The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles

 The Rocke elle Uni e si y P ess, 0021-9525/2000/03/925/6 $5.00 The Jou nal o Cell Biology, Volume 148, Numbe 5, Ma ch 6, 2000 925–930 h p://www.jcb.o g 925 The Emp24 Complex Rec ui s a Speci ic Ca go Molecule in o Endoplasmic Re iculum–de i ed Vesicles Manuel Muñiz, Claude Nuo e , Hans-Pe e Hau i, and Howa d Riezman Biozen um o he Uni e si y o Basel, CH-4056 Basel, Swi ze land Abs ac . Membe s o he yeas p24 amily, including Emp24p and E 25p, o m a he e ome ic complex e- qui ed o he e icien anspo o selec ed p o eins om he endoplasmic e iculum (ER) o he Golgi ap- pa a us. The speci ic unc ions and si es o ac ion o his complex a e unknown. We show ha Emp24p is di- ec ly equi ed o e icien packaging o a lumenal ca go p o ein, Gas1p, in o ER-de i ed esicles. Emp24p and E 25p can be di ec ly c oss-linked o Gas1p in ER-de i ed esicles. Gap1p, which was no a - ec ed by emp24 mu a ion, was no c oss-linked. These esul s sugges ha he Emp24 complex ac s as a ca go ecep o in esicle biogenesis om he ER. Key wo ds: COPII-coa ed esicles • ER • E 25p • Saccha omyces ce e isiae • p o ein so ing In oduc ion In euka yo ic cells, p o ein anspo be ween he o - ganelles o he sec e o y pa hway is media ed by esicles ha bud om a dono compa men and use wi h an ap- p op ia e accep o compa men (Palade, 1975). The s a - ing poin o he exocy ic ou e is he ER. The e, co ec ly olded and assembled ca go molecules can en e COPII- coa ed esicles o anspo o he cis -Golgi compa men (Schekman and O ci, 1996). Many cy osolic and ans- memb ane p o eins ha e been iden i ied ha media e his esicula anspo s ep (Ro hman and Wieland, 1996; Schekman and O ci, 1996). p24 p o eins a e p esen in he e ome ic complexes ha ha e been p oposed o cycle be ween ER and Golgi com- pa men s because hey a e ound in bo h COPII and COPI esicles in addi ion o ER and Golgi memb anes (Schimmölle e al., 1995; S amnes e al., 1995; Belden and Ba lowe, 1996; Sohn e al., 1996; Rojo e al., 1997; Dominguez e al., 1998; Füllek ug e al., 1999; Ma zioch e al., 1999). In mammals, unc ional da a sugges a ole o hese p o eins in he o ma ion o ER exi si es (La oie e al., 1999). In yeas , eigh genes encode p24 amily mem- be s: EMP24 , ERV25 , and ERP1 – ERP6 (Ma zioch e al., 1999). Mu an s in se e al o hese genes show selec i e p o ein anspo de ec s. S ains wi h a single dele ion o EMP24 , ERV25 , ERP1 , o ERP2 show a delay in he ER o Golgi anspo o Gas1p (Schimmölle e al., 1995; Belden and Ba lowe, 1996; Ma zioch e al., 1999), a gly- cosylphospha idylinosi ol (GPI) 1 -ancho ed p o ein. The emp24 and e 25 mu an s also show a anspo de ec o in e ase, a soluble sec e ed p o ein. Se e al o he ca go molecules, including ␣ ac o , a sec e ed phe omone, do no equi e p24 p o eins o e icien anspo . Mu a ion o se e al yeas p24 genes esul s in sec e ion o he ER lu- menal p o ein, Ka 2p (El od-E ickson and Kaise , 1996; Ma zioch e al., 1999). The Emp24 complex in yeas comp ises Emp24p, E 25p, and mos likely E p1p and E p2p. Dele ion o EMP24 causes a s ong educ ion in he le els o he o he h ee p o eins o he complex (Belden and Ba lowe, 1996; Ma - zioch e al., 1999). In his s udy we p o ide e idence ha he Emp24 complex is di ec ly equi ed o e icien pack- aging o Gas1p in o ER-de i ed esicles. Two subuni s o his complex can be di ec ly c oss-linked o he ca go p o- ein in pu i ied ER-de i ed esicles, consis en wi h he hypo hesis ha he Emp24 complex plays a ole as a ca go ecep o in ER o Golgi anspo . Ma e ials and Me hods S ains An myc epi ope was in oduced a he NH 2 e minus o ma u e Emp24p. The myc- agged emp24-E178A mu an was cons uc ed by subs i u ing an app op ia e agmen o EMP24 wi h he sequenced mu an e sion ob- ained by PCR echniques. RH4443 ( MAT ␣ , u a3, leu2, his4, ba 1, emp24:: KanMx ) was ob ained by eplacing he en i e EMP24 coding sequence o RH1959 ( MAT ␣ , u a3, leu2, his4, ba 1 ) wi h a KanMx casse e. EMP24 al- leles we e cloned in o a YCplac111 (CEN/ARS) plasmid. The S. ce e isiae s ain RH696-2B ( MAT ␣ , sec18-20 gap1 ⌬ ::LEU2 u a3 ade2 leu2 lys2 ⌬ 201 pPL269) was ob ained by c ossing PLY129 (pPL269) ( MAT ␣ gap1 ⌬ :: LEU2 u a3 ade2 leu2 lys2 ⌬ 201 pPL269) (Kuehn e al., 1996), wi h RH478 ( MAT ␣ sec18-20 leu2 his4 ). Cy osols we e p epa ed om RH732 ( MAT ␣ his4 leu2 u a3 lys2 pep4::URA3 ba 1 ) and RH2043 ( MAT ␣ sec18-20 his4 leu2 u a3 pep4::URA3 ba 1 ). Add ess co espondence o Howa d Riezman, Biozen um o he Uni e - si y o Basel, Klingelbe gs asse 70, CH-4056 Basel, Swi ze land. Tel.: 41- 61-267-2160. Fax: 41-61-267-2149. E-mail: [email p o ec ed] 1 Abb e ia ions used in his pape : DSG, disuccinimidyl glu a a e; DSP, di hiobis(succinimidylp opiona e); gp ␣ F, glycosyla ed p o ␣ ac o ; GPI, glycosylphospha idylinosi ol. on Ap il 19, 2016jcb. up ess.o gDownloaded om Published Ma ch 6, 2000 The Jou nal o Cell Biology, Volume 148, 2000 926 P o ein Techniques P o ein le els we e de e mined by ex ac ion o log phase cul u es and Wes e n blo ing (Sü e lin e al., 1997) using an ibodies aised agains he Emp24p cy osolic ail o he myc epi ope. Analysis o Gas1p anspo in i o was by a pulse-chase p o ocol (Sü e lin e al., 1997). Gas1p ma u a- ion was analyzed using a 4-min pulse and subsequen chase a 30 ⬚ C, ol- lowed by immunop ecipi a ion, SDS-PAGE, and luo og aphy. Imma u e (105 kD) and ma u e (125 kD) o ms o Gas1p we e quan i ied using a densi ome e . The pe cen age o ma u e Gas1p was used as an indica ion o anspo o he Golgi appa a us. De e mina ion o pa i ioning o Gas1p be ween aqueous and de e gen phases o T i on X-114 was pe - o med as desc ibed (Nuo e e al., 1993), excep ha he media ac ions we e no analyzed. A e sepa a ion in o de e gen and aqueous phases, Gas1p was dena u ed, immunop ecipi a ed, esol ed by SDS-PAGE, i- sualized, and quan i ied using a Phospho Image . In Vi o ER-budding Assay The pe meabilized, cell-based, ER ca go packaging assay was pe o med as desc ibed (Kuehn e al., 1996) wi h a ew modi ica ions. S ains RH1959, RH4438, and RH696-2B we e ans o med wi h pCNYG1 o o e exp ess Gas1p (app oxima ely i e old) (Nuo e e al., 1991). O e - exp ession did no a ec Gas1p anspo kine ics o dependence on EMP24 . 10 mM 2-me cap oe hanesul onic acid eplaced 10 mM di hio- h ei ol. Budding eac ions con ained 30 ␮ l o memb anes ( om 12 ⫻ 10 7 cells), 300 ␮ g c ude cy osol, 3 ␮ g Sa 1p, 1 ⫻ ATP mix, and 0.2 mM GTP in a 150- ␮ l olume. A e 2 h a 25 ⬚ C, a po ion o he sample was emo ed o analysis ( o al), he emaining aliquo was sedimen ed (14,000 pm, 2 min, 4 ⬚ C), and 125 ␮ l o he supe na an collec ed and subjec ed o lo a- ion on a Nycodenz ® s ep g adien (Ba lowe e al., 1994). 100 ␮ l om he op was disca ded and he nex 800 ␮ l ans e ed o a new ube. 400 ␮ l was dilu ed h ee old wi h B88 and cen i uged (100,000 g , 1 h, 4 ⬚ C). The memb ane pelle was dissol ed in 1% SDS in TEPI bu e (Sü e lin e al., 1997) o 10 min a 55 ⬚ C, subjec ed o immunop ecipi a ion, and analyzed by SDS-PAGE wi h subsequen exposu e and quan i a ion using a Phos- pho Image . Vesicle Immunoisola ion Vesicles we e p oduced in a budding eac ion using sec18 memb anes and cy osol. The sec18 memb anes we e p epa ed as abo e, excep ha he las 5 min o deple ion and he pulse-labeling we e a 32 ⬚ C. The sec18 cy- osol was p eincuba ed (32 ⬚ C, 10 min) be o e use. Vesicles we e immu- noisola ed wi h o wi hou Emp24p an i- ail an ibody and p ocessed ac- co ding o Kuehn e al. (1996). C oss-Linking Nycodenz-pu i ied esicles p oduced in a budding eac ion we e adjus ed o 2.5 M u ea in B88 and incuba ed wi h 1 mM di hiobis(succinimidylp o- piona e) (DSP) o a ious amoun s o disuccinimidyl glu a a e (DSG; Pie ce) (20 ⬚ C, 20 min). The c oss-linking eac ion was quenched by addi- ion o glycine (50 mM inal, 5 min, 20 ⬚ C). Vesicles we e sedimen ed a 100,000 g (1 h, 4 ⬚ C), dissol ed wi h 1% SDS in TEPI (5 min, 95 ⬚ C o Gas1p and glycosyla ed p o ␣ ac o [gp ␣ F], o 55 ⬚ C o Gap1p), and im- munop ecipi a ed wi h Emp24p an i- ail an ibody o E 25 an ibody (Belden and Ba lowe, 1996) and p o ein A–Sepha ose. P ecipi a ed ma e- ial was elu ed om he Sepha ose beads by incuba ion wi h 1% SDS in TEPI (5 min, 95 o 55 ⬚ C) and eimmunop ecipi a ed wi h an i-Gas1p o an i-Gap1p an ibody. Resul s Emp24p Is Requi ed o E icien Rec ui men o Gas1p in o ER-de i ed Vesicles To in es iga e whe he Emp24p plays a ole in ca go exi om he ER we quan i ied he packaging o di e en sec e o y p o eins in o esicles ha we e gene a ed om wild- ype and emp24 mu an ER memb anes in i o. Bud- ding o Gas1p was much less e icien ( ⬎ 70% less) om emp24 ⌬ memb anes han om wild- ype memb anes, whe eas he budding e iciencies o gp ␣ F and an amino acid pe mease (Gap1p) we e simila om he wo mem- b ane sou ces (Fig. 1). gp ␣ F is an es ablished con ol o selec i i y, because i s anspo om he ER o he Golgi is no a ec ed in emp24 ⌬ cells (Schimmölle e al., 1995). Gap1p was also in es iga ed because i has been shown o be e icien ly inco po a ed in o COPII-coa ed esicles in i o (Kuehn e al., 1996). The e o e, Emp24p is equi ed o e icien packaging o a leas one sec e o y p o ein in o COPII-coa ed esicles. The magni ude o he de ec in Gas1p budding om he ER is consis en wi h he ER o Golgi anspo de ec seen in i o (Schimmölle e al., 1995) and could, he e o e, be he en i e explana ion o he anspo delay. I has been p oposed ha Emp24p may be a nega i e egula o o esicle o ma ion (El od-E ickson and Kaise , 1996) and ha i s absence would esul in inc eased Ka 2p exi om he ER. To add ess his poin , we compa ed he inco po a ion o Ka 2p in o esicles using wild- ype and emp24 ⌬ memb anes. Ka 2p was poo ly packaged in o ER- de i ed esicles using ei he memb ane sou ce (Fig. 1). The e o e, i does no seem ha Emp24p is equi ed o excluding Ka 2p om ER-de i ed esicles. This sugges s, bu does no p o e, ha he eason o Ka 2p sec e ion in he emp24 mu an is a de ec in e og ade anspo . Figu e 1. In i o budding o ER-de i ed esicles om wild- ype and emp24⌬ memb anes. Sphe oplas s om wild- ype (EMP24) and mu an (emp24⌬) cells we e pulse-labeled (3 min), pe me- abilized, and incuba ed wi h an ene gy sou ce wi h o wi hou cy- osol (2 h, 25⬚C). Vesicles we e pu i ied by di e en ial cen i- uga ion and lo a ion on Nycodenz® g adien s. Budding o indica ed p o eins was assayed by immunop ecipi a ion, SDS- PAGE, isualiza ion, and quan i ica ion using a Phospho Image . The budding e iciency o a gi en p o ein e e s o he pe cen - age o he o al adiolabeled p o ein ha was eco e ed in he pu i ied esicula ac ion. on Ap il 19, 2016jcb. up ess.o gDownloaded om Published Ma ch 6, 2000 Muñiz e al. Ca go Rec ui men in o ER-de i ed Vesicles 927 Emp24p Is Di ec ly In ol ed in he Selec i e Packaging o Ca go Molecules Since Emp24p could play a ole in e og ade anspo i is possible ha he ine icien budding o Gas1p om emp24 ⌬ memb anes is an indi ec consequence o a e o- g ade anspo de ec . To add ess his poin , we used an i- bodies agains he cy osolic ail o Emp24p o inhibi he unc ion o Emp24p in wild- ype memb anes. P eincuba- ion o memb anes wi h an ibodies on ice educed he budding e iciency o Gas1p g ea e han h ee old (Fig. 2 A). This inhibi ion was speci ic o Gas1p, because he packaging o Gap1p and gp ␣ F was no a ec ed. As a con- ol o he speci ici y o ou an i- ail an ibodies, we used an emp24 mu an wi h an E178A subs i u ion in he cy o- solic ail. This poin mu a ion does no a ec he unc ion o Emp24p (Fig. 2 B), bu he an i- ail an ibodies no longe ecognize he mu an p o ein (Fig. 2 B). P eincuba- ion o memb anes de i ed om emp24 - E178A cells wi h an i- ail an ibodies had no e ec on budding e iciencies o Gas1p, gp ␣ F, o Gap1p (Fig. 2 A). The e o e, we conclude ha Emp24p is equi ed di ec ly o he e icien inco po- a ion o Gas1p in o ER-de i ed esicles. One equi emen o Gas1p exi om he ER is he a - achmen o he GPI ancho (Doe ing and Schekman, 1996). To es whe he he GPI ancho is e icien ly a - ached o Gas1p in he emp24 ⌬ mu an , we pe o med a pulse-chase analysis ollowed by sepa a ion o he p o ein ex ac s in o de e gen and aqueous phases using T i on X-114. GPI-ancho ed Gas1p pa i ions e icien ly in o he de e gen phase, whe eas he unancho ed p o ein pa i- ions app oxima ely equally be ween de e gen and aque- ous phases (Nuo e e al., 1993). A e he 4-min pulse- labeling, ⵑ 90% o he Gas1p was al eady ound in he de e gen phase in bo h wild- ype and emp24 ⌬ mu an s ains (Fig. 2 C), demons a ing ha he GPI ancho is e - icien ly a ached o Gas1p in he emp24 ⌬ mu an . The e- o e, a de ec in GPI ancho ing canno explain he ans- po de ec o Gas1p in he emp24 ⌬ mu an . Emp24p and Gas1p Can be Found in he Same P o ein Complex in ER-de i ed Vesicles Since Emp24p is e icien ly inco po a ed in o ER-de i ed esicles (Schimmölle e al., 1995) and is di ec ly equi ed o Gas1p ec ui men , we in es iga ed i Gas1p could be ound in a p o ein complex wi h Emp24p in ER-de i ed esicles. ER-de i ed esicles we e gene a ed in i o and uncoa ed esicles we e exposed o he clea able mem- b ane-pe meable c oss-linke DSP. A e lysis and dena- u a ion, he samples we e immunop ecipi a ed using an i- Emp24p ail an ibodies. The p ecipi a es we e dena u ed and subjec ed o a second immunop ecipi a ion using an i- bodies agains Gas1p. The c oss-linke was clea ed and he samples we e analyzed by SDS-PAGE . Gas1p was c oss-linked o Emp24p wi h good e iciency (Fig. 3). No Gas1p was eco e ed i c oss-linke was omi ed. Mo e- o e , Gas1p could no be de ec ed when he c oss-linking was pe o med on esicles gene a ed om emp24 - E178A memb anes (Fig. 3), p o ing ha Gas1p was eco e ed as pa o a complex con aining Emp24p. This complex is speci ic because ano he ca go molecule, Gap1p, whose budding was no educed om emp24 mu an memb anes, Figu e 2. Inhibi ion o Gas1p budding by an i-Emp24p ail an i- bodies. (A) Pe meabilized sphe oplas s we e p epa ed om wild- ype (EMP24) and emp24-E178A (mE178A) cells as in Fig. 1. They we e p eincuba ed in he p esence o absence o an ibod- ies agains he cy osolic domain o Emp24p (1 h, 4⬚C). Budding eac ions we e pe o med and analyzed as in Fig. 1. (B) The a e o Gas1p ma u a ion in wild- ype and emp24 mu an s was ana- lyzed a e a pulse-chase immunop ecipi a ion p o ocol. Emp24p was isualized by Wes e n blo ing using an ibodies agains he c-myc epi ope o agains he cy osolic ail o Emp24p. Mu an (emp24⌬) cells we e ans o med wi h emp y ec o (emp24⌬), he wild- ype allele (EMP24), myc- agged Emp24p (mEMP24), o he myc- agged emp24-E178A (mE178A) allele. (C) A e pulse-chase labeling o wild- ype (EMP24) and mu an (emp24⌬) s ains, cells we e lysed and ex ac ed wi h T i on X-114 ollowed by de e gen phase sepa a ion. Aqueous (A) and de e gen (D) phases we e subjec ed o immunop ecipi a ion and SDS-PAGE. Gas1p (bo h o ms combined) was quan i ied using a Phospho - Image . Imma u e Gas1p (105) was con e ed o he ma u e o m (125) wi h delayed kine ics in he emp24⌬ mu an , bu was e icien ly ex ac ed in o he de e gen phase. on Ap il 19, 2016jcb. up ess.o gDownloaded om Published Ma ch 6, 2000 The Jou nal o Cell Biology, Volume 148, 2000 928 was no c oss-linked o Emp24p. These da a clea ly dem- ons a e ha Emp24p and Gas1p can be ound in he same p o ein complex in ER-de i ed esicles and sugges ha his complex is speci ic. An al e na i e explana ion o he absences o a ole o Emp24p in Gap1p budding and o Gap1p om he c oss- linked Emp24 complex would be i Emp24p and Gap1p we e ound in di e en ER-de i ed esicles. To es his we p oduced esicles using memb anes and cy osol om a sec18 mu an . SEC18 encodes yeas N-e hylmaleimide– sensi i e usion p o ein, and i s inac i a ion unde he con- di ions o his expe imen blocks usion o ER-de i ed esicles wi h he Golgi appa a us (Muñiz, M., and H. Riez- man, manusc ip in p epa a ion). These condi ions ensu e ha we a e analyzing p ima y ER-de i ed esicles. The esicles we e immunoisola ed using an ibody agains he cy osolic ail o Emp24p. 83% o he Gas1p and 96% o he Gap1p copu i ied wi h he immunoisola ed esicles (Fig. 4). Vi ually no ca go p o eins we e isola ed when Emp24p ail an ibody was omi ed. The e o e, Emp24p is ound in he same esicles as Gap1p and Gas1p. The lack o e ec o he emp24 mu a ion on Gap1p budding and Gap1p absence om he c oss-linked Emp24 complex canno be due hei p esence in dis inc esicles. Emp24p and E 25p Can be Di ec ly C oss-Linked o Gas1p in ER-de i ed Vesicles The c oss-linking esul s p esen ed abo e sugges ha Emp24p may be pa o a ca go ecep o ha imp o es he e iciency o ca go en y in o ER-de i ed esicles. How- e e , hey do no show ha Emp24p binds he ca go mole- cule di ec ly. To add ess his issue we used a nonclea able c oss-linke , DSG, and pe o med simila c oss-linking s udies on ER-de i ed esicles. Emp24p was c oss-linked o Gas1p as seen by a smea s a ing a a ound 25,000 highe appa en molecula mass han he ER o m o Gas1p (Fig. 5 A). Addi ional bands we e seen a highe molecula weigh s. The band a 130 kD mus ep esen a di ec ly c oss-linked p oduc be ween Emp24p (24 kD) and Gas1p (105 kD). All o he p ecipi a ed ma e ial was speci ic because i was no seen when he esicles we e gene a ed om emp24-E178A mu an memb anes. Nex , we immunop ecipi a ed he c oss-linked ma e ial wi h an ibodies agains E 25p, ollowed by an ibodies agains Gas1p. A s ong band appea ed a he posi ion ex- pec ed o a di ec ly c oss-linked p oduc be ween he wo molecules. The e o e, we conclude ha E 25p can also be di ec ly and e icien ly c oss-linked o Gas1p in isola ed ER-de i ed esicles. We could also de ec his c oss-linked p oduc in esicles de i ed om emp24-E178A mu an (da a no shown), con i ming ha esicle budding oc- cu ed using hese memb anes. Finally, we pe o med a i a ion o DSG on he isola ed esicles. When c oss-linke was omi ed, no E 25p-Gas1p p oduc could be de ec ed (Fig. 5 B). A low concen a- ions o c oss-linke he di ec ly c oss-linked p oduc was by a he main p oduc seen. A highe concen a ions o c oss-linke mo e c oss-linked p oduc s we e seen and he bands a highe molecula weigh became mo e appa en . The c oss-linking s udies shown abo e demons a e ha Gas1p can be speci ically and di ec ly c oss-linked o he Figu e 3. C oss-linking o Emp24p and Gas1p. (A) ER-de i ed esicles gene a ed in i o om wild- ype (EMP24) and emp24- E178A mu an (mE178A) memb anes we e incuba ed wi h DSP. The samples we e dena u ed and immunop ecipi a ed wi h an i- bodies agains he ail o Emp24p. The p ecipi a es we e dena- u ed and ep ecipi a ed wi h an ibody agains Gas1p o Gap1p (C). 10 o 20% o he esicle p epa a ion was immunop ecipi- a ed di ec ly wi h an ibodies agains Gas1p o Gap1p, wi hou addi ion o DSP, o use as a s anda d (S) o eco e y. In con ol expe imen s, c oss-linking wi h DSP did no a ec eco e y o Gap1p by immunop ecipi a ion. Samples we e incuba ed wi h 5% 2-me cap oe hanol, analyzed by SDS-PAGE, and isualized using a Phospho Image . (B) Vesicles gene a ed om wild- ype memb anes we e incuba ed wi h o wi hou DSP and p ocessed as abo e. Figu e 4. Immunoisola ion o Emp24p con aining esicles. ER- de i ed esicles gene a ed in i o om sec18 memb anes and cy- osol we e immunoisola ed wi h o wi hou Emp24p an i- ail an- ibody. The supe na an s (S) and pelle s (P) we e p ocessed o immunop ecipi a ion. on Ap il 19, 2016jcb. up ess.o gDownloaded om Published Ma ch 6, 2000 Muñiz e al. Ca go Rec ui men in o ER-de i ed Vesicles 929 Emp24 complex. These esul s a e consis en wi h he hy- po hesis ha he Emp24 complex is a ca go ecep o e- qui ed o he e icien inco po a ion o Gas1p in o ER- de i ed esicles. Discussion A majo inding o his s udy is ha Emp24p is di ec ly equi ed o e icien inco po a ion o Gas1p in o ER- de i ed esicles in i o. Two independen assays we e used o show his. One used mu an memb anes de- oid o Emp24p. The o he used an ibodies o inhibi Emp24p unc ion di ec ly in wild- ype memb anes. Bo h assays showed ha Gas1p budding depended s ongly on Emp24p, whe eas budding o o he p o eins did no . This s iking speci ici y sugges s ha his complex has a highly speci ic ole in packaging o Gas1p in o ER-de i ed esi- cles. This di ec ole o Emp24p in selec i e budding om he ER can be explained in h ee ways. Fi s , Emp24p could be equi ed o e icien ancho a achmen o Gas1p. We ha e shown ha his is no he case. Second, Emp24p could be equi ed o he o ma ion o ER-de i ed esi- cles. I has been sugges ed ecen ly ha ␣2p24, a mamma- lian membe o he p24 amily, plays a ole in he o ma- ion o ER ca go exi si es (La oie e al., 1999). Howe e , in yeas , he absence o Emp24p o he inhibi ion o i s unc ion by he ail an ibody does no lead o a gene al de- ec in esicle o ma ion because budding o o he ca go p o eins was no a ec ed. Also, mu an memb anes e i- cien ly package he SNARE Sec22p (Belden and Ba - lowe, 1996). Mo eo e , since Emp24p is ound in esicles wi h Gas1p as well as wi h Gap1p, i is highly unlikely ha Emp24p is equi ed only o o ma ion o esicles con ain- ing Gas1p. The hi d explana ion o he di ec ole o Emp24p in selec i e budding om he ER is o conside Emp24p as pa o a ca go ecep o ha is equi ed o he packaging o a e y small subse o ca go p o eins. This ca go ecep- o would ec ui speci ic p o eins in o he ER-de i ed esicles, inc easing he e iciency o packaging. A second majo inding o ou s udy, ha Emp24p and E 25p can be di ec ly c oss-linked o Gas1p, suppo s his hypo hesis. This model aises wo issues. Why does mu a ion o EMP24 o o he genes encoding membe s o he p24 am- ily a ec he anspo o so ew p o eins? And why is anspo o hese p o eins no comple ely de ec i e in emp24 mu an cells? One possibili y o explain he small numbe o p o eins a ec ed is ha he e could be a la ge and he e ogeneous amily o ca go ecep o s wi h o e lapping unc ion. E i- dence exis s o ano he ype o ca go ecep o ac ing in ER o Golgi anspo , namely ERGIC 53, a lec in ha also appa en ly acili a es anspo o some glycop o eins (Appenzelle e al., 1999). I is in e es ing o no e ha mu- a ion o ei he ecep o only a ec s a small numbe o ca go molecules in a highly speci ic manne . The e is a la ge numbe o p o eins ha cycle be ween ER and Golgi compa men s. Many o hese could ha e ca go ecep o unc ions in di ec o indi ec ways. Ano he way o explain he small numbe o p o eins a - ec ed in a ca go ecep o mu an would be ha only a small subse o p o eins equi es a ecep o a his s ep. I seems clea ha some e y abundan p o eins in special- ized sec e o y cells a e no concen a ed in COPII-coa ed esicles (Ma ínez-Mená guez e al., 1999). The e o e, no all sec e o y p o eins equi e a ecep o o e icien ER exi . So why would some p o eins equi e ecep o s o his anspo s ep? Some p o eins may bind wi h low a - ini y o o he p o eins ha a e e ained in he ER lumen. These ca go molecules would equi e a highe a ini y in- e ac ion o libe a e hem om he ER. Al e na i ely, some p o eins may equi e high a es o sec e ion, o ex- ample, a p o ein wi h an ac i i y ha would be de imen al in he ER. Gas1p p obably has a unc ion in c oss-linking ␤ 1,3 glucans wi h ␤ 1,6 glucans and chi in in he cell wall (Popolo and Vai, 1999). Such an ac i i y may ha e nega- i e consequences in he ER. P e iously, a bulk low mechanism (Wieland e al., 1987) whe e p o eins lea e he ER simply by being a ail- able o en y in o he o ming esicle, simila o he p o- cess o luid-phase pinocy osis, has been p oposed. This could explain why mu a ion o genes encoding membe s o he p24 amily a ec s anspo o some ca go p o eins s ongly, bu no comple ely. Wi hou a ecep o , he ca go p o ein would s ill be able o en e ER-de i ed esicles by bulk low. When he unc ion o o he ca go ecep- o s, such as he mannose-6-phospha e ecep o (Ko n- eld, 1992), o yeas Vps10p (Ma cusson e al., 1994) is blocked, he lysosomal o acuola p o eins ha a e a - ec ed s ill exi he Golgi. Howe e , hey a e sec e ed a he han a ge ed o he lysosome o acuole. Since, as a as we know, he e is only one des ina ion o ER- de i ed esicles, namely he Golgi appa a us, p o ein mis- so ing would no occu in he absence o ecep o s in he ER. This may also help o explain why only a small subse o p o eins ac ually needs a ecep o . As long as hey can exi he ER hey will go o he Golgi compa men . A ole in speci ic ca go ec ui men is no likely o be he only ole o he Emp24 complex. Sec e ion o Ka 2p caused by mu a ions in subuni s o his complex implies a ole in he e og ade anspo om he Golgi compa - men o he ER (El od-E ickson and Kaise , 1996; Ma zi- och e al., 1999). Consis en wi h his, p24 ails om animal Figu e 5. Di ec c oss-linking o Emp24p and E 25p wi h Gas1p. (A) Vesicles gene a ed in i o om wild- ype (EMP24) and emp24-E178A mu an (mE178A) memb anes we e incu- ba ed wi h 0.25 mM o DSG and p ocessed as in Fig. 3. (B) Vesi- cles gene a ed om wild- ype memb anes we e incuba ed wi h he indica ed amoun s o DSG, dena u ed, immunop ecipi a ed wi h an ibody agains E 25p, hen ep ecipi a ed wi h an ibody agains Gas1p and p ocessed as in Fig. 3. on Ap il 19, 2016jcb. up ess.o gDownloaded om Published Ma ch 6, 2000 The Jou nal o Cell Biology, Volume 148, 2000 930 cells can bind COPI p o eins (Fiedle e al., 1996; Sohn e al., 1996; Dominguez e al., 1998) and when displayed on liposomes hey can s imula e he o ma ion o COPI- coa ed esicles (B emse e al., 1999). Quali y con ol in he ER in ol es ecycling om he Golgi compa men o he ER o mis olded p o eins (Hammond and Helenius, 1994). In e es ingly, a mu a ion in a p24 p o ein om C. el- egans, SEL-9, allows some mis olded p o eins o escape he quali y con ol mechanisms in he ea ly sec e o y pa h- way (Wen and G eenwald, 1999) which could pe haps be explained by a e og ade de ec . S udies ha can di ec ly add ess he e og ade o o he pu a i e unc ions o p24 p o eins in esicula a ic would be desi able. We hank C. Ba lowe, M.J. Kuehn, and R. Schekman o ma e ials and ad ice; B. And é o an ibody; Riezman labo a o y membe s and M.I. Geli o c i ical eading o he manusc ip ; and T. Aus and T. Ebe le o echnical assis ance. This wo k was suppo ed by Swiss Na ional Founda ion g an s ( o H. Riezman and H.-P. Hau i), Fede a ion o Eu opean Biochemical Socie ies and Human F on ie s Science P og am O ganiza ion ellowships ( o M. Muñiz). Submi ed: 29 No embe 1999 Re ised: 14 Janua y 2000 Accep ed: 19 Janua y 2000 Re e ences Appenzelle , C., H. Ande sson, F. Kappele , and H.P. Hau i. 1999. 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