Protein O-mannosyltransferases participate in ER protein quality control

144 Resea ch A icle In oduc ion A e ansloca ion ac oss o inse ion in o he endoplasmic e iculum (ER), memb ane p o eins ha e o old, a p ocess ha is assis ed by a a ie y o ER esiden chape ones ( o a e iew see Ellgaa d and Helenius, 2003). Folding is moni o ed by ER quali y con ol mechanisms, and co ec ly olded p o eins a e allowed o exi he ER by esicula budding (Ba lowe, 2003). Assis ance in p o ein olding and quali y con ol a e o en o e lapping unc ions o ER chape ones. A olding in e media e is ecognized by a chape one h ough such uni e sal signa u es as exposed hyd ophobic pa ches, unpai ed cys eines, and p oneness o agg ega ion (F a e al., 1993; Hellman e al., 1999; Zhang e al., 1997). I ini ial olding is slow o ails, hese ea u es will con inue o be exposed and will ensu e immedia e e-binding o a chape one. By his mechanism, slow- olding p o eins o p o eins ha mis old will be e ained inside he ER o ex ended pe iods o ime. Some ER e en ion mechanisms ha e addi ional complexi y, such as he calnexin–cal e iculin cycle ound in mammalian cells, whe e se e al chape ones and enzymes oge he moni o he olding o glycop o eins (Hammond e al., 1994; Pa odi, 2000). As well as aiding in p o ein olding and ER e en ion, some uni e sal chape ones such as calnexin and BiP possess a hi d unc ion in ha hey can a ge e minally mis olded p o eins o deg ada ion (B odsky e al., 1999; Denic e al., 2006; McC acken and B odsky, 1996; Plempe e al., 1997). Deg ada ion o abe an ER p o eins occu s mos ly by a p ocess called ER-associa ed p o ein deg ada ion (ERAD), whe eby p o eins a e ‘ e o ansloca ed’ in o he cy osol o be deg aded by he ubiqui in– p o easome sys em ( o a e iew see Meusse e al., 2005). Co e componen s o he esponsible machine ies o e o ansloca ion a e he ER memb ane-embedded E3 ubiqui in ligases. In yeas , he e a e wo such ubiqui in ligases, Doa10p and H d1p, which a e ound in dis inc memb ane p o ein complexes (Ca alho e al., 2006; Denic e al., 2006; Swanson e al., 2001). Toge he hey p omo e e o ansloca ion and deg ada ion o mos , i no all, mis olded subs a es in he ER. Whe eas he Doa10p complex a ge s memb ane p o eins wi h lesions in hei cy osolic po ions o he p o easome, a ou e e med he ERAD-C pa hway, he H d1p complex e o ansloca es memb ane p o eins wi h mis olded ansmemb ane domains (ERAD-M pa hway) o memb ane and soluble p o eins wi h de ec s in hei luminal po ion (ERAD-L pa hway) (Ca alho e al., 2006; Vashis e al., 2001). All pa hways con e ge a he cy osolic Cdc48p ATPase complex, which p obably p o ides he ene gy o e o ansloca ion and o subs a e ans e o he p o easome (Ca alho e al., 2006; Rabino ich e al., 2002; Ye e al., 2001). Whe eas he as majo i y o mis olded p o eins o he sec e o y pa hway a e ecognized ea ly while s ill inside he ER and will ul ima ely be a ge ed o ERAD, he e a e cases whe e a ac ion o hese species exi s he ER no mally. This occu s i de ec i e p o eins a e exp essed in la ge quan i ies o i expo signals a e dominan enough ha hey compe e wi h e en ion mechanisms (Caldwell e al., 2001; Haynes e al., 2002; Kincaid and Coope , 2007; Vashis e al., 2001). In ei he case, i becomes mo e e iden ha he cell possesses addi ional, albei less well-cha ac e ized, quali y con ol mechanisms in pos -ER compa men s ha can a ge abe an p o eins o deg ada ion (He ema e al., 2004; Hong e al., 1996; Reggio i and Pelham, 2002; Wang and Ng, 2010). One in e es ing bu poo ly unde s ood p o ein modi ica ion ha occu s inside he ER is ha o p o ein O-mannosyla ion, which is one o a a ie y o possible O-glycosyla ion e en s ha occu h oughou he sec e o y pa hway ( o a e iew see Spi o, 2002). O-mannosyla ion is media ed by membe s o he p o ein O- mannosyl ans e ase (PMT) amily (Lussie e al., 1995; S ahl- Bolsinge e al., 1993). PMTs a e mul ispanning memb ane p o eins Accep ed 9 No embe 2010 Jou nal o Cell Science 124, 144-153 © 2011. Published by The Company o Biologis s L d doi:10.1242/jcs.072181 Summa y In euka yo ic cells, p o eins en e he sec e o y pa hway a he endoplasmic e iculum (ER) as linea polypep ides and old a e ansloca ion ac oss o inse ion in o he memb ane. I co ec olding ails, many p o eins a e O-mannosyla ed inside he ER by an O-mannosyl ans e ase, he Pm 1p–Pm 2p complex. The consequences o his modi ica ion a e con o e sial and he cellula ole o he Pm 1p–Pm 2p complex in his espec is unclea . He e, we ha e iden i ied he binding pa ne s o yeas Pm 1p and Pm 2p. These include ER chape ones in ol ed in oxida i e p o ein olding; he H d1p complex, which is in ol ed in ER-associa ed p o ein deg ada ion (ERAD); and he p24 p o ein complex in ol ed in ER expo . The esul s sugges ha he Pm 1p–Pm 2p complex pa icipa es in hese p ocesses. We es ed his assump ion in a unc ional assay and ound ha whe eas he Pm 1p–Pm 2p complex p omo es as ER expo o he GPI-ancho ed p o ein Gas1p, i e ains he mis olded e sion Gas1*p and a ge s i o he H d1p complex o subsequen deg ada ion. Ou esul s e eal p e iously unknown cellula oles o he Pm 1p–Pm 2p complex in connec ion wi h he ERAD machine y and show i s pa icipa ion in ER p o ein quali y con ol. Key wo ds: Endoplasmic e iculum, ER p o ein quali y con ol, ER-associa ed p o ein deg ada ion, Pm complex P o ein O -mannosyl ans e ases pa icipa e in ER p o ein quali y con ol Vei Gode * and Alejand o Mele o Depa men o Gene ics, Uni e si y o Se ille, A e Reina Me cedes 6, 41012 Se ille, Spain *Au ho o co espondence ([email p o ec ed]) Jou nal o Cell Science wi h se en ansmemb ane domains and wo la ge luminal loops, which oge he a e needed o he enzyma ic ac i i y (Gi bach e al., 2000; S ahl-Bolsinge and Scheinos , 1999). They a e conse ed om yeas o humans al hough hey appea o be missing in plan s (Wille e al., 2003). Single mannose esidues a e a ached o side chains o one o se e al se ine o h eonine esidues. I was epo ed ha many p o eins will be O-mannosyla ed inside he ER only in cases whe e hey mis old (Ha y e al., 2001; Vashis e al., 2001). The a e o mis olded p o eins ha ha e been O- mannosyla ed is con o e sial. Whe eas some o hem seem inc easingly p o ec ed om deg ada ion, o he s a e epo ed o be deg aded by he cy osolic p o easome o which hey a e a ge ed by an unknown mechanism (Ha y e al., 2001; Hi ayama e al., 2008). In e es ingly, like mos ER chape ones o membe s o he ERAD machine ies, PMTs a e up egula ed du ing ER s ess by he un olded p o ein esponse (UPR) (T a e s e al., 2000). This s udy aimed a iden i ying he cellula ole o he PMTs wi h espec o mis olded p o eins. We iden i ied he cellula binding pa ne s o yeas Pm 1p and Pm 2p, which a e known o o m an ac i e s ochiome ic complex (Pm 1p–Pm 2p) (Gi bach and S ahl, 2003). Binding pa ne s include ER chape ones in ol ed in oxida i e p o ein olding; he H d1p complex, which is in ol ed in ERAD; and he p24 p o ein complex, which is in ol ed in p o ein ER expo (Muniz e al., 2000; Schimmolle e al., 1995). These indings sugges ha he Pm 1p–Pm 2p complex migh pa icipa e in all hese cellula p ocesses. In a subsequen unc ional assay, we es ed his hypo hesis and showed ha he Pm 1p–Pm 2p complex is equi ed o as ER expo o he GPI-ancho ed p o ein Gas1p, whe eas i e ains he mis olded e sion Gas1*p and a ge s i o he H d1p complex o subsequen deg ada ion. Ou esul s pu he Pm 1p–Pm 2p complex in a ca ego y wi h ER chape ones ha unc ion in ER p o ein quali y con ol. We p o ide a model o how he machine ies o p o ein O-mannosyla ion, ER expo and ERAD a e connec ed on a molecula le el. Finally, ou esul s uni y p e iously con adic ing da a o he ole o he Pm 1p– Pm 2p complex in ERAD. Resul s In e ac ion pa ne s o Pm 1p and Pm 2p To e eal he cellula unc ion o he Pm 1p–Pm 2p complex, we i s asked which componen s i is associa ed wi h. We sepa a ely agged Pm 1p and Pm 2p ch omosomally wi h a usion ag ha con ained a P o ein A moie y and a calmodulin binding pep ide (CA ag) and exp essed he p o eins om hei endogenous p omo e s. These agged p o eins we e ully unc ional (supplemen a y ma e ial Fig. S1). Yeas cells we e g own in olumes o 3 l. A e cell lysis, memb ane ac ions we e isola ed and solubilized wi h 1% digi onin. Tagged p o eins we e a ini y pu i ied oge he wi h hei binding pa ne s using IgG-coupled magne ic beads. The elua e was subjec ed o SDS-PAGE ollowed by Coomassie Blue s aining (Fig. 1A). The isualized bands we e cu ou and hei iden i y de e mined by andem mass spec ome y (Table 1 and supplemen a y ma e ial Table S1). Al e na i ely, we p ecipi a ed he en i e elua e wi h ichlo oace ic acid (TCA) and iden i ied he pool o bound p o eins by andem mass spec ome y (Table 1 and supplemen a y ma e ial Table S1). A wild- ype s ain wi hou agged p o eins was used as a con ol in all expe imen s. As shown in Fig. 1A, Pm 1p and Pm 2p we e isola ed oge he bu no abundan addi ional binding pa ne s we e isible (Fig. 1A, lanes 2 and 3, bands 1 and 2; Table 1). Howe e , a ain smea was seen con aining p o eins o he p24 p o ein complex (Fig. 1A, 145 Pm 1p–Pm 2p complex in ER quali y con ol lanes 2 and 3, band 5; Table 1). In Fig. 1A, bands 3 and 4 con ained esidual IgG hea y and ligh chain om incomple e coupling o magne ic beads. Using TCA p ecipi a ion, we iden i ied addi ional and less abundan binding pa ne s o he Pm 1p–Pm 2p complex. Along wi h p o eins o he p24 p o ein complex, we ound Cdc48p, H d1p, Usa1p and Yos9p (componen s o he ERAD-media ing H d1p complex), E o1p and Pdi1p (p o eins in ol ed in oxida i e p o ein olding), Ub 1p and Cue4p (p o eins in ol ed in p o ein ubiqui yla ion), D m1p (a dis inc co- ac o o he Cdc48p ATPase) and Ted1p (a p o ein linked o GPI-ancho emodeling) (see Table 1). In summa y, he Pm 1p–Pm 2p complex associa es weakly and hus p obably ansien ly, wi h ER componen s ha ha e es ablished oles in p o ein olding and ER expo as well as in ERAD. We Fig. 1. The Pm 1p–Pm 2p complex associa es wi h ER machine ies in ol ed in ER p o ein expo and in ERAD. (A)  In e ac ion pa ne s o Pm 1p and Pm 2p. Wild- ype yeas cells (con) o cells exp essing ei he Pm 1- CA o Pm 2-CA we e lysed and memb ane ac ions solubilized wi h 1% digi onin. The ex ac was incuba ed wi h IgG-coupled magne ic beads and bound ma e ial analyzed by SDS-PAGE and Coomassie Blue s aining. Visualized bands (lanes 2 and 3) and egions om he con ol eac ion (lane 1) we e numbe ed, cu ou and hei p o ein con en de e mined by mass spec ome y (Table 1; supplemen a y ma e ial Table S1). (B)  Yeas cells exp essing Emp24-3H wi h o wi hou exp essing Pm 2-CA we e lysed. Samples we e analyzed di ec ly (inpu ; 5% o o al ma e ial) o a e immunop ecipi a ion using IgG-coupled magne ic beads (IP:IgG; 95% o o al ma e ial). All samples we e sepa a ed by SDS-PAGE (4-20% g adien gel) and analyzed by immunoblo ing (IB) wi h he indica ed an ibodies. The a owhead indica es he co-immunop ecipi a ed ac ion. The s a indica es Pm 2-CA ha was also ecognized by he seconda y an ibody. (C)  As o B, bu using cells exp essing Pm 1-3HA wi h o wi hou exp essing H d1-CA. The SDS-PAGE was pe o med using a 7% s anda d gel. The a owhead indica es he co-immunop ecipi a ed ac ion. The s a indica es H d1-CA ha was also ecognized by he seconda y an ibody. Jou nal o Cell Science hus hypo hesized ha he Pm 1p–Pm 2p complex i sel ac i ely pa icipa es in hese cellula p ocesses. Be o e es ing ou hypo hesis expe imen ally we wan ed o con i m and isualize he in e ac ions o he Pm 1p–Pm 2p complex wi h he p edominan binding pa ne s ha we iden i ied: he H d1p complex and he p24 p o ein complex. To his end, we cons uc ed s ains in which wo p o eins we e di e en ially agged. As can be seen in Fig. 1B, a ac ion o HA- agged Emp24p (Emp24-3HA, a membe o he p24 p o ein complex) co-p ecipi a es wi h CA- agged Pm 2p (Pm 2-CA). A con ol s ain wi hou Pm 2-CA did no b ing down agged Emp24p (Fig. 1B, compa e lanes 5 and 6). As can be seen in Fig. 1C, we could co-isola e a ac ion o HA- agged Pm 1p (Pm 1- 3HA) wi h CA- agged H d1p (H d1-CA, he E3-ligase o he H d1p complex). Only ain backg ound s aining was isible i H d1p was un agged (Fig. 1C, compa e lanes 5 and 6). To assess whe he deg ada ion o he agged Pm 1p–Pm 2p complex by he H d1p complex leads o hei obse ed associa ion, we pe o med a cycloheximide (CHX)-chase analysis o CA- agged Pm 2p. Su p isingly, Pm 2-CA was ela i ely uns able and was deg aded wi h an app oxima e hal li e o 1.5 hou s (supplemen a y ma e ial Fig. S2). Howe e , he u no e was independen o H d1p and hus he physical in e ac ion was no due o deg ada ion by he H d1p complex (supplemen a y ma e ial Fig. S2). Gas1*p is a subs a e o he Pm 1p–Pm 2p complex and is la gely deg aded by he ERAD-L pa hway To add ess ou hypo hesis ha he Pm 1p–Pm 2p complex pa icipa es in p o ein olding, ER expo and ERAD, we nex looked o a sui able model subs a e. Ideally, such a subs a e should no mally be expo ed om he ER in a p24 p o ein complex- dependen manne , whe eas a mu an should be deg aded ia H d1p complex-media ed ERAD. The GPI-ancho ed p o ein Gas1p comes closes o hese c i e ia. In i s wild- ype o m, he p o ein lea es 146 Jou nal o Cell Science 124 (1) he ER depending on he p24 p o ein complex and is a ge ed o he plasma memb ane (Muniz e al., 2001; Muniz e al., 2000; Schimmolle e al., 1995). Impo an ly, a mu an e sion o Gas1p, Gas1*p, is uns able and deg aded by an uniden i ied p o easome- dependen pa hway (Fuji a e al., 2006). In e es ingly, whe eas Gas1p is O-glycosyla ed by Pm 4p and Pm 6p alone, Gas1*p is u he O-glycosyla ed by Pm 1p and Pm 2p (Hi ayama e al., 2008). Thus, he wild- ype p o ein Gas1p and i s mu an e sion Gas1*p appea well sui ed o ou analysis. We i s con i med ha Gas1*p is O-mannosyla ed by he Pm 1p–Pm 2p complex as epo ed p e iously (Hi ayama e al., 2008). To his end, we exp essed ch omosomally HA- agged species o wild- ype Gas1p [Gas1p(HA)] o mu an Gas1*p [Gas1*p(HA)] om hei endogenous p omo e s in wild- ype o in Pm 1–Pm 2 dele ion mu an (pm 1pm 2) cells. We gene ally obse ed a lowe p o ein exp ession le el in pm 1pm 2 cells han in wild- ype cells, which was also e lec ed in a educed g ow h a e (da a no shown). As can be seen in Fig. 2A, Gas1*p showed an inc ease in elec opho e ic mobili y in pm 1pm 2 cells as compa ed wi h wild- ype cells, consis en wi h i being O-mannosyla ed (Fig. 2A, compa e lanes 3 and 4, a ows). We occasionally obse ed a smea abo e he majo p o ein bands, bu because he phenomenon was a e we conside ed i unspeci ic. In con as o Gas1*p, bo h he p ecu so and he ma u e o m o Gas1p mig a ed wi h simila elec opho e ic mobili y in pm 1pm 2 and in wild- ype cells (Fig. 2A, compa e lanes 1 and 2). To exclude he possibili y ha he inc ease in elec opho e ic mobili y o Gas1*p in pm 1pm 2 cells esul s om de ec i e N-glycosyla ion a he han om lack o O-mannosyla ion, we pe o med an addi ional se o expe imen s using he de-N-glycanase PNGase F o emo e N-linked glycans p io o SDS-PAGE (supplemen a y ma e ial Fig. S3) and co- immunop ecipi a ion wi h he mannose-speci ic lec in Concana aline A (supplemen a y ma e ial Fig. S4B). Finally, he Table 1. Summa y o in e ac ing p o eins ha we e immunop ecipi a ed wi h CA- agged Pm 1p (Pm 1-CA) o CA- agged Tagged p o ein (bai ) Pm 1-CA Pm 2-CA In e ac ing p o ein Func ional ca ego y Indi idual To al Indi idual To al Pm 1p 33[1] 36/32 35[2] 39/34 Pm 2p O-mannosyla ion 28[2] 25/22 20[1] 22/21 Cdc48p ERAD – 1/2 – 18/3 E o1p P o ein olding – 2/2 – 3/2 Pdi1p P o ein olding – 2/1 – –/1 H d1p ERAD – 1/– – 2/– Usa1p ERAD – 2/– – 2/2 Yos9p ERAD – –/– – 1/– Ub 1p Ubiqui in ligase – –/5 – –/– D m1p Cdc48p co ac o – –/– – 5/2 Cue4p Ubiqui in binding – –/2 – 1/3 Ted1p GPI emodeling – –/3 – –/2 IgG hc [3] [3] IgG lc [4] [4] Emp24p – 2/2 – 2/2 E 25p 1[5] 6/4 – 5/4 E p1p 1[5] 5/5 2[5] 4/3 E p2p ER expo (p24 complex) – 2/2 4[5] 1/1 Bound p o eins we e iden i ied using mass spec ome y. We analyzed ei he indi idual p o ein bands a e hei sepa a ion using SDS-PAGE and s aining wi h Coomassie Blue (indi idual) o he o al p o ein elua e a e p ecipi a ion wi h ichlo oace ic acid ( o al). Numbe s indica e he numbe o pep ides iden i ied by mass spec ome y. Fo mul iple expe imen s, numbe s a e sepa a ed by a solidus. The numbe s in squa e b acke s co espond o he bands in Fig. 1A. See supplemen a y ma e ial Table S1 o he comple e se o mass spec ome y da a. Al hough Yos9p was only iden i ied wi h one indi idual pep ide and would no pass ou c i e ia as a ue hi , we show i because i is a well-known H d1p complex componen . IgG hc, immunoglobulin hea y chain; IgG lc, immunoglobulin ligh chain. Pm 2p (Pm 2-CA) Jou nal o Cell Science a o emen ioned expe imen o es o he unc ionali y o agged Pm 2p showed a Pm 2p-dependen change in elec opho e ic mobili y o Gas1*p o e ime (supplemen a y ma e ial Fig. S1). Toge he , hese da a con i m ha Gas1*p bu no Gas1p is modi ied in a Pm 1p–Pm 2p complex-dependen manne , s ongly sugges ing ha Gas1*p in e ac s di ec ly wi h he Pm 1p–Pm 2p complex and is O-mannosyla ed. Nex , we asked whe he Gas1*p is deg aded ia he H d1p complex-dependen ERAD-L pa hway as p edic ed on he basis o he ac ha he mis olded GPI-ancho ed p o ein is exposed en i ely o he lumen o he ER. To his end, we exp essed Gas1*p in wild- ype and dele ion mu an s o he H d1p complex and pe o med a CHX-chase expe imen . As can be seen in Fig. 2B, lanes 1–4 and g aph, when exp essed in wild- ype cells Gas1*p is deg aded wi h a hal -li e o oughly 1.5 hou s, consis en wi h p e ious da a (Fuji a e al., 2006). When we es ed dele ion mu an s o all memb ane-bound H d1p complex componen s (H d1p, H d3p, De 1p,and Usa1p), we ound ha hey s abilized Gas1*p whe eas 147 Pm 1p–Pm 2p complex in ER quali y con ol dele ion o Doa10p ( he cen al componen o he ERAD-C pa hway) did no (Fig. 2B,C). Dele ions o D m1p, Ub 1p o Cue4p (o he binding pa ne s o he Pm 1p–Pm 2p complex; Table 1) did no in luence Gas1*p u no e (Fig. 2C and da a no shown). These esul s show ha Gas1*p is indeed a ge ed o deg ada ion by he ERAD-L pa hway. Ou esul s a e di e en om hose published p e iously epo ing ha dele ing H d1p had no e ec on Gas1*p u no e (Fuji a e al., 2006). I has been epo ed ha he p24 p o ein complex has a ole in deg ada ion o Gas1*p (Fuji a e al., 2006). Because he p24 p o ein complex has an es ablished unc ion in ER expo o wild- ype Gas1p, he simples scena io would be consis en wi h a p24 p o ein complex-dependen ER exi o a ac ion o Gas1*p and i s a ge ing o deg ada ion om a pos -ER compa men . Howe e , ou ea lie esul s showed ha bo h he H d1p complex and he p24 p o ein complex a e p esen as associa es o he Pm 1p–Pm 2p complex. I could hus be ha he p24 p o ein complex in e ac s di ec ly wi h he H d1p complex and has a ole in a ge ing o Gas1*p o he Fig. 2. The mis olded model p o ein Gas1*p is a subs a e o he Pm 1p–Pm 2p complex and is in pa deg aded by he ERAD-L pa hway. (A)  Gas1p(HA) o Gas1*p(HA) we e indi idually exp essed in wild- ype o in pm 1pm 2 cells. Equal amoun s o cells we e lysed and he p o eins analyzed by SDA-PAGE (5% s anda d gel) ollowed by an i-HA immunoblo ing. A ows indica e he O-mannosyla ed ac ion o Gas1*p: p, p ecu so o m; m ma u e o m. (B,C)  The deg ada ion o Gas1*p(HA) was measu ed in wild- ype cells o in cells ca ying he designa ed dele ions a e inhibi ion o p o ein syn hesis by addi ion o 200  g/ml cycloheximide. A he indica ed ime poin s, equal aliquo s o cells we e emo ed, lysed and he amoun o emaining Gas1*p(HA) was analyzed by SDS-PAGE (4–20% g adien gel) ollowed by an i-HA immunoblo ing. The bands we e quan i ied by densi ome y and he esul s o a leas h ee independen se s o expe imen s we e plo ed ( igh panels). Jou nal o Cell Science ERAD machine y. To check his idea, we es ed whe he we could co-immunop ecipi a e membe s o he ERAD machine y di ec ly wi h Emp24p. As can be seen in Fig. 3A, we could no co- p ecipi a e Usa1p (a membe o he H d1p complex) wi h Emp24- CA, whe eas he con ol eac ion using CA- agged De 1p (De 1-CA), ano he membe o he H d1p complex, eadily b ough down Usa1p (Fig. 3A, compa e lanes 4–6). Fu he mo e, we did no ind H d1p complex componen s as binding pa ne s o Emp24- CA when pe o ming a la ge-scale pull-down expe imen ollowed by mass spec ome ic analysis, as desc ibed in Fig. 1 (V.G. and A.M., unpublished da a). We hen di ec ly measu ed he deg ada ion o Gas1*p in p24 p o ein complex dele ion mu an s. Dele ions o ei he Emp24p o o E 25p led o s abiliza ion o Gas1*p (Fig. 3B, lanes 1–12), consis en wi h p e ious da a (Fuji a e al., 2006). This ac ion was no sec e ed o a ge ed o he acuole (V.G. and A.M., unpublished da a). When we addi ionally disabled he ERAD-L pa hway using h d1emp24 dele ion mu an cells we ound maximal s abiliza ion o Gas1*p (Fig. 3B, lanes 13–16 and g aph). Toge he , hese da a sugges ha Gas1*p can be deg aded by wo sepa a e pa hways: one is he ERAD-L pa hway, which depends on he H d1p complex, and he o he depends on he p24 p o ein complex and is consis en wi h leading o p o ein deg ada ion om a pos -ER compa men . The Pm 1p–Pm 2p complex e ains Gas1*p and ul ima ely a ge s i o he H d1p complex o deg ada ion Nex , we di ec ly add essed he ole o he Pm 1p–Pm 2p complex in deg ada ion o Gas1*p. The physical in e ac ion o he Pm 1p– Pm 2p complex wi h he H d1p complex sugges s ha he Pm 1p– 148 Jou nal o Cell Science 124 (1) Pm 2p complex migh a ge Gas1*p o he H d1p complex. In ha case, absence o he Pm 1p–Pm 2p complex would lead o a s abiliza ion o Gas1*p. I , al e na i ely, he Pm 1p–Pm 2p complex e ains Gas1*p and p e en s i s e o ansloca ion by he H d1p complex, i s absence should esul in as e deg ada ion o Gas1*p. To dis inguish be ween hese possibili ies, we pe o med a CHX- chase expe imen wi h dele ion mu an s o ei he Pm 1p o Pm 2p o bo h. As can be seen in Fig. 4, dele ion o he Pm 1p–Pm 2p complex d as ically inc eases he u no e a e o Gas1*p, bes isible o he pm 1pm 2 cells, in which he hal li e was educed o app oxima ely 45 minu es. This indica es ha he Pm 1p–Pm 2p complex has an inhibi o y unc ion o he deg ada ion o Gas1*p, consis en wi h an ER e en ion unc ion o he mis olded p o ein. As men ioned ea lie , some classical ER chape ones such as BiP and calnexin a e known o ha e mul iple unc ions and play a ole in p o ein olding, p o ein ER e en ion and in a ge ing o p o ein deg ada ion. A simila unc ion o he Pm 1p–Pm 2p complex o a ge subs a es o he ERAD machine y o o he p24 p o ein complex could be masked by he e en ion e ec ha we obse ed. We hus cons uc ed iple mu an s in which we dele ed H d1p o Emp24p in a pm 1pm 2 backg ound and measu ed he deg ee o s abiliza ion o Gas1*p. This allowed us o compa e he amoun o Gas1*p deg aded by ei he pa hway in he p esence and absence o he Pm 1p–Pm 2p complex. As can be seen in Fig. 4B, bo h iple mu an s s abilized Gas1*p as compa ed wi h pm 1pm 2 cells. Howe e , he ela i e amoun o Gas1*p deg aded ia he ERAD-L pa hway was ma kedly educed in he absence o he Pm 1p–Pm 2p complex, whe eas mos o he Gas1*p was deg aded ia a p24 complex-dependen pa hway (Fig. Fig. 3. The p24 p o ein complex does no in e ac wi h he H d1p complex and is pa o an ERAD-L independen deg ada ion pa hway o a ac ion o Gas1*p. (A)  Wild- ype cells (lanes 1 and 4) o cells exp essing a agged e sion o De 1p (De 1-CA; lanes 2 and 5) o Emp24-CA (lanes 3 and 6) we e lysed and samples we e analyzed as o Fig. 1B excep ha memb anes we e immunos ained wi h an i-Usa1p an ibodies. The SDS-PAGE was pe o med using a 7% s anda d gel. The a owhead indica es co-immunop ecipi a ed ac ion (no e ha he signal o Usa1p in he inpu ac ions is low; lanes 1–3). S a s indica e Emp24-CA and De 1-CA ha we e also ecognized by he seconda y an ibody. The b acke indica es IgG hea y chains ha we e ecognized by he seconda y an ibody. (B)  The deg ada ion o Gas1*p(HA) was measu ed, quan i ied and plo ed in wild- ype cells o in cells ca ying he designa ed dele ions as desc ibed in Fig. 2. The SDS- PAGE was pe o med using a 7% s anda d gel. Jou nal o Cell Science 4C). Toge he wi h ou da a o physical in e ac ions (Fig. 1C), hese esul s sugges ha he Pm 1p–Pm 2p complex has a dual ole in he a e o Gas1*p: i s , i e ains he p o ein and p e en s i s o he wise apid deg ada ion; and second, i ul ima ely deli e s he p o ein o he H d1p complex-dependen ERAD-L pa hway o deg ada ion. The Pm 1p–Pm 2p complex is equi ed o he as ER exi o wild- ype Gas1p Because o he associa ion o he Pm 1p–Pm 2p complex wi h he p24 p o ein complex, we p edic ed ha he Pm 1p–Pm 2p complex plays a ole in ER exi o e en wild- ype Gas1p. I he Pm 1p– Pm 2p complex possesses a chape one unc ion, i should p omo e ER exi o Gas1p. We i s es ed whe he Gas1p would be s able in he absence o he Pm 1p–Pm 2p complex, which would be an indica ion o i s p ope olding. To his end, we exp essed ch omosomally HA- agged Gas1p in wild- ype cells and in pm 1pm 2 cells and pe o med a CHX-chase expe imen (Fig. 5A, lanes 5–12). As can be seen, Gas1p ma u ed and was s able o e a pe iod o se e al hou s in wild- ype and in pm 1pm 2 cells, which was consis en wi h he wild- ype p o ein being olded p ope ly e en in he absence o he Pm 1p–Pm 2p complex. As a con ol, we show he ypical pa e n o Gas1*p ha is being O- mannosyla ed and deg aded in wild- ype cells (Fig. 5A, lanes 1– 4). We we e awa e o a minu e dec ease in elec opho e ic mobili y o he ma u e o m o Gas1p in pm 1pm 2 cells compa ed wi h wild- ype cells, he eason o which is cu en ly unknown (Fig. 5A, compa e lanes 5–8 wi h 9–12). Nex , we di ec ly add essed he ER exi kine ics o Gas1p in wild- ype cells and in pm 1pm 2 cells. We pe o med a adioac i e pulse-labeling and chase 149 Pm 1p–Pm 2p complex in ER quali y con ol expe imen ollowed by immunop ecipi a ion o Gas1p (Fig. 5B). As can be seen, he ER exi o Gas1p was ma kedly dec eased in pm 1pm 2 cells compa ed wi h wild- ype cells, as judged by he slowe con e sion o he p ecu so o m in o he ma u e o m. Whe eas in wild- ype cells abou hal o he Gas1p was con e ed in o he ma u e o m wi hin 10 minu es, he same p ocess ook abou 22 minu es in pm 1pm 2 cells (Fig. 5B). By con as , he ER exi o endogenous ca boxypep idase Y (CPY) was undis u bed in pm 1pm 2 cells compa ed wi h wild- ype cells, showing ha he o e all kine ics o ER exi we e no gene ally a ec ed (Fig. 5C). These esul s demons a e ha he Pm 1p–Pm 2p complex p omo es ER exi o wild- ype Gas1p. Discussion We ha e shown ha he Pm 1p–Pm 2p complex possesses p e iously unknown cellula unc ions ha a e eminiscen o hose collec i ely e med ‘ER p o ein quali y con ol’. The iden i ica ion o i s binding pa ne s and subsequen unc ional da a p o ide a ela i ely simple pic u e and sugges a model o how he Pm 1p– Pm 2p complex pe o ms quali y con ol o ER p o eins. Ou da a also cla i y some puzzling esul s wi h ega d o he deg ada ion o mis olded GPI-ancho ed p o eins. Fo ins ance, al hough i was epo ed ha Gas1*p is la gely deg aded by he p o easome, a pa hway o he p o easome could no be iden i ied (Fuji a e al., 2006). Fu he mo e, al hough i was shown ha he Pm 1p–Pm 2p complex O-mannosyla es Gas1*p, i has emained unclea why and whe e O-mannosyla ed Gas1*p is a ge ed o deg ada ion (Hi ayama e al., 2008). In he model depic ed in Fig. 6, he Pm 1p–Pm 2p complex plays he cen al and mo e uni e sal ole, whe eas he unc ion o Fig. 4. The Pm 1p–Pm 2p complex has a dual ole in he a e o Gas1*p: ini ial e en ion o he p o ein and i s ul ima e a ge ing o he H d1p complex. (A,B)  Deg ada ion o Gas1*p(HA) in cells ca ying he indica ed dele ions was measu ed, quan i ied and plo ed as desc ibed in Fig. 2. The SDS-PAGE was pe o med using a 4–20% g adien gel (A) o 5% s anda d gel (B). (C)  The deg ee o Gas1*p(HA) s abiliza ion when deple ing he H d1p complex was compa ed wi h ha when deple ing he p24 p o ein complex, bo h in he absence and p esence o he Pm 1p–Pm 2p complex (see Ma e ials and Me hods o calcula ion). Jou nal o Cell Science he p24 p o ein complex is es ic ed o GPI-ancho ed p o eins, like ou model p o eins Gas1p and Gas1*p. We base he la e assump ion on he ac ha he p24 p o ein complex is associa ed wi h Gas1p h oughou i s ER esidence ime and also du ing i s ER exi (Manuel Muñiz, pe sonal communica ion). Acco dingly, we p opose ha he Pm 1p–Pm 2p complex binds o Gas1p ha is al eady associa ed wi h he p24 p o ein complex. Because he p esence o he Pm 1p–Pm 2p complex p omo es as ER exi o Gas1p (Fig. 5B), he simples scena io would be ha he complex di ec ly o indi ec ly aids in p o ein olding (Fig. 6A). This is suppo ed by wo obse a ions: i s , E o1p and Pdi1p, which media e oxida i e p o ein olding, a e amongs he binding pa ne s o he Pm 1p–Pm 2p complex (Table 1); and second, mammalian cells exp ess a highly UPR- egula ed soluble p o ein in he ER wi h homology o he la ges luminal loop, loop 5, o he amily o PMTs (Fukuda e al., 2001; Hamada e al., 1996). This p o ein, SDF2L1 in mouse, is also ound in complex wi h o he ER esiden chape ones (Meunie e al., 2002). Ve y in e es ingly, on he basis o he egion o homology be ween SDF2L1, Pm 1p and Pm 2p and on unc ional s udies wi h Pm 1p (Gi bach e al., 2000), SDF2L1 should be incapable o media ing O-mannosyla ion. This s ongly sugges s ha he p oposed chape one-like unc ion o he Pm 1p–Pm 2p complex is dis inc om i s O-mannosyla ion ac i i y. Finally, p24 p o ein complex-dependen ER expo o co ec ly olded Gas1p occu s a e dissocia ion om he Pm 1p– 150 Jou nal o Cell Science 124 (1) Pm 2p complex, which, in con as o he p24 p o ein complex, emains in he ER (Haselbeck and Tanne , 1983; Huh e al., 2003). Like Gas1p, Gas1*p binds o he p24 p o ein complex and subsequen ly o he Pm 1p–Pm 2p complex (Fig. 6B). Howe e , since Gas1*p canno be olded p ope ly i con inuously ebinds o he Pm 1p–Pm 2p complex and is hus e ained by i in he ER (Fig. 4 and Fig. 6B). This e en ion has a leas wo consequences: i s , Gas1*p is inc easingly O-mannosyla ed wi h ime in a Pm 1p– Pm 2p complex-dependen manne (Fig. 2, Fig. 5A, Fig. 6B; supplemen a y ma e ial Fig. S1 and Fig. S3); and second, he Pm 1p–Pm 2p complex e en ually a ge s Gas1*p o he H d1p complex o ERAD (Fig. 4 and Fig. 6B). The p ecise molecula ole o O-mannans in Pm 1p–Pm 2p complex-media ed p o ein e en ion and H d1p complex-media ed p o ein deg ada ion is an exci ing subjec o be add essed nex . Because he Pm 1p–Pm 2p complex can associa e wi h he H d1p complex p o ein, O- mannosyla ion should ha e a unc ion di e en om a ge ing p o eins o he ERAD machine y. Consis en ly, O-mannosyla ion is no s ic ly equi ed o ERAD-dependen deg ada ion o Gas1*p because deg ada ion ia he H d1p complex s ill occu s in he absence o he Pm 1p–Pm 2p complex (Fig. 4B). We es ed whe he he Pm 1p–Pm 2p complex migh egula e subs a e deg ada ion by O-mannosyla ing ERAD machine y componen s bu ound ha his is no he case (supplemen a y ma e ial Fig. S4). In ano he model, inc easing O-mannosyla ion o he subs a e migh help o Fig. 5. The Pm 1p–Pm 2p complex p omo es ER exi o Gas1p. (A)  Gas1*p(HA) o Gas1p(HA) we e indi idually exp essed in wild- ype o in pm 1pm 2 cells and analyzed as desc ibed in Fig. 2. p, p ecu so o m; m, ma u e o m. The SDS-PAGE was pe o med using a 5% s anda d gel. (B)  Yeas cells exp essing Gas1p(HA) in wild- ype o in pm 1pm 2 cells we e labeled wi h [35S]me hionine o 10 minu es and chased o he indica ed imes. The cells we e lysed and Gas1p(HA) was immunop ecipi a ed using an i- HA an ibodies and analyzed by SDS-PAGE (5% s anda d gel) and au o adiog aphy. P o ein ma u a ion was plo ed o e ime on he basis o he ob ained ac ion o ma u e p o ein o m (m) om o al p o ein a indica ed imes ( igh panel). Values we e ob ained using a Phospo Image . The appa en a e o ma u a ion o Gas1p was calcula ed and shown wi h linea eg ession a e inclusion o he alue ze o a ime –10 (s a o pulse). (C)  The same expe imen wi h iden ical cells was pe o med bu he acuola pep idase CPY was immunop ecipi a ed using an i-CPY an ibodies. p1, p ecu so o m 1, p2, p ecu so o m 2. Plo ing and calcula ions we e done as o B. Jou nal o Cell Science dissocia e i om he Pm 1p–Pm 2p complex and allow ans e o he associa ed ERAD machine y. This model can be es ed in he u u e h ough he gene a ion and u iliza ion o sui able O- mannosyla ion mu an s o he Pm 1p–Pm 2p complex. Deple ion o he Pm 1p–Pm 2p complex leads o an ‘escape’ o mos o he Gas1*p om H d1p complex-media ed ERAD o a p24-dependen , p obably pos -ER, deg ada ion (Figs 4 and 6). Redundan quali y con ol in he ER, al hough abundan , is p obably no oo s ingen o a oid cos ly des uc ion o olding in e media es. Howe e , i becomes clea ha he cell possesses addi ional and less well cha ac e ized quali y con ol sys ems in he sec e o y pa hway downs eam o he ER ha can a ge p o eins o deg ada ion (Caldwell e al., 2001; Haynes e al., 2002; Vashis e al., 2001; Wang and Ng, 2010). Al hough we canno ule ou wi h ce ain y ha he p24 complex is a membe o such a pos -ER 151 Pm 1p–Pm 2p complex in ER quali y con ol quali y con ol sys em, we did no ind componen s wi h known unc ional links o he p o easome o o he acuole when we analyzed he binding pa ne s o Emp24p (V.G. and A.M., unpublished da a). Thus, he pa hway o p24 p o ein-dependen Gas1*p deg ada ion emains o be iden i ied. Apa om Gas1*p, he Pm 1p–Pm 2p complex has been shown o O-mannosyla e se e al o he mis olded soluble o memb ane- bound p o eins. Examples include KHN, KWW, mu an aspa ic p o ease I and mu an - ac o , none o which a e GPI-ancho ed bu all o which a e subjec ed o H d1p complex-dependen ERAD (Ha y e al., 2001; Naka sukasa e al., 2004; Vashis e al., 2001; Wahlman e al., 2007). An o e all inc ease in p o ein O- mannosyla ion has also been obse ed upon inhibi ion o N- glycosyla ion, which induces p o ein mis olding (Ha y e al., 2001). We he e o e sugges ha he Pm 1p–Pm 2p complex can a ge a wide a ie y o mis olded p o eins o he H d1p complex. One explana ion why his conclusion has been missed so a and why he e is con o e sy abou he a e o O-mannosyla ed mis olded p o eins is ha classical es s o in ol emen o cellula componen s in p o ein deg ada ion measu e he deg ee o inhibi ion o subs a e deg ada ion in dele ion mu an s. Howe e , ou da a clea ly show ha he Pm 1p–Pm 2p complex has a e en ion unc ion on op o i s a ge ing unc ion o he ERAD machine y, which complica es ma e s. In addi ion, he cell possesses mul iple and appa en ly e y dynamic pa hways o p o ein deg ada ion, which can lead o he a ge ing o mis olded p o eins o di e en ou es upon deple ion o one pa hway. Conside ing his, a mo e combina o ial app oach using mu an s wi h dis up ions in mul iple pa hways was needed o ob ain be e insigh in o he se e al unc ions o he Pm 1p–Pm 2p complex (Fig. 4). The same a gumen s eadily explain he kine ic al e a ions obse ed o ER expo o Gas1p and o he deg ada ion o Gas1*p in he absence o he Pm 1p–Pm 2p complex. Whe eas he expo a e o Gas1p was dec eased (Fig. 5), he appa en deg ada ion a e o Gas1*p was inc eased (Fig. 4). Wi h espec o Gas1p, he delay o ER expo in he absence o he Pm 1p–Pm 2p complex is consis en wi h loss o a chape one ha helps apid olding o Gas1p (Fig. 5B). Wi h espec o Gas1*p, as e deg ada ion by a pa hway o he han ERAD in he absence o he Pm 1p–Pm 2p complex is consis en wi h loss o bo h ER e en ion and a ge ing o ERAD (Fig. 4). Besides i s gene al ole in ER p o ein e en ion, ERAD and ER expo , he Pm 1p–Pm 2p complex migh possess an addi ional and mo e speci ic unc ion o he olding and ER expo o GPI- ancho ed p o eins. This is sugges ed by he physical link o he p24 p o ein complex, as only one o many known ER expo ac o s. In e es ingly, we also iden i ied Ted1p as a binding pa ne o he Pm 1p–Pm 2p complex bu no o he p24 p o ein complex (Table 1; V.G. and A.M., unpublished da a). Ted1p was i s iden i ied in a sc een o p o eins impai ing he su ace exp ession o mammalian G-p o ein-ac i a ed Ki channel GIRK2 in yeas (Haass e al., 2007). Likewise, he p24 p o ein complex componen s Emp24p and E 25p we e among he only six o he hi s om he 376 es ed dele ions. I was p e iously shown ha Ted1p and he p24 complex p o eins clus e in an epis asis mini-a ay (E-MAP), which sugges ed a common biological unc ion (Schuldine e al., 2005). Indeed, he ma u a ion o Gas1p was delayed in a Ted1p dele ion s ain simila o an Emp24p dele ion s ain (Haass e al., 2007). The mammalian o holog o Ted1p, PAGP5, is a GPI- ancho emodeling enzyme and was ecen ly shown o emo e he Fig. 6. A model o he ole o he Pm 1p–Pm 2p complex in ER p o ein quali y con ol. The scheme illus a es he p oposed ole o he Pm 1p–Pm 2p complex in ER p o ein quali y con ol o he es ed model p o eins Gas1p and Gas1*p. The basic p inciple should also be applicable o o he subs a es (see Discussion). (A)  ER ma u a ion o Gas1p (solid black a ows). A e ER ansloca ion and GPI-ancho ans e and p io o ER expo , Gas1p olds and has i s GPI ancho emodeled. Gas1p is p obably associa ed o he p24 p o ein complex h oughou i s ER esidence ime. Because he Pm 1p–Pm 2p complex possesses some cha ac e is ics o classical chape ones and aids in apid ER expo o Gas1p, i could be in ol ed in p o ein olding and/o GPI- ancho emodeling ( hick g ay a ows). (B)  ER e en ion and deg ada ion o Gas1*p (dashed black a ows). Like Gas1p, Gas1*p associa es wi h he p24 p o ein complex. Howe e , an ER expo compe en old canno be achie ed, which esul s in ex ended associa ion wi h he Pm 1p–Pm 2p complex (“Re en ion”), p o ein O-mannosyla ion (“O-mannosyla ion”) and subsequen ans e o he H d1p complex (“Ta ge ing o ERAD”) o e o ansloca ion and p o easomal deg ada ion (see Discussion o de ails). Those Gas1*p a ian s ha can escape his p ima y ER quali y con ol ( he amoun will d as ically inc ease when he Pm 1p–Pm 2p complex is absen ) will be subjec ed o a second, as ye uncha ac e ized, con ol sys em, p obably in a pos -ER compa men (ques ion ma k). Jou nal o Cell Science 152 Jou nal o Cell Science 124 (1) side-chain e hanolamine phospha e o he second mannose a ached o he GPI-ancho backbone. This ac i i y is p e equisi e o e icien ER exi o GPI-ancho ed p o eins (Fuji a e al., 2009). In a specula i e model, he Pm 1p–Pm 2p complex migh egula e access o he Gas1p–p24 p o ein complex o GPI-ancho emodeling enzymes and hus couple p o ein olding wi h GPI- ancho emodeling. This scena io could be pa o Pm 1p–Pm 2p complex-media ed Gas1p ‘ olding’, as shown in Fig. 6A. Ma e ials and Me hods Yeas s ains and plasmids The s ains used we e isogenic o W303 (MATa leu2-3,112 p1-1 can1-100 u a3-1 ade2-1 his3-11,15) excep hose used in expe imen s shown in Fig. 1, Fig. 3A and supplemen a y ma e ial Fig. S2, which we e isogenic o BY4741 (MATa his3leu2 u a3). Tagging o p o eins o genomic gene dele ions we e pe o med using s anda d PCR-based homologous ecombina ion echniques. Gas1*p(HA) was in eg a ed in o he URA3 locus o yeas cells using he in eg a i e plasmid pMF616, which was a gi om Mo ihisa Fuji a (Fuji a e al., 2006). Gas1p(HA) was de i ed om pMF616 by back mu a ion o he single base exchange using s anda d PCR- based mu agenesis and in eg a ed in o he URA3 locus o yeas cells. All cons uc s we e sequenced. P o ein complex pu i ica ion and co-immunop ecipi a ion Fo mass spec ome y analysis, app oxima ely 15 g o cells we e lysed by g inding in liquid ni ogen and he memb anes sedimen ed. Memb anes we e solubilized in bu e con aining 1% digi onin (Calbiochem). The ex ac was incuba ed o se e al hou s wi h IgG-coupled magne ic beads (Dynal). A e washing and elu ion, he elua e was ei he p ecipi a ed wi h ichlo oace ic acid (TCA) o sepa a ed by SDS- PAGE, ollowed by Coomassie Blue s aining and excision o indi idual bands. In bo h cases, he p o eins we e iden i ied by mass spec ome y. Fo co- immunop ecipi a ion, essen ially he same p o ocol was u ilized, wi h he excep ion ha ma e ial om 200 ml cul u es we e used and cells we e lysed using bead bea ing. A e SDS-PAGE, p o eins we e analyzed by immunblo ing wi h an ibodies o HA (Roche) o Usa1p (Ca alho e al., 2006), o wi h abbi IgG (Sigma). Gas1*p(HA) and Gas1p(HA) deg ada ion expe imen s Cycloheximide shu -o expe imen s we e pe o med in exponen ially g owing cells. The shu -o was s a ed by addi ion o CHX o a inal concen a ion o 200 g/ml. Equal olume aliquo s o cell cul u e we e emo ed a indica ed ime poin s and mo ed o ice. Cells we e lysed using 150 mM NaOH ollowed by addi ion o sample bu e con aining 1% SDS and hea ing. Cellula Gas1*p(HA) o Gas1p(HA) con en was p obed by SDS-PAGE ollowed by wes e n blo ing using an i-HA an ibodies. To quan i y bands wi h he Odyssey In a ed Imaging Sys em (LI-COR Biosciences) a dye-coupled seconda y an ibody was used (Fig. 2B,C, Fig. 4A; supplemen a y ma e ial Fig. S2). To quan i y bands wi h a LAS-3000 Imaging Sys em (Fuji ilm Li escience) and Mul i-Gauge So wa e, a pe oxidase-coupled seconda y an ibody was used (Fig. 3B, Fig. 4B; supplemen a y ma e ial Fig. S1). Radioac i e pulse-chase and immunop ecipi a ion Fo in i o pulse labeling, an o e nigh yeas cul u e was dilu ed and g own o an op ical densi y (OD) o ~0.8 a 600 nm. Cells equi alen o 5 OD we e esuspended in 1 ml medium lacking me hionine, incuba ed o 15 minu es a 30°C, and labeled o 10 minu es wi h 100 Ci/ml [35S]me hionine (Pe kin Elme s). Cells we e dilu ed o OD 0.8, supplemen ed wi h me hionine and 200 g/ml CHX. Aliquo s we e aken a indica ed imes, cells we e mo ed o ice and supplemen ed wi h 10 mM azide, pelle ed, esuspended in 50 mM T is pH 7.5, 5 mM EDTA, 1 mM PMSF, and lysed wi h glass beads o 7 minu es in a bead-bea e , supplemen ed wi h 1% SDS, and hea ed a 65°C o 10 minu es. Cell emnan s we e emo ed by cen i uga ion o 10 minu es in a mic o uge, and he supe na an used o immunop ecipi a ion using an i-HA an ibodies. Immune complexes we e isola ed wi h P o ein G-Sepha ose (GE Heal hca e) and analyzed by SDS-gel elec opho esis and au o adiog aphy using a Phospho Image (Fuji). Calcula ing Gas1*p s abiliza ion in h d1 and emp24 cells in p esence o absence o he Pm 1/2p complex as shown in Fig. 4C Gas1*p u no e in wild- ype cells was de e mined om a leas h ee indi idual expe imen s by measu ing emaining Gas1*p by wes e n blo ing as shown in Fig. 2B. The ob ained alues o indi idual imepoin s we e used o linea eg ession using he leas squa e me hod (LINEST unc ion in EXCEL). We ob ained R2 alues o 0.9344 o highe o each da a se . The m alues (ymx+b) o each indi idual eg ession se we e calcula ed, as well as he mean alue and i s s anda d de ia ion. We hen compa ed he m alue ob ained wi h wild- ype cells wi h hose ob ained wi h single dele ion mu an s (emp24 and h d1). The de ia ion in m alues o mu an s om hose ob ained wi h wild- ype cells was aken as deg ee o s abiliza ion (s abmmu /mw ). Because we ound Gas1*p is maximally s able in emp24h d1 cells compa ed o he indi idual mu an s (Fig. 3B), we assumed sepa a e deg ada ion pa hways and combined he indi idual de e mined deg ees o s abiliza ion o 100% (s abemp24+s abh d1100). Las ly, we plo ed he deg ee o Gas1*p s abiliza ion o each mu an as a pe cen age o o al, ei he in he p esence o he Pm 1p–Pm 2p complex (h d1 s emp24) o in i s absence (h d1pm 1pm 2 s emp24pm 1pm 2). We would like o hank Ped o Ca alho (CRG, Ba celona, Spain) and Manuel Muñiz (Uni e si y o Se ille, Se ille, Spain) o an ibodies, Mo ihisa Fuji a (Osaka Uni e si y, Osaka, Japan) o plasmids and Ross Tomaino (Ha a d Medical School, Bos on, MA) o excellen mass spec ome ic analysis, Alex Palazzo, Tom Rapopo , Ma in Spiess and Ka l E landson o c i ical eading o an ea lie e sion o he manusc ip ; and Manuel Muñiz and Le icia Lemus o e y ui ul discussions h oughou he wo k. V.G. is suppo ed by a G an o he Spanish Minis y o Science, BFU2009-07290. V.G. is a Ramon y Cajal ellow. Supplemen a y ma e ial a ailable online a h p://jcs.biologis s.o g/cgi/con en / ull/124/1/144/DC1 Re e ences Balli , B. A., Roux, P. P., Ge be , S. A., MacKeigan, J. P., Blenis, J. and Gygi, S. P. (2005). Quan i a i e phospho yla ion p o iling o he ERK/p90 ibosomal S6 kinase- signaling casse e and i s a ge s, he ube ous scle osis umo supp esso s. P oc. Na l. Acad. Sci. USA 102, 667-672. Ba lowe, C. (2003). Signals o COPII-dependen expo om he ER: wha ’s he icke ou ? T ends Cell. Biol. 13, 295-300. B achmann, C. B., Da ies, A., Cos , G. J., Capu o, E., Li, J., Hie e , P. and Boeke, J. D. (1998). Designe dele ion s ains de i ed om Saccha omyces ce e isiae S288C: a use ul se o s ains and plasmids o PCR-media ed gene dis up ion and o he applica ions. Yeas 14, 115-132. B odsky, J. L., We ne , E. D., Dubas, M. E., Goeckele , J. L., K use, K. B. and McC acken, A. A. (1999). The equi emen o molecula chape ones du ing endoplasmic e iculum-associa ed p o ein deg ada ion demons a es ha p o ein expo and impo a e mechanis ically dis inc . J. Biol. Chem. 274, 3453-3460. Caldwell, S. R., Hill, K. J. and Coope , A. A. (2001). Deg ada ion o endoplasmic e iculum (ER) quali y con ol subs a es equi es anspo be ween he ER and Golgi. J. Biol. Chem. 276, 23296-23303. Ca alho, P., Gode , V. and Rapopo , T. A. (2006). Dis inc ubiqui in-ligase complexes de ine con e gen pa hways o he deg ada ion o ER p o eins. Cell 126, 361-373. Denic, V., Quan, E. M. and Weissman, J. S. (2006). A luminal su eillance complex ha selec s mis olded glycop o eins o ER-associa ed deg ada ion. Cell 126, 349-359. Dieguez-Acuna, F. J., Ge be , S. A., Kodama, S., Elias, J. E., Beausoleil, S. A., Faus man, D. and Gygi, S. P. (2005). Cha ac e iza ion o mouse spleen cells by sub ac i e p o eomics. Mol. Cell P o eomics 4, 1459-1470. Ellgaa d, L. and Helenius, A. (2003). Quali y con ol in he endoplasmic e iculum. Na . Re . Mol. Cell Biol. 4, 181-191. F a, A. M., Fagioli, C., Finazzi, D., Si ia, R. and Albe ini, C. M. (1993). Quali y con ol o ER syn hesized p o eins: an exposed hiol g oup as a h ee-way swi ch media ing assembly, e en ion and deg ada ion. EMBO J. 12, 4755-4761. Fuji a, M., Yoko, O. T. and Jigami, Y. (2006). Inosi ol deacyla ion by Bs 1p is equi ed o he quali y con ol o glycosylphospha idylinosi ol-ancho ed p o eins. Mol. Biol. Cell 17, 834-850. Fuji a, M., Maeda, Y., Ra, M., Yamaguchi, Y., Taguchi, R. and Kinoshi a, T. (2009). GPI glycan emodeling by PGAP5 egula es anspo o GPI-ancho ed p o eins om he ER o he Golgi. Cell 139, 352-365. Fukuda, S., Sumii, M., Masuda, Y., Takahashi, M., Koike, N., Teishima, J., Yasumo o, H., I amo o, T., Asaha a, T., Dohi, K. e al. (2001). Mu ine and human SDF2L1 is an endoplasmic e iculum s ess-inducible gene and encodes a new membe o he Pm / p o ein amily. Biochem. Biophys. Res. Commun. 280, 407-414. Gi bach, V. and S ahl, S. (2003). Membe s o he e olu iona ily conse ed PMT amily o p o ein O-mannosyl ans e ases o m dis inc p o ein complexes among hemsel es. J. Biol. Chem. 278, 12554-12562. Gi bach, V., Zelle , T., P iesmeie , M. and S ahl-Bolsinge , S. (2000). S uc u e- unc ion analysis o he dolichyl phospha e-mannose: p o ein O-mannosyl ans e ase ScPm 1p. J. Biol. Chem. 275, 19288-19296. Gode , V., Ca alho, P. and Rapopo , T. A. (2008). The ER-associa ed deg ada ion componen De 1p and i s homolog D m1p a e con ained in complexes wi h dis inc co ac o s o he ATPase Cdc48p. FEBS Le 582, 1575-1580. Haass, F. A., Jonikas, M., Wal e , P., Weissman, J. S., Jan, Y. N., Jan, L. Y. and Schuldine , M. (2007). Iden i ica ion o yeas p o eins necessa y o cell-su ace unc ion o a po assium channel. P oc. Na l. Acad. Sci. USA 104, 18079-18084. Hamada, T., Tashi o, K., Tada, H., Inazawa, J., Shi ozu, M., Shibaha a, K., Nakamu a, T., Ma ina, N., Nakano, T. and Honjo, T. (1996). Isola ion and cha ac e iza ion o a no el sec e o y p o ein, s omal cell-de i ed ac o -2 (SDF-2) using he signal sequence ap me hod. Gene 176, 211-214. Hammond, C., B aakman, I. and Helenius, A. (1994). Role o N-linked oligosaccha ide ecogni ion, glucose imming, and calnexin in glycop o ein olding and quali y con ol. P oc. Na l. Acad. Sci. USA 91, 913-917. Jou nal o Cell Science