Orm family proteins mediate sphingolipid homeostasis

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1 Vol Februry 2 doi:.38/nture8787 ARTICLES Orm fmily proteins medite sphingolipid homeostsis Dvid K. Breslow,2,3,4, Sen R. Collins,2,4 {, Bernd Bodenmiller 5 {, Ruedi Aebersold 5,6,7, Ki Simons 8, Andrej Shevchenko 8, Christer S. Ejsing 8,9 & Jonthn S. Weissmn,2,4 Despite the essentil roles of sphingolipids both s structurl components of membrnes nd criticl signlling molecules, we hve limited understnding of how cells sense nd regulte their levels. Here we revel the function in sphingolipid metbolism of the ORM genes (known s ORMDL genes in humns) conserved gene fmily tht includes ORMDL3, which hs recently been identified s potentil risk fctor for childhood sthm. Strting from n unbised functionl genomic pproch in Scchromyces cerevisie, we identify Orm proteins s negtive regultors of sphingolipid synthesis tht form conserved complex with serine plmitoyltrnsferse, the first nd rte-limiting enzyme in sphingolipid production. We lso define regultory pthwy in which phosphoryltion of Orm proteins relieves their inhibitory ctivity when sphingolipid production is disrupted. Chnges in ORM gene expression or muttions to their phosphoryltion sites cuse dysregultion of sphingolipid metbolism. Our work identifies the Orm proteins s criticl meditors of sphingolipid homeostsis nd rises the possibility tht sphingolipid misregultion contributes to the development of childhood sthm. As cells grow, divide nd respond to their environment, they must synthesize lipids to meet metbolic demnds while ensuring the correct blnce of wide rry of structurlly nd functionlly diverse lipid species. At the sme time, cells need to mintin the correct distribution of lipids within the membrne bilyers of different orgnelles nd the plsm membrne. Regultory mechnisms tht ensure proper levels of some lipid species including sterols 3 nd vrious glycerolipids 4 6 hve been identified, with loss of such controls leding to vriety of disese sttes,7. Although remrkble progress hs been mde in defining the protein mchinery responsible for synthesizing 8,9 nd trnsporting sphingolipids 3, insights into how cells sense nd mintin their levels re only now emerging 4,5. The need for precise regultion of sphingolipids my be prticulrly cute s, in ddition to the structurl roles of the terminl products, severl biosynthetic intermedites including sphingosine, cermide nd their phosphorylted derivtes re signlling molecules tht prticipte in key physiologicl nd pthologicl processes 6,7. Our investigtions into how cells chieve sphingolipid homeostsis stem from nlysis of the ORM (or ORMDL) genes, which hve been implicted recently in sthm. Asthm hs emerged s mjor helth problem, with rtes of childhood disese incresing mrkedly over the pst three decdes 8. There is strong heritble component tht modultes susceptibility to sthm, nd mny studies hve been undertken to identify genetic risk fctors 9. A brekthrough in these efforts cme with recent genome-wide ssocition study 2, which identified single nucleotide polymorphisms t chromosome 7q2 ner the ORMDL3 gene tht confer n incresed risk of childhood sthm. Subsequent studies hve reproduced this finding in multiple ethnic groups Furthermore, the disese-ssocited polymorphisms hve been shown to modulte expression of ORMDL3 nd the djcent gene GSDMB 2,24, suggesting tht dysregultion of one or both of these genes my contribute to childhood sthm. Thus genetic vrints ner ORMDL3 re widespred risk fctor for developing childhood sthm tht my rise the incidence of the disese by up to pproximtely 2% (refs 22, 25). However, trnslting these findings into n incresed understnding of how ORMDL3 my contribute to the pthogenesis of sthm hs been hmpered by the lck of informtion on the function of ORM-fmily genes. Members of the ORM gene fmily encode trnsmembrne proteins loclized in the endoplsmic reticulum (ER) nd include two genes in the budding yest S. cerevisie (ORM/2) nd three genes in humns (ORMDL/2/3) 26. The Orm proteins hve no known functionl domins, nd little is known bout their cellulr role; however, they re strongly conserved nd hve been proposed to shre common function 26. Here we use combintion of globl nd trgeted studies to identify Orm proteins s homeosttic regultors of the first nd rte-limiting step in sphingolipid biosynthesis. Orm/2 negtively regulte sphingolipid synthesis We begn our investigtion of S. cerevisie ORM nd ORM2 by using globl pproch to chrcterize gene function bsed on lrge-scle mesurements of genetic interctions, termed episttic mini-rry profiles (E-MAPs) 27. In this strtegy, the function of given gene cn be inferred without priori hypotheses by compring the spectrum of genetic interctions resulting from muttion of tht gene with interction ptterns cused by muttions in other genes of defined function. When we compred the genetic interction profile resulting from deletion of ORM2 with those obtined for collection of more thn,4 yest mutnts in study focused on ER biology (mnuscript in preprtion nd ref. 27), we observed strong nticorreltion with the interction ptterns seen for hypomorphic lleles tht reduce expression of LCB nd LCB2 (lcb-damp nd 27 ), thus suggesting tht ORM2 nd LCB/2 hve opposing cellulr roles (Fig. nd Supplementry Informtion). We lso Deprtment of Cellulr nd Moleculr Phrmcology, 2 Howrd Hughes Medicl Institute, 3 Grdute Progrm in Chemistry nd Chemicl Biology, 4 The Cliforni Institute for Quntittive Biomedicl Reserch, University of Cliforni, Sn Frncisco, 7 4th Street, Sn Frncisco, Cliforni 9458, USA. 5 Institute of Moleculr Systems Biology, ETH Zurich, 893 Zurich, Switzerlnd. 6 Institute for Systems Biology, Settle, Wshington 983, USA. 7 Fculty of Science, University of Zurich, 857 Zurich, Switzerlnd. 8 Mx Plnck Institute of Moleculr Cell Biology nd Genetics, Pfotenhuerstrsse 8, 37 Dresden, Germny. 9 Deprtment of Biochemistry nd Moleculr Biology, University of Southern Denmrk, Cmpusvej 55, 523 Odense, Denmrk. {Present ddresses: Chemicl nd Systems Biology, Bio-X Progrm, Stnford University, Stnford, Cliforni 9435, USA (S.R.C.); Deprtment of Microbiology nd Immunology, Bxter Lbortory in Genetic Phrmcology, Stnford University, 269 Cmpus Drive, Stnford, Cliforni 9435, USA (B.B.). 48

2 NATURE Vol Februry 2 ARTICLES Number of genes 8 5 lcb-damp 5 ptdh3-orm ptef2-orm ptdh3-orm2 ptef2-orm2 5 5 orm2 lcb-damp 4 5 orm2 lcb-damp Log 2 (fold chnge vs ) Correltion with orm Correltion with ORM overexpression Reltive growth rte Correltion with ORM2 overexpression b 4 c d FA-CoA Serine * 3 ptdh3-orm Lcb, Lcb2, Tsc3 Orm/2. * * ptdh3-orm2 FA-CoA 2 ormδ/orm2δ Fen/Sur4 Tsc sur4δ elongse.75 LCBs LCB-Ps VLCFA-CoA Lc,Lg Long-chin ldehyde.5 Cermides phospho-ethnolmine 2 3 YEPD YEPD YEPD Aur + myriocin IPC 4 Sur, Csg2, Csh 5 orm /orm2 / LCBs Cermides IPC MIPC M(IP) 2 C MIPC orm /orm2 M(IP) 2 C Figure Orm/2 re negtive regultors of sphingolipid synthesis., Genetic interction (E-MAP) profiles were generted for strins contining ORM2 deletion, ORM overexpression nd ORM2 overexpression lleles (orm2d, ptdh3-orm nd ptef2-orm, nd ptdh3-orm2 nd ptef2-orm2, respectively). Correltions between the E-MAP profiles of these strins nd those of more thn,4 other yest mutnts re shown in histogrms (see Methods nd Supplementry Informtion). b, Lipids were extrcted from the indicted strins nd nlysed using globl mss spectrometry pproch 3. Amounts of the indicted metbolites re shown reltive to wild type (; verge 6 s.d., n 5 4; see Methods nd Supplementry Informtion). c, Logrithmic-phse growth rtes for the indicted strins were mesured in stndrd medi (YEPD) or medi supplemented with myriocin t 5 ng ml 2. Growth rtes (verge 6 s.d., n $ 3) re shown normlized to wild type; sterisks denote sttisticl significnce (P,.5). d, Schemtic of the sphingolipid biosynthesis pthwy in S. cerevisie, with selected metbolites nd genes indicted. FA-CoA, ftty cid co-enzyme A; LCB-Ps, long-chin bse phosphtes; IPC, inositolphosphorylcermide; MIPC, mnnosylinositolphosphorylcermide; MIP 2 C, mnnosyl-diinositolphosphorylcermide; VLCFA-CoA, very long-chin ftty cid co-enzyme A. exmined the genetic interctions tht result from overexpression of ORM or ORM2 by using the strong promoters ptdh3 nd ptef2, s it ws shown tht the sthm-ssocited polymorphisms ner ORMDL3 re ssocited with incresed expression of this gene 2,24. Overexpression of ORM or ORM2 produced pttern of genetic interctions tht ws highly correlted with those seen for the lcb- DAmP nd strins, indicting tht incresed Orm expression phenocopies reduced Lcb/2 ctivity (Fig. ). LCB nd LCB2 encode serine plmitoyltrnsferse, n essentil heterodimeric enzyme tht ctlyses the first nd rte-limiting step in sphingolipid biosynthesis 9,28. The synthesis of sphingolipids begins in the ER, where two clsses of lipid metbolite, long-chin bses (LCBs) nd very long-chin ftty cids, re formed (Fig. d). LCBs re produced by the condenstion of serine with ftty cids by serine plmitoyltrnsferse (Lcb/2 in yest nd SPTLC/2/3 in mmmls), followed by Tsc-medited reduction 8,9,29. LCBs cn then be phosphorylted or N-cylted with very long-chin ftty cids to form long-chin bse phosphtes or cermides, respectively, with the ltter undergoing further modifiction to generte complex sphingolipids (Fig. d). Thus, our E-MAP dt showing correltion between incresed ORM expression nd decresed LCB/2 function implicte the Orm proteins s negtive regultors of sphingolipid synthesis nd highlight the rection performed by Lcb/2 s the possible step in the pthwy t which Orm/2 my ct (Fig., d). We therefore exmined the effects of ltering Orm levels on cellulr lipid composition using mss-spectrometry-bsed globl lipidomic technique 3. In greement with the genetic interction dt, overexpression of ORM or ORM2 resulted in chnges to the lipidome, including reduced levels of LCBs nd cermides, which closely resembled those seen for the mutnt (Fig. b nd Supplementry Informtion). Conversely, cells deleted for ORM/2 hd highly incresed levels of LCBs nd cermides. In principle, this ccumultion of LCBs cused by deletion of ORM/2 could result from filure to consume these species by rection with very long-chin ftty cids to form cermides, s is seen when SUR4 is deleted (Fig. b nd ref. 3). However, in contrst to the sur4d strin, the incresed LCB levels in the ormd/orm2d strin re lso ccompnied by incresed mounts of the terminl sphingolipid mnnosyl-diinositolphosphorylcermide. Thus, ORM/2 deletion cuses incresed flux throughout the sphingolipid pthwy. This incresed flux is lso the primry cuse of the growth defect seen in ormd/orm2d cells, s rtificilly decresing Lcb/2 ctivity, either through the use of myriocin, drug tht specificlly inhibits Lcb/2 (ref. 3), or through the llele, significntly suppresses the growth defect cused by loss of ORM/2 (Fig. c). A complex with Orm/2, Lcb/2 nd Sc We next explored how Orm proteins modulte sphingolipid production by identifying proteins bound by Orm nd Orm2 in vivo. Isoltion of functionl 33Flg-tgged forms of Orm nd Orm2 expressed from their endogenous loci reveled previously unchrcterized, roughly stoichiometric complex (Fig. 2). Mss spectrometry showed tht this complex comprises the serine plmitoyltrnsferse proteins Lcb, Lcb2 nd Tsc3, in ddition to the phosphoinositide phosphtse Sc (ref. 32) (Supplementry Tble ). To chrcterize physicl interctions mong these proteins further, we used 33Flg-tgged form of Lcb tht is ble to rescue the invibility seen upon loss of LCB (lthough this llele does exhibit mild sensitivity to myriocin). Purifiction of 33Flg Lcb yielded the sme components seen in the Orm/2 pull-downs (Fig. 2b), indicting tht the Orm-ssocited proteins re likely to exist in single complex. Orm proteins nd potentilly their binding prtners my lso form higher-order oligomers, s we detected Orm2 coimmunoprecipittion with 33Flg Orm (nd vice vers) nd self-ssocition of differently tgged copies of the sme Orm protein (Fig. 2 nd see lter). The presence of Sc in ssocition with Lcb/2 suggests new connection between this phosphoinositide phosphtse nd sphingolipid biosynthesis. Deletion of SAC leds to incresed levels of 49

3 ARTICLES NATURE Vol Februry 2 LCBs (Supplementry Fig. nd ref. 33) nd resistnce to the Lcb/2 inhibitor myriocin (Supplementry Fig. b). This resistnce ws lso seen in cells expressing the sc 8 (ref. 34) ctlyticlly inctive mutnt of SAC. Additionlly, we found tht Sc nd Orm/2 bind independently to Lcb/2 (Fig. 2b) nd tht SAC nd ORM/2 deletions exhibit synthetic lethlity (Supplementry Fig. c). These findings together suggest tht Sc modultes serine plmitoyltrnsferse ctivity directly, but in mode distinct from Orm/2. Finlly, we showed tht the functionl complex between Orm proteins nd serine plmitoyltrnsferse is conserved in humn cells. Immunoprecipittions from HEK293T cells expressing 33Flgtgged ORMDL3 led to prominent SPTLC bnd detected by western blot (Fig. 3). Furthermore, we found tht simultneous knockdown by RNA interference of ORMDL/2/3 in HeL cells cuses n pproximtely threefold increse in cermide levels (Fig. 3b, Supplementry Fig. 2 nd Supplementry Informtion). Anti-Flg Anti-hSPTLC Empty vector Flg ORMDL3 97 Sc 64 Sc Flg Lcb Lcb Lcb Sc Lcb2 5 Lcb2 Lcb2 Flg Orm Orm2 Tsc3 Flg Orm No tg kd Dye front ORMDL3 Flg Input IP nti-flg Input Flg Orm2 IP nti-flg Flg Orm2 Orm Orm, Orm2 Tsc3* b Cermides (reltive to untreted) 3 2 Untreted Control sirna ORMDL3 sirna ORMDL/2/3 sirna Figure 3 ORM gene function is conserved in humn cells., HEK293T cells were trnsfected with either n empty vector or vector encoding ORMDL3 fused to the 33Flg epitope (N- or crboxy-terminl fusion). Immunoprecipittions with nti-flg grose were nlysed by western blot ginst humn serine plmitoyltrnsferse (nti-hsptlc) nd ginst the Flg epitope (nti-flg). b, HeL cells were trnsfected with short interfering RNAs (sirnas) directed ginst the indicted genes. After 72 h, cells were collected nd their lipids were nlysed by mss spectrometry. Cermide levels normlized to phosphtidylcholine re shown (moles of cermide per mole of phosphtidylcholine, reltive to untreted cells; verge 6 s.d., n 5 3). b Tsc3 Flg Lcb sc * orm / orm2 Figure 2 Orm proteins form complex with serine plmitoyltrnsferse., Colloidl-stined gels re shown for proteins eluted fter ffinity purifictions from strins expressing 33Flg Orm or 33Flg Orm2, or from n untgged control strin. Immunoprecipitted proteins were identified by mss spectrometry (Supplementry Tble ; sterisk indictes the prtly obscured protein Tsc3, whose presence ws confirmed by mss spectrometry). b, Affinity purifictions of 33Flg Lcb from wild-type, scd nd ormd/orm2d strins were performed s in. Asterisks indicte bnds tht re lost in deletion bckgrounds. 5 * Together, these results estblish tht Orm proteins re components of novel nd conserved protein complex, which we term the SPOTS complex (serine plmitoyltrnsferse, Orm/2, Tsc3, nd Sc). Additionlly, our dt indicte tht Orm/2 negtively regulte sphingolipid synthesis by cting directly on Lcb/2 (for exmple by ltering Lcb/2 ctlytic ctivity, ccessibility of substrtes to the enzyme or its subcellulr locliztion). Homeosttic regultion of Orm/2 ctivity Why would cells include negtive regultors of serine plmitoyltrnsferse, Orm/2, s core components of this essentil enzyme complex? This seemingly prdoxicl rrngement prompted us to investigte whether Orm-medited inhibition of LCB production might be regulted in response to chnges in the cellulr need for sphingolipid synthesis. We therefore exmined how ORM/2 deletion ffects sensitivity to the Lcb/2 inhibitor myriocin. Growth in the presence of myriocin is strongly dependent on the cellulr cpcity for LCB production, s the strin is highly sensitive to myriocin (Fig. 4); in ddition, SAC deletion, which leds to incresed LCB levels, confers myriocin resistnce (Fig. 4). Thus, nively, we might expect deletion of ORM/2 lso to confer pronounced resistnce to this drug. However, we observed tht wildtype nd ormd/orm2d strins exhibit comprble growth in the presence of myriocin (Fig. 4). Thus, loss of ORM/2 strongly ffects cell growth under stndrd conditions but hs little pprent effect when sphingolipid synthesis is disrupted, suggesting tht Orm/2 my be inctivted in response to myriocin tretment. To investigte this possibility further, we compred the effects of myriocin on the LCB levels of wild-type nd ormd/orm2d strins. As described erlier, the ORM/2 deletion strin exhibits highly incresed LCB levels in the bsence of myriocin. However, with incresing doses of myriocin, LCB levels in the ormd/orm2d strin decrese strongly nd pproximtely converge with those of the wildtype strin (Fig. 4b), supporting our growth dt indicting tht Orm/2 my be inctivted in response to myriocin tretment (Fig. 4). Interestingly, we lso found tht wild-type cells mintin roughly constnt LCB levels even t intermedite concentrtions of myriocin (up to bout 4 ng ml 2 ). This robustness is not due to filure of myriocin to ffect Lcb/2 t these concentrtions, s LCB levels in the ormd/orm2d strin re reduced strongly by the sme drug concentrtions (Fig. 4b; for further evidence see Fig. 4d). Rther, these results indicte tht progressive inctivtion of Orm/2 my provide cells with homeosttic mechnism to mintin nerly constnt sphingolipid output in the fce of incresing Lcb/2 inhibition. Consistent with this hypothesis, LCB levels in the wild-type strin begin to decline only t myriocin concentrtions where Orm-medited inhibition of LCB synthesis ppers to be fully relieved, s evidenced by convergence of wild-type nd ormd/ orm2d LCB levels (Fig. 4b). Orm/2 regultion by phosphoryltion We next investigted the mechnism by which Orm/2 ctivity might be decresed in response to myriocin tretment. Growth in myriocin did not lter Orm/2 expression levels (Fig. 4c nd Supplementry Fig. 7) or bolish their bility to interct with Lcb/2 nd Sc (Supplementry Fig. 3). However, we observed notble reduction in locliztion of green fluorescent protein (GFP) Orm2 to the corticl but not perinucler ER fter tretment with myriocin (Supplementry Fig. 4), rising the possibility tht chnges in subcellulr locliztion contribute to regultion of SPOTS complex ctivity. We lso found strong reduction in Orm protein co-immunoprecipittion in cells treted with myriocin (Fig. 4c), both for Orm Orm2 interction nd for self-ssocition of Orm nd of Orm2. A wek reduction in Orm Lcb binding ws lso observed in response to myriocin (Fig. 4c). These findings suggest model in which Orm Lcb/2 sub-complexes cn self-ssocite into higher-order oligomers in

4 NATURE Vol Februry 2 ARTICLES orm /orm2 -A orm /orm2 -B c Input sc No myriocin Myriocin (2 ng ml ) Flg Orm Flg Orm2 Flg Orm Flg Orm2 HA Orm2 HA Orm HA Orm HA Orm2 Myriocin: Anti-HA Anti-Flg Myriocin (4 ng ml ) d Flg Orm b Cellulr LCBs (reltive to ) P-Orm Orm. 5 Myriocin (ng ml ) Myriocin: orm /orm2 +CIP Flg IP Anti-HA Anti-Flg Anti-Lcb Figure 4 Orm/2 re regulted in response to disruption of sphingolipid synthesis., Seril dilutions of the indicted strins were spotted on pltes contining, 2 or 4 ng ml 2 myriocin nd imged fter growth for h. b, LCBs were extrcted nd nlysed from wild-type nd ORM/2 deletion strins grown in medi supplemented with the indicted concentrtions of myriocin. The combined mounts of C8 dihydrosphingosine nd C8 phytosphingosine re shown (reltive to wildtype LCB levels in the bsence of myriocin; verge of n $ 2 experiments). c, Ntive immunoprecipittions of 33Flg-tgged Orm nd Orm2 were performed from strins grown in stndrd medi or medi supplemented Flg Orm2 P-Orm2 Orm2 Lyste with 5 ng ml 2 myriocin, nd nlysed by western blot. The indicted strins lso expressed 33HA Orm/2 from their endogenous loci (diploid strins were used to exmine self-ssocition of Orm nd Orm2). d, Lystes were prepred from yest expressing 33Flg Orm/2 fter growth in medi contining the indicted concentrtions of myriocin. Western blots show phosphorylted forms of 33Flg Orm (P-Orm) nd 33Flg Orm2 (P- Orm2) fter seprtion on phosphte-ffinity gels 35.An immunoprecipitted (IP) smple treted with clf intestine phosphtse (CIP) shows the position of unphosphorylted Orm/2. IP mnner tht is inhibited in response to disruptions to sphingolipid synthesis. We lso detected in our Orm/2 co-immunoprecipittions slight shift in the electrophoretic mobility of Orm isolted from myriocintreted cells, indicting tht Orm proteins my be post-trnsltionlly modified (see Fig. 4c, Flg Orm nd hemgglutinin (HA) Orm inputs). Anlysis of 33Flg Orm nd 33Flg Orm2 in SDS polycrylmide gel electrophoresis (SDS PAGE) gels tht incorported phosphte-binding gent to improve the seprtion of phosphorylted species 35 resolved multiple phosphorylted forms for both Orm nd Orm2 tht collpsed to fster-migrting species upon phosphtse tretment (Fig. 4d). Importntly, growth in myriocin induced dose-dependent shift to more highly phosphorylted forms, nd this response occurred over the sme concentrtions of myriocin tht led to homeosttic inctivtion of Orm/2, s determined by mesurements of LCBs (compre Fig. 4b nd 4d). We lso observed incresed Orm/2 phosphoryltion fter shutting off expression of downstrem sphingolipid synthetic enzymes such s Tsc or Lg (in lcd bckground; Supplementry Fig. 5), further indicting tht phosphoryltion of Orm proteins is homeosttic response to disruption of sphingolipid production (see Fig. 5c). Finlly, we did not observe substntil chnges in Orm/2 phosphoryltion upon shutting off expression of AUR or CSG2 (Supplementry Fig. 5), thus providing preliminry evidence tht metbolite upstrem of these enzymes, such s cermide, is n intermedite whose levels re sensed to regulte Orm/2 ctivity. To test our phosphoryltion-medited feedbck model directly, we identified residues of Orm nd Orm2 tht re phosphorylted. Through combintion of trgeted mutgenesis nd mss spectrometry, we found severl sites of Orm nd Orm2 phosphoryltion in the mino-terminl regions of these proteins (Fig. 5 nd Supplementry Fig. 6). Importntly, muttion of these sites to lnine blocks formtion of the slower-migrting phosphorylted species seen in phosphte-ffinity gels (Fig. 5). This loss of phosphoryltion is unlikely to be due to these muttions resulting in non-functionl Orm proteins, s the phospho-mutnts re expressed t levels comprble to wild type nd rescue the slow growth of the ormd/orm2d strin (Supplementry Fig. 7 nd dt not shown). Furthermore, immunoprecipittions of phospho-mutnt Orm nd Orm2 reveled tht these proteins not only mintin the bility to interct with Lcb nd ech other, but tht blocking phosphoryltion prevents the chnge in higher-order ssembly of the SPOTS complex normlly seen in response to myriocin (Fig. 5b). Thus Orm/2 phosphoryltion ppers to be the event tht triggers dynmic regultion of SPOTS complex oligomeriztion. We next investigted the in vivo role of Orm/2 phosphoryltion. Consistent with bsl level of Orm/2 phosphoryltion tht is importnt for stedy-stte LCB production, we observed substntil (twofold) reduction in LCB levels in the ORM/2 phospho-mutnt strin under stndrd growth conditions (Fig. 5d). Furthermore, we found tht this strin is highly sensitive to tretment with myriocin (Fig. 5e). This result therefore confirms key prediction of our homeosttic model nd indictes tht lthough cells my possess dditionl mechnisms to regulte sphingolipid biosynthesis, such pthwys cnnot compenste for disruption of the criticl feedbck control provided by Orm/2. Thus, Orm proteins re dynmic negtive regultors of serine plmitoyltrnsferse, whose inhibitory ctivity is dependent on dequte levels of downstrem sphingolipids (Fig. 5c). Discussion Our studies identify Orm proteins s homeosttic regultors of sphingolipid metbolism nd demonstrte tht ltertion of these controls leds to misregultion of sphingolipid production (Fig. 5c). Specificlly, loss of the Orm/2-medited brke on serine plmitoyltrnsferse ctivity results in toxic ccumultion of sphingolipids, wheres muttions tht prevent this brke from being removed render cells unble to survive disruptions to sphingolipid synthesis. A phosphoryltion-bsed feedbck loop provides mechnistic bsis for homeosttic regultion of sphingolipid production nd exhibits severl fetures tht mke it well suited to control sphingolipid levels precisely. First, phosphoryltion of direct regultor of serine plmitoyltrnsferse my enble cells to respond rpidly to chnging environments. Second, Orm/2 exhibit prtil phosphoryltion under stndrd growth conditions, which my position the 5

5 ARTICLES NATURE Vol Februry 2 ORM-: orm-p*-: orm-p*-2: orm-p*-3: orm-p*-4: b Flg IP Input d orm-p*- orm-p*-2 orm-p* Flg Orm FPSAGSASSSIKTTEPVKDHRRRRSSSII FPAAGAASSSIKTTEPVKDHRRRRSSSII FPAAGAAAAAIKTTEPVKDHRRRRSSSII FPSAGSASSSIKTTEPVKDHRRRRAAAII FPAAGAAAAAIKTTEPVKDHRRRRAAAII Flg Orm HA Orm2 orm-p*-4 orm2-p*-2 Flg Orm2 HA Orm orm-p*-4 orm2-p*-2 Myriocin: Anti-HA Anti-Flg Anti-HA Anti-Flg Anti-Lcb LCBs (%) orm-p*-4/ orm2-p*-2 e orm-p*-4 ORM2 -: orm2-p*-: orm2-p*-2: orm /orm2 orm-p*-4/ orm2-p*-2 Myriocin Orm/2-bsed sphingolipid rheostt for mximl sensitivity to chnges in lipid metbolism. Furthermore, multi-site phosphoryltion of Orm nd Orm2 hs the potentil to generte severl distinct nd stble phospho-sttes, which my provide cells with grded mechnism to djust lipid production finely to mtch cellulr needs. Finlly, severl phosphoryltion sites my lso render Orm/2 responsive to multiple physiologicl or metbolic cues. Our identifiction of the SPOTS complex revels new mechnism by which cells my be ble to coordinte vriety of importnt secretory pthwy processes. Although link between sphingolipids nd phosphoinositides hs been pprecited for some time 29,33,36, we provide evidence tht the phosphoinositide phosphtse Sc directly modultes sphingolipid metbolism, s it is ssocited physiclly with serine plmitoyltrnsferse. These results re of prticulr interest given the role of phosphoinositides, including the Sc substrte phosphtidylinositol-4-phosphte, in the trfficking of both proteins nd sphingolipids from the ER nd Golgi pprtus,,32. Additionlly, chnges in Sc locliztion from the ER to the Golgi orm2-p* Flg Orm2 orm2-p* NESPAFEESPLTP...ISTPVTDHRRRRSSSVI NESPAFEESPLTP...ISTPVTDHRRRRAAAVI NEAPAFEEAPLAP...ISAPVTDHRRRRAAAVI FA-CoA VLCFA-CoA FA-CoA Serine Lcb, Lcb2, Tsc3 LCBs Cermides IPC MIPC M(IP) 2 C LCB-Ps Orm/2 Myriocin + Myriocin P-Orm/2 Figure 5 Muttion of phosphorylted Orm/2 residues impirs sphingolipid homeostsis., Lystes were prepred from strins expressing wild-type or phospho-site mutnt 33Flg Orm or 33Flg Orm2 fter growth in stndrd medi or medi supplemented with 5 ng ml 2 myriocin. Western blots re shown fter protein seprtion on phosphteffinity gels. Residues mutted to lnine in the indicted phospho-mutnts re highlighted in red below. b, Ntive immunoprecipittions of 33Flgtgged wild-type or phospho-mutnt Orm nd Orm2 were performed from strins expressing wild-type or phospho-mutnt 33HA Orm/2 fter growth in stndrd medi or medi supplemented with ng ml 2 myriocin. Western blots were nlysed s in Fig. 4c. c, Model for homeosttic regultion of Orm/2, in which Orm proteins ct s negtive regultors of serine plmitoyltrnsferse (Lcb, Lcb2 nd Tsc3), whose inhibitory ctivity is dependent on levels of downstrem sphingolipids. See Fig. d for bbrevitions used. d, LCBs were extrcted nd nlysed from wild-type nd 33Flg-tgged ORM/2 double phospho-mutnt strins grown in stndrd medi. Dt were nlysed s in Fig. 4b (verge 6 s.d., n 5 3). e, Seril dilutions of wild-type, ORM/2 deletion nd 33Flg-tgged ORM/2 double-phospho-mutnt strins were spotted on pltes with or 28 ng ml 2 myriocin nd imged fter growth for h. 52 c hve been shown in both yest nd mmmls to regulte protein flux through the secretory pthwy in response to nutrient levels 37.We therefore suggest tht the SPOTS complex my represent dynmic coordinting centre tht couples chnges in sphingolipid nd nutrient levels to the ctivity nd locliztion of key enzymes of lipid metbolism nd trfficking. This model is consistent with our finding tht Orm proteins self-ssocite in regulted fshion nd with evidence tht mmmlin serine plmitoyltrnsferse is prt of high-moleculrmss complex 38. Finlly, our chrcteriztion of ORM gene function provides frmework for investigting whether ltertions in ORMDL3 ctivity nd sphingolipid metbolism ply role in the pthogenesis of sthm. Severl studies support role for sphingolipids such s cermide nd sphingosine--phosphte in sthm-ssocited inflmmtory processes, including mst-cell degrnultion, irwy hyper-responsiveness nd immune-cell trfficking Our findings now link this growing body of evidence to the identifiction of single nucleotide polymorphisms ner ORMDL3 tht re ssocited with childhood sthm 2. Specificlly, our observtion tht ltered Orm levels strongly impct sphingolipid metbolism rises the testble hypothesis tht misregultion of sphingolipids my hve direct nd custive role in the development of sthm. A broder potentil role for sphingolipids in inflmmtory disese is lso suggested by reports tht single nucleotide polymorphisms ner ORMDL3 re ssocited with Crohn s disese 44, type I dibetes 45 nd primry biliry cirrhosis 46. Finlly, the existence of rich set of phrmcologicl tools trgeting the sphingolipid pthwy 3 rises the possibility of therpeutic intervention to counterct the potentil effects of chnges in ORMDL3 ctivity. METHODS SUMMARY Phosphoryltion nlysis by phosphte-ffinity SDS PAGE. Lystes for phosphoryltion nlysis were prepred by pelleting nd immeditely resuspending logrithmiclly growing yest in SDS lysis buffer (5 mm Tris-Cl, ph 6.8, 2% SDS, % glycerol) with protese inhibitors (Complete EDTA-free Protese Inhibitor Tblets; Roche) t 65 uc. SDS PAGE gels with phosphte ffinity regent 35 (synthesized ccording to stndrd synthetic procedures) were prepred with 7.5% crylmide resolving gel with 5 mm MnCl 2 nd 25 mm phosphte ffinity regent (375 mm Tris, ph 8.8,.% SDS). Gels were run t 7 V for 2.5 h, rinsed twice for 5 min in trnsfer buffer with mm EDTA nd rinsed twice more for 5 min in trnsfer buffer without EDTA before trnsfer to nitrocellulose membrnes. Western blots. Proteins were trnsferred from SDS PAGE gels to nitrocellulose membrnes nd probed for proteins of interest by using stndrd western blot procedures. Antibodies used were s follows: rbbit nti-flg (Sigm Aldrich), mouse nti-flg (M2; Sigm Aldrich), mouse nti-ha ntibodies 2CA5 (Roche) nd HA. (Covnce), mouse nti-ypgk (Moleculr Probes), rbbit nti-ylcb (rised ginst peptide ntigen SYIKKSHHDDPYRTTC; Abgent), mouse nti-hsptlc (Becton Dickinson) nd rbbit nti-ormdl/2/3 (rised ginst peptide ntigen KGTPFETPDQGKARLC; Abgent). Western blots were scnned using n Odyssey fluorescent scnner (Li-Cor Biosciences). Full Methods nd ny ssocited references re vilble in the online version of the pper t Received 23 August; ccepted 7 December 29.. Brown, M. S. & Goldstein, J. L. Cholesterol feedbck: from Schoenheimer s bottle to Scp s MELADL. J. Lipid Res. 5 (suppl.), S5 S27 (29). 2. Hmpton, R. Y. & Grz, R. M. Protein qulity control s strtegy for cellulr regultion: lessons from ubiquitin-medited regultion of the sterol pthwy. Chem. Rev. 9, (29). 3. Burg, J. S. et l. Insig regultes HMG-CoA reductse by controlling enzyme phosphoryltion in fission yest. Cell Metb. 8, (28). 4. Loewen, C. J. et l. Phospholipid metbolism regulted by trnscription fctor sensing phosphtidic cid. Science 34, (24). 5. Mrtin, C. E., Oh, C. S. & Jing, Y. Regultion of long chin unsturted ftty cid synthesis in yest. Biochim. Biophys. Act 77, (27). 6. Kurt, C. F. et l. Cdk/Cdc28-dependent ctivtion of the mjor tricylglycerol lipse Tgl4 in yest links lipolysis to cell-cycle progression. Mol. Cell 33, (29). 7. Fischer, J. et l. The gene encoding dipose triglyceride lipse (PNPLA2) is mutted in neutrl lipid storge disese with myopthy. Nture Genet. 39, 28 3 (27).

6 NATURE Vol Februry 2 ARTICLES 8. Dickson, R. C., Sumnseker, C. & Lester, R. L. Functions nd metbolism of sphingolipids in Scchromyces cerevisie. Prog. Lipid Res. 45, (26). 9. Cowrt, L. A. & Obeid, L. M. Yest sphingolipids: recent developments in understnding biosynthesis, regultion, nd function. Biochim. Biophys. Act 77, (27).. Hnd, K. et l. Moleculr mchinery for non-vesiculr trfficking of cermide. Nture 426, (23).. D Angelo, G. et l. Glycosphingolipid synthesis requires FAPP2 trnsfer of glucosylcermide. Nture 449, (27). 2. Kumgi, K., Kwno, M., Shinki-Ouchi, F., Nishijim, M. & Hnd, K. Interorgnelle trfficking of cermide is regulted by phosphoryltion-dependent coopertivity between the PH nd START domins of CERT. J. Biol. Chem. 282, (27). 3. Klemm, R. W. et l. Segregtion of sphingolipids nd sterols during formtion of secretory vesicles t the trns-golgi network. J. Cell Biol. 85, 6 62 (29). 4. Aronov, S. et l. Regultion of cermide biosynthesis by TOR complex 2. Cell Metb. 7, (28). 5. Vcru, A. M. et l. Sphingomyelin synthse-relted protein SMSr controls cermide homeostsis in the ER. J. Cell Biol. 85, 3 27 (29). 6. Hnnun, Y. A. & Obeid, L. M. Principles of bioctive lipid signlling: lessons from sphingolipids. Nture Rev. Mol. Cell Biol. 9, 39 5 (28). 7. Lebmn, D. A. & Spiegel, S. Cross-tlk t the crossrods of sphingosine-- phosphte, growth fctors, nd cytokine signling. J. Lipid Res. 49, (28). 8. Akinbmi, L. J., Moormn, J. E., Grbe, P. L. & Sondik, E. J. Sttus of childhood sthm in the United Sttes, Peditrics 23 (suppl. 3), S3 S45 (29). 9. Vercelli, D. Discovering susceptibility genes for sthm nd llergy. Nture Rev. Immunol. 8, (28). 2. Mofftt, M. F. et l. Genetic vrints regulting ORMDL3 expression contribute to the risk of childhood sthm. Nture 448, (27). 2. Glnter, J. et l. ORMDL3 gene is ssocited with sthm in three ethniclly diverse popultions. Am. J. Respir. Crit. Cre Med. 77, 94 2 (28). 22. Wu, H. et l. Genetic vrition in ORM-like 3 (ORMDL3) nd gsdermin-like (GSDML) nd childhood sthm. Allergy 64, (29). 23. Bouzigon, E. et l. Effect of 7q2 vrints nd smoking exposure in erly-onset sthm. N. Engl. J. Med. 359, (28). 24. Verln, D. J. et l. Allele-specific chromtin remodeling in the ZPBP2/GSDMB/ ORMDL3 locus ssocited with the risk of sthm nd utoimmune disese. Am. J. Hum. Genet. 85, (29). 25. Tvendle, R., Mcgregor, D. F., Mukhopdhyy, S. & Plmer, C. N. A polymorphism controlling ORMDL3 expression is ssocited with sthm tht is poorly controlled by current medictions. J. Allergy Clin. Immunol. 2, (28). 26. Hjelmqvist, L. et l. ORMDL proteins re conserved new fmily of endoplsmic reticulum membrne proteins. Genome Biol. 3, doi:.86/gb reserch27 (22). 27. Schuldiner, M. et l. Explortion of the function nd orgniztion of the yest erly secretory pthwy through n episttic minirry profile. Cell 23, (25). 28. Hnd, K. Serine plmitoyltrnsferse, key enzyme of sphingolipid metbolism. Biochim. Biophys. Act 632, 6 3 (23). 29. Beeler, T. et l. The Scchromyces cerevisie TSC/YBR265w gene encoding 3-ketosphingnine reductse is identified in screen for temperture-sensitive suppressors of the C 2 -sensitive csg2d mutnt. J. Biol. Chem. 273, (998). 3. Ejsing, C. S. et l. Globl nlysis of the yest lipidome by quntittive shotgun mss spectrometry. Proc. Ntl Acd. Sci. USA 6, (29). 3. Delgdo, A., Css, J., Llebri, A., Abd, J. L. & Fbris, G. Inhibitors of sphingolipid metbolism enzymes. Biochim. Biophys. Act 758, (26). 32. Strhl, T. & Thorner, J. Synthesis nd function of membrne phosphoinositides in budding yest, Scchromyces cerevisie. Biochim. Biophys. Act 77, (27). 33. Brice, S. E., Alford, C. W. & Cowrt, L. A. Modultion of sphingolipid metbolism by the phosphtidylinositol-4-phosphte phosphtse Scp through regultion of phosphtidylinositol in Scchromyces cerevisie. J. Biol. Chem. 284, (29). 34. Kerns, B. G. et l. Essentil role for dicylglycerol in protein trnsport from the yest Golgi complex. Nture 387, 5 (997). 35. Kinoshit, E., Kinoshit-Kikut, E., Tkiym, K. & Koike, T. Phosphte-binding tg, new tool to visulize phosphorylted proteins. Mol. Cell. Proteomics 5, (26). 36. Tbuchi, M., Audhy, A., Prsons, A. B., Boone, C. & Emr, S. D. The phosphtidylinositol 4,5-biphosphte nd TORC2 binding proteins Slm nd Slm2 function in sphingolipid regultion. Mol. Cell. Biol. 26, (26). 37. Blgoveshchensky, A. & Myinger, P. SAC lipid phosphtse nd growth control of the secretory pthwy. Mol. Biosyst. 5, (29). 38. Hornemnn, T., Wei, Y. & von Eckrdstein, A. Is the mmmlin serine plmitoyltrnsferse high-moleculr-mss complex? Biochem. J. 45, (27). 39. River, J., Proi, R. L. & Oliver, A. The llince of sphingosine--phosphte nd its receptors in immunity. Nture Rev. Immunol. 8, (28). 4. Ammit, A. J. et l. Sphingosine -phosphte modultes humn irwy smooth muscle cell functions tht promote inflmmtion nd irwy remodeling in sthm. FASEB J. 5, (2). 4. Msini, E. et l. Cermide: key signling molecule in guine pig model of llergic sthmtic response nd irwy inflmmtion. J. Phrmcol. Exp. Ther. 324, (28). 42. Ryn, J. J. & Spiegel, S. The role of sphingosine--phosphte nd its receptors in sthm. Drug News Perspect. 2, (28). 43. Idzko, M. et l. Locl ppliction of FTY72 to the lung brogtes experimentl sthm by ltering dendritic cell function. J. Clin. Invest. 6, (26). 44. Brrett, J. C. et l. Genome-wide ssocition defines more thn 3 distinct susceptibility loci for Crohn s disese. Nture Genet. 4, (28). 45. Brrett, J. C. et l. Genome-wide ssocition study nd met-nlysis find tht over 4 loci ffect risk of type dibetes. Nture Genet. 4, (29). 46. Hirschfield, G. M. et l. Primry biliry cirrhosis ssocited with HLA, IL2A, nd IL2RB2 vrints. N. Engl. J. Med. 36, (29). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We cknowledge A. Flick, S. Zhou nd D. King for identifiction of proteins by mss spectrometry; M. Schuldiner nd S. Wng for E-MAP dt collection; D. Fiedler nd K. Shokt for providing the phosphte-binding crylmide regent; E. Griffis nd M. D Ambrosio for ssistnce with fluorescence microscopy; N. Ingoli for codon-optimized mcherry-tgging plsmid; E. Burchrd nd J. Glnter for discussions of sthm genetics; I. Poser nd A. Hymn for dvice nd providing the HeL-Kyoto cell line; nd M. Bssik, G. Brr, V. Denic, A. Frost, N. Ingoli, E. Qun, B. Toym nd other members of the Weissmn lbortory for discussions. This work ws supported by funding from Deutsche Forschungsgemeinschft SFB/TR 3 projects A (K.S.) nd D (A.S.), EUFP6 PRISM (K.S.), ETH Zurich (R.A.), the Ntionl Hert, Lung, nd Blood Institute, Ntionl Institute of Helth (N-HV-2879) (R.A.), SystemsX.ch, the Swiss inititive for systems biology (R.A.), the Boehringer Ingelheim Fonds nd the Swiss Ntionl Science Foundtion (B.B.), the Howrd Hughes Medicl Institute (J.S.W.), the University of Cliforni, Sn Frncisco Strtegic Asthm Bsic Reserch Center (J.S.W.), the Ntionl Science Foundtion Grdute Reserch Fellowship Progrm (D.K.B.) nd the Fnnie nd John Hertz Foundtion (D.K.B.). Author Contributions D.K.B. designed, performed nd interpreted experiments. S.R.C. oversw E-MAP dt collection nd nlysis. B.B. performed nd nlysed protein mss spectrometry experiments to identify sites of phosphoryltion under the supervision of R.A. C.S.E. performed nd nlysed lipidomic mesurements with the support of A.S. nd K.S. J.S.W. designed nd interpreted experiments. D.K.B. nd J.S.W. prepred the mnuscript. Author Informtion Reprints nd permissions informtion is vilble t The uthors declre no competing finncil interests. Correspondence nd requests for mterils should be ddressed to J.S.W. (weissmn@cmp.ucsf.edu). 53

7 doi:.38/nture8787 METHODS Growth medi, plsmids nd strin construction. All yest experiments were conducted in strin BY474 (ATCC), relted strin suitble for E-MAP experiments 27, nd derivtives thereof. Strins were grown in YEPD medi or medi supplemented with myriocin (Sigm Aldrich; t lest 2 h growth in myriocin in ll cses), with the exception of smples prepred for lipidome nlysis, which were grown in synthetic defined medi. Deletion, DAmP, overexpression (ptef2 nd pthd3), mcherry fusion nd tetrcycline-repressible promoter strins were constructed by stndrd PCRbsed methods (see Supplementry Informtion). Strins expressing N- terminlly epitope-tgged or GFP-tgged forms of wild-type nd mutnt Orm, Orm2, Lcb nd Sc were mde by integrtion of DNA frgments encoding these gene vrints into strins in which these genes hd been deleted with the URA3 counter-selectble mrker. For the essentil gene LCB, diploid strin ws used for integrtion of the tgged llele contining the 33Flg epitope inserted between codons 9 nd, s previously reported 47. ORM nd ORM2 mutnts, s well s the previously described sc 8 muttion 34, were creted by stndrd PCR mutgenesis. HEK293T cells (ATCC) nd HeL-Kyoto cells ( gift from A. Hymn) were grown in DMEM high-glucose medi supplemented with penicillin, streptomycin, glutmine nd % het-inctivted fetl clf serum. ORMDL3 ws cloned from HEK293T cdna into expression plsmids contining N- or C-terminl 33Flg epitopes (p3xflag-cmv- nd p3xflag-cmv-4; Sigm Aldrich). E-MAP dt collection nd nlysis. E-MAP dt were collected nd nlysed s described previously 27 (mnuscript in preprtion). Genetic interction ptterns were compred for pirs of mutnts by first identifying those genetic bckgrounds for which E-MAP dt were vilble for both mutnts of interest. The similrity of the genetic interction scores for the two mutnts ws then clculted by computing the cosine correltion between their interction scores cross these shred mutnt bckgrounds. For given mutnt of interest such s orm2d, these pirwise comprisons were performed with ll other mutnt strins hving t lest 2 shred genetic interction dt points. For the ORM nd ORM2 overexpression mutnts, n verge of the ptdh3 nd ptef2 interction profiles ws used s the query profile for correltion clcultions. Lipidome nlysis by mss spectrometry. Lipidome nlyses were conducted s described previously 3 with minor modifictions bsed on n existing method 48 for LCB nlysis. Smples were collected from 2-ml cultures of yest cells growing logrithmiclly in synthetic defined medi or from bout to HeL cells grown s described erlier. Cells were wshed in wter or 55 mm mmonium bicrbonte t 4 uc, nd cell pellets were then frozen immeditely in liquid nitrogen. Yest dt re from two independent smples ech nlysed twice; HeL cell dt re from smples nlysed in triplicte. Growth rte mesurements. Logrithmic growth rtes were mesured by OD 6 from n $ 3 replicte smples. Sturted cultures of the relevnt strins were diluted into fresh medi nd grown for more thn 7 h before growth nlysis. Myriocin-treted cultures were grown for 3 h in the presence of the drug before nlysis of growth rte. Ntive ffinity purifictions of solubilized membrne proteins (lrge scle, yest). ER-derived microsomes were prepred from yest cells grown in YEPD (,4, OD 6 units collected t OD 6,.4). Cells were collected, wshed in wter t 4 uc, resuspended in lysis buffer (5 mm HEPES-KOH, ph 6.8, 5 mm potssium cette (KOAc), 2 mm MgOAc, mm CCl 2, 2 mm sorbitol) supplemented with protese inhibitors (Complete EDTA-free Cocktil; Roche) nd frozen in drop-by-drop fshion in liquid nitrogen. Frozen cells were then pulverized in bll mill (6 3 3 min t 3 Hz; Retsch), thwed in lysis buffer t 4 uc nd dounced ten times to homogeneity. After clrifiction by two consecutive centrifugtions t,g, microsomes were pelleted t 44,g, resuspended in.5 ml lysis buffer nd then diluted with 4 ml immunoprecipittion buffer (5 mm HEPES-KOH, ph 6.8, 5 mm KOAc, 2 mm MgOAc, mm CCl 2, 5% glycerol) with 2% digitonin (Clbiochem) supplemented with protese inhibitors. After nutting for.5 h t 4 uc, unsolubilized mteril ws removed by centrifugtion t 44,g, nd the remining superntnt ws dded to nti-flg resin (5 ml bed volume; Sigm Aldrich). Immunoprecipittions were nutted for 3 h t 4 uc nd then wshed 4 3 ml with immunoprecipittion buffer with.% digitonin. Bound proteins were eluted twice with 5 ml immunoprecipittion buffer with.25% digitonin nd 2 mg ml 2 33Flg peptide (Sigm Aldrich). Elutes were combined, seprted on SDS PAGE gels nd stined with colloidl blue stining kit (Invitrogen). Smples were prepred for phospho-site mpping using the sme protocol, except tht phosphtse inhibitors (Hlt Phosphtse Inhibitor Cocktil; Pierce) were included in the buffers used. Ntive ffinity purifictions of solubilized membrne proteins (smll scle, yest). Lystes for smll-scle immunoprecipittions were prepred from logrithmiclly growing yest cells (,25 OD 6 units) grown in YEPD. Cultures were collected, wshed in wter t 4 uc nd resuspended in 5 ml immunoprecipittion buffer (see bove) with.% digitonin nd supplemented with protese nd phosphtse inhibitors. Cell lystes were prepred by bed-beting t 4 uc, followed by ddition of 5 ml immunoprecipittion buffer with.9% digitonin to rise the finl digitonin concentrtion to %. Membrne proteins were solubilized by nutting lystes t 4 uc for 4 min. Unsolubilized mteril ws next removed by centrifugtion t,g, nd clrified lystes were incubted with nti-flg resin (25 ml bed volume) for 2.5 h. The nti-flg resin ws then wshed four times with ml immunoprecipittion buffer contining.% digitonin, nd bound proteins were eluted by ddition of 3 ml immunoprecipittion buffer with.25% digitonin nd 2 mg ml 2 33Flg peptide. Ntive ffinity purifictions of solubilized membrne proteins (smll scle, humn cells). HEK293T cells grown in DMEM high-glucose medi supplemented with penicillin, streptomycin, glutmine nd % het-inctivted fetl clf serum were trnsfected using Fugene6 trnsfection regent (Roche) ccording to the mnufcturer s instructions. After pproximtely 48 h growth, trnsfected cells were collected by trypsiniztion nd wshed with PBS. Cell pellets were then resuspended t 4 uc in ml immunoprecipittion buffer (see bove) with 2% digitonin nd protese inhibitors nd nutted for 45 min to solubilize membrne proteins. Insoluble mteril ws removed by centrifugtion t,g, nd the clrified superntnt ws incubted with nti-flg resin (25 ml bed volume) for 2.5 h. The reminder of the immunoprecipittion ws done in the sme fshion s the yest smll-scle ffinity purifictions (see erlier). Denturing immunoprecipittions nd phosphtse tretment. A 5-ml yest culture in logrithmic phse growth ws collected nd resuspended in ml 5% tricholorocetic cid (TCA) nd incubted for min t 4 uc. Cell pellets were wshed once in ml 5% TCA, wshed twice in ml cetone nd then ir-dried. Acid-wshed glss beds (2 ml) nd 5 ml SDS lysis buffer (5 mm Tris-Cl, ph 7.8, mm EDTA, % SDS, 2 M ure) supplemented with protese inhibitors were dded to the dried pellets, which were then lysed by bed-beting (3 3 6 s t 4 uc). Lystes were diluted by ddition of,35 ml Tween immunoprecipittion buffer ( mm Tris-Cl, ph 7.8, 2 mm NCl,.5% Tween-2, lso supplemented with protese inhibitors) nd clrified t 2,g. Superntnts were then dded to nti-flg grose (25 ml bed volume) nd immunoprecipitted for 2.5 h t 4 uc. The nti-flg resin ws then wshed 2 3 ml with Tween immunoprecipittion buffer, 3 ml with Tween/ure wsh buffer ( mm Tris-Cl, ph 7.8, mm NCl,.5% Tween-2, 2 M ure), nd 2 3 ml in phosphtse buffer (NEBuffer 3; New Englnd Biolbs), with.% Tween-2. The wshed resin ws then resuspended in 54 ml phosphtse buffer plus 6 ml clf intestine phosphtse (New Englnd Biolbs) nd incubted for h t 37 uc. After phosphtse tretment, the resin ws sequentilly wshed with ml Tween immunoprecipittion buffer nd ml SDS DOC wsh buffer (5 mm Tris-Cl, ph 7.8,.% SDS,.% N-deoxycholte). Bound proteins were then eluted by ddition of 6 ml SDS buffer ( mm Tris-Cl, ph 6.8, 4% SDS, 2% glycerol) nd incubtion for min t 65 uc. RNA interference in HeL cells. HeL cells grown in DMEM high-glucose medi supplemented with penicillin, streptomycin, glutmine nd % hetinctivted fetl clf serum were trnsfected using HiPerFect trnsfection regent (Qigen) ccording to the mnufcturer s instructions. After pproximtely 72 h growth, trnsfected cells were collected by trypsiniztion for lipidomic nd gene expression nlysis. For ech trgeted gene, pool of four sirnas ws used (ON- TARGETplus SMARTpool; Dhrmcon), with sirna sequences s follows: ORMDL: 59-UGGCAAGUUUCUAUACGAA-39, 59-GGAGUUGGCUUGCU UCAUA-39, 59-GCUUAUAAGUUAAAGGGCA-39, 59-GGACCAGCUUAACU UAUAA-39; ORMDL2: 59-UGGUAGGAUUGCUGCAUAU-39, 59-AGUUUAA UCAGGAUGGGUA-39, 59-GUGUACUGCUGCCGAAGUU-39, 59-AGUAUG AUGCUGCGCACUU-39; ORMDL3: 59-GAACAUGGACCACGCAGUU-39, 59-ACACUAAGUACGACCAGAU-39, 59-CGGUACGGCUUCUGGAUUG-39, 59-UGGGUAGGGAGCUGUCUAA-39. RT PCR nlysis of gene expression in HeL cells. RNA ws extrcted from HeL cells using the RNesy kit (Qigen) fter cell homogeniztion (QIAshredder; Qigen). Extrcted RNA ws reverse-trnscribed using AMV RT (Promeg) nd mplified with Tq polymerse (GoTq; Promeg). Rel-time SYBR green fluorescence ws monitored using DNA Engine Opticon System (MJ Reserch), nd the following primers were used: RPL9:59-ATGTATCACAGCCTGTACCTG-39, 59-TTCTTGGTCTCTTCCTCCTTG-39; ORMDL: 59-CTGACCAGGGTAAAGC AAGG-39, 59-TCCAAAGATCCGAACACCAT-39; ORMDL2: 59-CATACGGTGA AAGGGACACC-39, 59-CGGCAGCAGTACACTTAGCA-39; ORMDL3: 59-AACC CCTGCTCACTTGTTTG-39,59-CAAAGACCCATCCCACACTT-39. Live-cell yest fluorescence microscopy. Yest cells expressing Sec63 mcherry nd GFP Orm or GFP Orm2 were grown in rich medi with or without myriocin nd spotted on concnvlin-a-coted mm coverslip covered with mm coverslip. Cells were imged through the mm coverslip using Zeiss Axiovert 2M microscope (Crl Zeiss Microimging) equipped

8 doi:.38/nture8787 with spinning disk confocl scnhed nd lser system (Yokogw CS, Yokogw Electronics; Solmere). Imges were collected s Z-stcks with.5 mm spcing between frmes using mmnger softwre nd n ImgEM C9-3 EM-CCD cmer (Hmmtsu Photonics). Seril-dilution cell-spotting ssys. Logrithmiclly growing yest were spotted onto gr pltes in tenfold seril dilutions from strting OD 6 density of.3 to finl OD 6 density of Pltes were imged fter growth for h. Pltes with myriocin were stndrd YEPD supplemented with.% tergitol nd 2 mm citric cid. LCB extrction nd nlysis by high-performnce liquid chromtogrphy. LCB extrction nd nlysis ws performed s described 48. Myriocin-treted smples were grown for 4 6 h in the presence of drug before extrctions. For extrctions, 5 ml of logrithmiclly growing culture ws collected nd resuspended in ml 5% TCA t 4 uc. After min incubtion, cells were wshed once in 5% TCA nd three times in H 2 Ot4uC. Cell pellets were then resuspended in 75 mlh 2 O followed by ddition of 425 ml 7.6 mm TEA/ethnol. Lipids were extrcted by bth soniction ( s, 3 W) nd incubtion t 65 uc for 3 min. Of this extrct, 5 ml ws derivtized by rection for 4 min t room temperture with 3 ml of AccQ Fluor regent (Wters Corportion). Ester-linked lipids were de-cylted by ddition of 22.5 ml of.5 M KOH/ methnol. After 3 min incubtion t 37 uc, the rection ws neutrlized by ddition of 22.5 ml.74 M cetic cid/methnol. Smples were nlysed on n Agilent Chemsttion high-performnce liquid chromtogrphy using reversephse C8 column (ZORBAX Eclipse XDB-C8; Agilent). Protein identifiction by mss spectrometry. Gel slices were trypsinized nd mss spectr were cquired on Bruker Reflex III MALDI-TOF mss spectrometer. Proteins were identified by serching the NCBInr dtbse using the MS- Fit progrm on Protein Prospector (University of Cliforni, Sn Frncisco, Identifiction of phosphoryltion sites by mss spectrometry. Proteins eluted from ntive ffinity purifictions of 33Flg Orm or 33Flg Orm2 (see erlier) were precipitted using ice-cold cetone. After incubtion overnight t 22 uc, proteins were pelleted t 4 uc by centrifugtion. Protein pellets were then processed, nd the resulting peptide mixtures were nlysed on hybrid LTQ-Orbitrp mss spectrometer, s described previously 5. Dtbse serches were performed using the Scchromyces Genome Dtbse non-redundnt dtbse, s described Gble, K., Slife, H., Bcikov, D., Monghn, E. & Dunn, T. M. Tsc3p is n 8-mino cid protein ssocited with serine plmitoyltrnsferse nd required for optiml enzyme ctivity. J. Biol. Chem. 275, (2). 48. Lester, R. L. & Dickson, R. C. High-performnce liquid chromtogrphy nlysis of moleculr species of sphingolipid-relted long chin bses nd long chin bse phosphtes in Scchromyces cerevisie fter derivtiztion with 6-minoquinolyl- N-hydroxysuccinimidyl crbmte. Anl. Biochem. 298, (2). 49. Jiménez, C. R., Hung, L., Qiu, Y. & Burlingme, A. L. in Current Protocols in Protein Science (eds Colign, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W. & Wingfield, P. T.) (John Wiley, 998). 5. Huber, A. et l. Chrcteriztion of the rpmycin-sensitive phosphoproteome revels tht Sch9 is centrl coordintor of protein synthesis. Genes Dev. 23, (29).