roun the Y21 sie hin, while the C18 hin ontinues own roun the helix n pks in the groove etween V13, T16 n L17 (Fig. 2 n Supplementry Movie 1). The sphingosine hin pks in the groove elow V13. The other sphingomyelin moleulr speies lso pproh the TMD, ut steri effets seem to mke intertions more iffiult in these ses. For instne, the SM 14 lipi he group rther interts with Y21 y pking mostly elow the sie hin, whih rottes oth lipi hins wy from the helix n prevents effiient pking (Supplementry Movie 2). The lifetime of the SM 18 (TMD) omplex in the moleulr ynmis simultion ws of the orer of 2 ns, whih is five times longer thn oserve for the other sphingomyelin speies. Likewise, s ompre to SM 18, the reltive issoition onstnt of SM 14 ws 6.5-fol higher (Supplementry Fig. 9). Notly, lthough the TMD of is highly onserve in higher eukryotes, it is not onserve in yest, in greement with the sene of sphingomyelin in this orgnism. To serh for other nite sphingolipi-interting proteins, we efine ining signture where either -rnhe resiue (I, T, V) or Leu is llowe in ny of the first four positions, n n romti resiue (F, W, Y) is llowe in the lst position (Fig. 3). The signture represents unique sequene motifs, 13 of whih were foun to e overrepresente in set of mmmlin memrne proteins (see Full Methos in Supplementry Informtion). These 13 motifs ientifie 48 nite proteins (Supplementry Tle 1), mostly lolize to the plsm memrne (Fig. 3). Three reominnt nites n, s negtive ontrol, the nonsignture ontining silo-glyoprotein reeptor, were expresse in i = 13 ± 3 NH 2 V/I/T/L F/W/Y i i+3 i+7 i+4 VVLWSFFEALVLVAMTLGQIY i+8 CCOOH Per ent Plsm memrne Not nnotte Enoplsmi retiulum Golgi Mitohonri Lysosomes 2 1 IFN-γ INGR1 + 1, U ml 1 IFN-γ I17RB B3GT2 ASGR1 I17RB B3GT Figure 3 A onserve sphingolipi ining signture., Signture ptterns use for oth rnomiztion/shuffling pproh n sreening for potentil sphingolipi-ining proteins (upper pnel). All possile omintions were nlyse for signifint overrepresenttions in TMDs. Motifs overrepresente with P-vlue #.5 were use to generte the sequene logo (lower pnel). The letter size orrespons to the proility to fin this mino i t tht position. Eh ot elow the line represents n mino i position., Intrellulr istriution of signture-ontining trnsmemrne protein nites., In vivo lelling of -tgge onstruts of humn INGR1, I17RB, B3GT2 n ASGR1. Cells were lelle with [ 3 H]-photo-sphingosine, ultrviolet-irrite, lyse n sujete to IP using n ntioy ginst the tg. Riotivity reovere with protein nites ws visulize y utoriogrphy. The orresponing proteins were etete y western lot nlysis. Asterisk, expete size of proteins. 26 JANUARY 12 VOL 481 NATURE Mmilln Pulishers Limite. All rights reserve

4 RESEARCH LETTER HeL ells (Supplementry Fig. ). In in vivo lelling ll three nites showe strong ining to sphingolipi (Fig. 3). Interestingly, INGR1 ins to sphingolipi only upon tivtion y its lign interferon. Wht my e the funtion of the highly speifi intertion of SM 18 with? As ts s memrne mhinery for the formtion of COPI vesiles, we nlyse if loss of ining of SM 18 woul ffet trnsport of iosyntheti rgo 18,19. We nlyse trnsport of vesiulr stomtitis G (VSV-G) protein,21 to etermine the trnsport rtes in HeL ells stly trnsfete with full-length wil-type or L17F, N-terminlly fuse to YFP. Expression of L17F use n elertion of VSV-G protein trnsport (Fig. 4), resulting in 2.5- fol inrese of the frtion of totl VSV-G tht rehe the plsm memrne t t 5 45 min (see lso Supplementry Fig. 11, ). How n mrke elertion of trnsport e use y lk of ining of SM 18 to? A erese in reyling effiieny woul use n inrese nterogre trnsport of this rgo, n shoul ffet Golgi export. To test this possiility, we mesure Golgi export kinetis using fluoresene loss in photolehing (FLIP). Wheres Golgi-ssoite fluoresene of YFP wil-type ws reue to % within 4.5 min, the hlf-life of Golgi resiene of YFP L17F ws signifintly prolonge to 7 min (Fig. 4, for ontrols see Supplementry Fig. 11). To nlyse if inee retrogre trnsport from the Golgi to the ER is ffete we monitore trffiking to the CFP VSV-G (plsm memrne/totl) Dimers (%) f ox L17F ox + ox L17F + ox 4 6 Time (min) 6 4 Hlf life (min) e Dimers (%) Egg Brin SM 18: Egg Brin SM 18: YFP YFP L17F Protein synthesis (%) YFP / YFP L17F/ YFP YFP YFP L17F PE-A + + Figure 4 Bining of SM 18 to (TMD) ffets protein trnsport n triggers imeriztion., Expression of L17F elertes trnsport of ts-o45-g protein. For eh time point n experiment (n 5 3), t lest 6 ells were nlyse. CFP VSV-G, yn-fluoresent-protein-tgge ts-o45-g protein., Comprison of the verge hlf lives of ey in fluoresene in Golgi of YFP n YFP L17F. Dt represent the men of n experiments 6 s.e. P-vlue of two-tile, unpire t-test,.1 () is given., Expression of YFP L17F reues toxiity of PE-A. Error rs, s.e.m. Sttistis: two-tile, unpire t-test., SM 18: inues imeriztion of (TMD) (white rs) ut not of p23(tmd) (lk rs). TMDs reonstitute into proteoliposomes with inite sphingomyelin speies sujete to hemil rosslinking (n 5 4). Results of pire, two-tile t-tests re given. e, Quntifition of homoimers in CHO ells in situ. Error rs, s.e.m. Sttistis: two-tile, unpire t-test. P,.1 f, Moel of (TMD) SM 18 omplexes. Left, sie view; right, top view. enoplsmi retiulum of Pseuomons eruginos exotoxin A (PE-A). The toxin is trnsporte from the Golgi to the enoplsmi retiulum in COPI-epenent mnner 22. In ontrst to ells expressing YFP, YFP L17F-ells showe signifint reution of PE-A-epenent inhiition of protein iosynthesis, the reout for exotoxin trnsport to the enoplsmi retiulum (Fig. 4, for ontrols see Supplementry Fig. 11) Together, these t imply tht effiient retrogre COPIepenent trnsport epens on ining of to sphingomyelin. To test if ining to SM 18 might help orgnize the imeri, trnsport tive stte of, y ffeting its monomer/oligomer equilirium vi the TMD, we use hemil rosslinking ssy with proteoliposomes reonstitute from fmily TMD fusion proteins n sphingomyelins of vrious speies ompositions. Inee, imeriztion is signifintly inue only in liposomes ontining oth SM 18 n (Fig. 4 n Supplementry Fig. 12 ). To nlyse if imeriztion of is lso ffete in vivo in the presene of full-length L17F, we monitore imeriztion of using n in situ protein protein intertion ssy. Signls were signifintly reue in the presene of L17F (Fig. 4e, for representtive imges n western lots see Supplementry Fig. 12). Similr results were otine when ells expressing either YFP or YFP L17F were sujete to hemil rosslinking (Supplementry Fig. 12e). How might omplex of imeri n SM 18 e orgnize? We performe moleulr ynmis simultions strting from ifferent moels se on the sphingomyelin-oun monomeri strutures from the initil simultions. The most stle moel ws one with rther polr imeriztion interfe not overlpping with the sphingomyelin ining site (Fig. 4f); this omplex remine intt for t lest ns. Besies iret role of sphingomyelin in stilizing imer, other mehnisms of sphingomyelin-triggere imeriztion might pply, suh s sphingomyelin-epenent onformtionl hnge of or uil-up of high onentrtions of in SM 18 miroomins. In summry, we hve unovere n unpreeente speifiity of intertion etween n iniviul sphingolipi speies n TMD, n hve efine struturl signture within the TMD for this ining. Speifi ining of iniviul lipi speies to TMDs of memrne proteins my serve ifferent funtions n help to unerstn the nee for the omplexity of memrne lipi ompositions t funtionl level. METHODS SUMMARY In vivo photoffinity lelling of CHO ells. Cells were lelle with the ifferent phototivtle preursors s esrie 7,8. Briefly, ells were wshe with PBS, followe y ition of freshly prepre elipite meium ontining lipi preursors. After lelling, the meium ws remove n ells were wshe twie with PBS. After ultrviolet irrition, ells were ollete n lyse in lysis uffer ( mm HEPES-NOH, ph 7.4, mm NCl, 5 mm EDTA, 1% Triton X- (v/v),.5% eoxyholte (w/v) n protese inhiitor oktil). After lysis, smples were sujete to immunopreipittion. After SDS PAGE n western lotting, riotively lelle proteins were etete y igitl utoriogrphy (-Imger, Biospe). FRET ssy. FRET ws use to proe the intertion of TMDs with pentenoyllipis iretly. FRET experiments were onute on Jso 6 unit spetrofluorometer (Jso). Proteoliposomes (protein/lipi, 1:5,) were ilute in qurtz uvette in uffer ( mm HEPES/KOH ph 7.4) to finl onentrtion of.1 mm. The proteoliposomes were inute t uc for 5 min uner ontinuous stirring. The extent of FRET etween TMD n pentenoyl-lipis ws etermine y reoring emission spetr from 3 to 54 nm. Emission spetr were ollete, exiting t 28 nm the Trp present in the TMD. Slit withs of 5 nm were use for oth exittion n emission. Full Methos n ny ssoite referenes re ville s Supplementry informtion. Reeive 12 Jnury; epte 3 Novemer 11. Pulishe online 9 Jnury Coskun, U. & Simons, K. Cell memrnes: the lipi perspetive. Struture 19, (11). 528 NATURE VOL JANUARY 12 Mmilln Pulishers Limite. All rights reserve 12

5 RESEARCH 2. Popoff, V., Aolf, F., Brugger, B. & Wieln, F. COPI uing within the Golgi stk. Col Spring Hr. Perspet. Biol. oi:.11/shperspet.5231 ( August 11). 3. Béthune, J. et l. Cotomer, the ot protein of COPI trnsport vesiles, isrimintes enoplsmi retiulum resients from proteins. Mol. Cell. Biol. 26, (6). 4. Reinhr, C. et l. Reeptor-inue polymeriztion of otomer. Pro. Ntl A. Si. USA 96, (1999). 5. Brügger, B. et l. Evienefor segregtionof sphingomyelinnholesterol uring formtion of COPI-ote vesiles. J. Cell Biol. 1, (). 6. Bek, R., Rvet, M., Wieln, F. T. & Cssel, D. The COPI system: moleulr mehnisms n funtion. FEBS Lett. 583, (9); orrigenum 583, 3541 (9). 7. Herknt, P. et l. Protein-sphingolipi intertions within ellulr memrnes. J. Lipi Res. 49, (8). 8. Thiele, C., Hnnh, M. J., Fhrenholz, F. & Huttner, W. B. Cholesterol ins to synptophysin n is require for iogenesis of synpti vesiles. Nture Cell Biol. 2, (). 9. Kuershner, L. et l. Polyene-lipis: new tool to imge lipis. Nture Methos 2, (5).. Russ, W. P. & Engelmn, D. M. The GxxxG motif: frmework for trnsmemrne helix-helix ssoition. J. Mol. Biol. 296, (). 11. Senes, A., Gerstein, M. & Engelmn, D. M. Sttistilnlysis of mino i ptterns in trnsmemrne helies: the GxxxG motif ours frequently n in ssoition with -rnhe resiues t neighoring positions. J. Mol. Biol. 296, (). 12. Jones, D. T., Tylor, W. R. & Thornton, J. M. A muttion t mtrix for trnsmemrne proteins. FEBS Lett. 339, (1994). 13. Contrers, F. X., Bsnez, G., Alonso, A., Herrmnn, A. & Goni, F. M. Asymmetri ition of ermies ut not ihyroermies promotes trnsilyer (flip-flop) lipi motion in memrnes. Biophys. J. 88, (5). 14. Bkr, B. et l. A toxin-se proe revels ytoplsmi exposure of Golgi sphingomyelin. J. Biol. Chem. 285, ().. Li, X. M., Smy, J. M., Momsen, M. M., Brokmn, H. L. & Brown, R. E. Sphingomyelin interfil ehvior: the impt of hnging yl hin omposition. Biophys. J. 78, (). 16. Niemelä, P., Hyvönen, M. T. & Vttulinen, I. Struture n ynmis of sphingomyelin ilyer: insight gine through systemti omprison to phosphtiylholine. Biophys. J. 87, (4). 17. Niemelä, P. S., Hyvönen, M. T. & Vttulinen, I. Influene of hin length n unsturtion on sphingomyelin ilyers. Biophys. J. 9, (6). 18. Presley, J. F. et l. ER-to-Golgi trnsport visulize in living ells. Nture 389, (1997). 19. Sles, S. J., Pepperkok, R. & Kreis, T. E. Visuliztion of ER-to-Golgi trnsport in living ells revels sequentil moe of tion for COPII n COPI. Cell 9, (1997).. Keller, P., Toomre, D., Diz, E., White, J. & Simons, K. Multiolour imging of post- Golgi sorting n trffiking in live ells. Nture Cell Biol. 3, (1). 21. Simpson, J. C. et l. An RNAi sreening pltform to ientify seretion mhinery in mmmlin ells. J. Biotehnol. 129, (7). 22. Jkson, M. E. et l. The KDEL retrievl system is exploite y Pseuomons exotoxin A, ut not y Shig-like toxin-1, uring retrogre trnsport from the Golgi omplex to the enoplsmi retiulum. J. Cell Si. 112, (1999). Supplementry Informtion is linke to the online version of the pper t Aknowlegements The uthors woul like to thnk T. Shsenheimer for tehnil ssistne, A. Broe for help with lipi synthesis, D. Cssel for omments on the mnusript, n the memers of the Wieln lortory for isussion. This work ws supporte y grnt of the Germn reserh fountion (DFG, TRR83) to B.B. n F.W. n y ERC grnts to E.L. (98) n G.v.H. (AG232648); F.-X.C. ws supporte y FEBS fellowship n A.M.E. y the Peter n Trul Engelhorn fountion. Author Contriutions F.-X.C., A.M.E. n P.H. esigne n performe the experiments. P.B. performe the ioinformtis nlyses uner the supervision of A.E., G.v.H. n A.M.E.; E.L. esigne, performe n interprete moleulr ynmis simultion experiments. B.G. performe in vivo rosslinking experiments. C.Th. provie regents n helpe to estlish photolelling n FRET experiments. C.Ti. n R.P. provie regents n tools n supporte A.M.E. onerning VSV-G experiments. F.W. n B.B. esigne the experiments n wrote the mnusript. Author Informtion Reprints n permissions informtion is ville t The uthors elre no ompeting finnil interests. Reers re welome to omment on the online version of this rtile t Corresponene n requests for mterils shoul e resse to F.W. (felix.wieln@zh.uni-heielerg.e) n B.B. (ritt.ruegger@zh.uni-heielerg.e). 26 JANUARY 12 VOL 481 NATURE Mmilln Pulishers Limite. All rights reserve