Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum

INTRODUCTION Du ing in e phase he cis e nal o ganiza ion o he Golgi complex is main ained by a con olled balance o memb ane inpu and ou pu . The inpu pa hway consis s o an e og ade endoplasmic e iculum (ER)-Golgi memb ane anspo . The ou pu depends on bo h he p oduc ion o so ing and exocy ic esicles om he ans-Golgi ne wo k (TGN) and he exis ence o a Golgi-ER e og ade pa hway (Klausne e al., 1992; Lip- pinco -Schwa z, 1993; Ro hman and O ci, 1992). Cu en ly he e is g ea in e es in he e og ade pa hway because i is assumed o ul ill he ole o e u ning escaped ER esiden p o eins, which e en ually each he Golgi complex (Lewis and Pelham, 1992; Pelham, 1988) as well as mis olded p o eins ha a e no sui able o p oceed along he exocy ic pa hway (Hammond and Helenius, 1994). On he basis o obse a ions made on cells ea ed wi h he ungal me aboli e b e eldin A (BFA), he e og ade pa hway has been desc ibed o occu by he mic o ubule-dependen ex ension o ubule p ocesses which emana e om he Golgi cis e nae and use wi h he ER (Lippinco -Schwa z e al., 1990). By con as , an e og ade memb ane anspo is hough o be media ed by ca ie esicles which anspo bo h p o eins and lipids om he ER o he cis-Golgi and be ween he di e en Golgi compa men s (Ro hman and O ci, 1992; Takizawa and Malho a, 1993). Condi ions ha al e he no mal balance exis ing be ween an e og ade and e og ade memb ane pa hways lead o dis- up ion o he Golgi o ganiza ion. Thus, ea men wi h BFA inhibi s he ER-Golgi an e og ade anspo bu no he e o- g ade pa hway and consequen ly he Golgi complex is disas- sembled and edis ibu es in o he ER (Klausne e al., 1992). These e ec s a e also obse ed in wo o he si ua ions: o e - exp ession o a Golgi ecep o o ER lumenal p o eins (Hsu e al., 1992) and ADP- ibosyla ion o ho, a small GTP-binding p o ein (Sugai e al., 1992a,b). In addi ion, cell mu an s ha e been desc ibed ha exhibi a BFA-like pheno ype (Kao and D ape , 1992; Zube e al., 1991). Taken oge he hese s udies sugges ha Golgi-ER e og ade anspo is a highly egula ed p ocess ha in luences bo h Golgi s uc u al main enance and unc ion. The mechanism unde lying an e og ade ans e be ween memb ane compa men s o he exocy ic pa hway has been ex ensi ely in es iga ed using bo h biochemical and gene ic app oaches (Ro hman and O ci, 1992). By con as , li le a en ion has been gi en o he ac o s ha egula e e og ade anspo . S udies using BFA ha e indica ed ha edis ibu ion o Golgi memb anes o he ER is inhibi ed by bo h he non- hyd olyzable analog o GTP, GTP γS, and he ac i a o o ime ic G p o eins, AlF(3-5), sugges ing ha GTP-binding p o eins migh be in ol ed (Donaldson e al., 1991b; Tan e al., 1992). Howe e , bo h GTPγS and AlF(3-5) p ima ily in e e e wi h he associa ion/disassocia ion cycle o he coa p o eins o Golgi memb anes and he e o e hey also inhibi an e og ade anspo as well (Donaldson e al., 1991a; Melançon e al., 1987). Pa o he di icul y in unde s anding he basic mechanism o e og ade anspo is due o he lim- 1805 S ep olysin O-pe meabilized cells incuba ed wi h a high concen a ion (5-10 mg/ml) o cy osolic p o eins and ATP- gene a ing sys em exhibi edis ibu ion in o he endoplas- mic e iculum (ER) o Golgi in eg al p o eins (mannosidase II, galac osyl ans e ase, TGN 38), de ec ed by immuno- luo escence. In addi ion, mannosidase II is de ec ed in he ER o cells exposed o a high concen a ion o cy osolic p o eins and p ocessed o immunolec on mic oscopy by immunope oxidase. The edis ibu ion p ocess equi es ATP and is no a ec ed by p e ious mic o ubule depoly- me iza ion. Ul as uc u al obse a ions indica e ha Golgi disassembly occu s by budding o coa ed esicles. This s age o he p ocess is inhibi ed by GTPγS, AlF(3-5), ansducin βγ subuni s, and mas opa an, indica ing he in ol emen o ime ic G p o eins. A a la e s age, esicles lose hei coa s and use wi h he ER. This pa o he p ocess does no occu in cells incuba ed a ei he 15°C o 20°C, o exposed o N-e hylmaleimide. In cells ea ed wi h ei he chole a o pe ussis oxin Golgi edis ibu ion in o he ER shows a 50- old lowe equi emen o cy osolic ac o s han in un ea ed cells. These da a sugges a egu- la o y ole o bo h αsand αi ime ic G p o eins in he no mal Golgi-ER e og ade anspo aking place in in ac cells. Key wo ds: Golgi complex, e og ade anspo , G p o ein SUMMARY T ime ic G p o eins egula e he cy osol-induced edis ibu ion o Golgi enzymes in o he endoplasmic e iculum Jose ina Hidalgo, Manuel Muñiz and Angel Velasco* Depa men o Cell Biology, Facul y o Biology, Uni e si y o Se ille, A d. Reina Me cedes s/n, 41012-Se ille, Spain *Au ho o co espondence Jou nal o Cell Science 108, 1805-1815 (1995) P in ed in G ea B i ain © The Company o Biologis s Limi ed 1995 1806 i a ions imposed by he use o BFA, a d ug ha also induces ubule p ocesses om he TGN, endosomes, and lysosomes (Lippinco -Schwa z e al., 1991; Tooze and Hollinshead, 1992; Wood e al., 1991). Thus he e is no di ec e idence ha Golgi-ER e og ade anspo in he absence o BFA is ubule- media ed (Pelham, 1991). Recen ly ubule o ma ion om Golgi cis e nae has been epo ed o occu in i o in he absence o BFA and unde condi ions o low ATP o cy osol supply (Clue e al., 1993; Weidman e al., 1993). Al hough hese ubule p ocesses ha e been assimila ed o hose o med in i o du ing e og ade anspo hei physiological ele ance is unce ain. In he p esen s udy we show disas- sembly o he Golgi complex and edis ibu ion in o he ER in S ep olysin O (SO)-pe meabilized cells incuba ed wi h a high concen a ion (5-10 mg/ml) o cy osolic p o eins. This p ocess equi es ATP, is independen o ubule o ma ion, and is egula ed by ime ic G p o eins. MATERIALS AND METHODS Ma e ials Tissue cul u e eagen s we e ob ained om GIBCO BRL (Gai he s- bu g, MD). SO was pu chased om Wellcome Diagnos ics (Beckenham, UK). N-e hylmaleimide (NEM), GTPγS, nocodazole, 3,3′-diaminobenzidine e ahyd ochlo ide, saponin, and componen s o he ATP-gene a ing sys em we e om Sigma Chemical Co (S Louis, MO). Mas opa an was om Fluka Chemika (Buchs, Swi ze - land). Bac e ial oxins we e pu chased om Calbiochem (San Diego, CA). An ibodies agains α-mannosidase II, β-COP, KDEL, galac o- syl ans e ase, and TGN38 we e kindly p o ided by D s M. G. Fa quha (Uni e si y o Cali o nia San Diego, CA), T. K eis (Uni- e si é de Genè e, Swi ze land), S. Fulle (Eu opean Molecula Biology Labo a o y, Heidelbe g, Ge many), E. Be ge (Uni e si ä Zü ich, Swi ze land), and G. Ban ing (Uni e si y o B is ol, UK), espec i ely. FITC-, TRITC-, and pe oxidase-conjuga ed goa seconda y an ibodies agains abbi and mouse IgG we e pu chased om TAGO (Bu lingame, CA). Pu i ied ansducin βγ subuni s we e kindly p o ided by D Y. K. Ho (Uni e si y o Illinois a Chicago, IL) (Ja e e al., 1993). Cy osol p epa a ion Cy osol was p epa ed as desc ibed (Taylo e al., 1992). B ie ly, bo ine b ains we e homogenized a 4°C in 25 mM T is-HCl (pH 8.0), 500 mM KCl, 250 mM suc ose, 1 mM DTT, 1 mM PMSF and cen- i uged a 125,000 g o 90 minu es. Supe na an was ex ensi ely dialysed agains 25 mM Hepes-KOH (pH 7.2), 50 mM KAcO, 1 mM Mg(AcO)2 and hen cen i uga ed a 10,000 g o 20 minu es o emo e p ecipi a es. Aliquo s (15-20 mg p o ein/ml) we e ozen in liquid ni ogen and s o ed a −70°C. Cell cul u e, pe meabiliza ion, and incuba ion condi ions No mal a kidney (NRK) and HeLa cells we e cul u ed in high glucose DME con aining 2 mM glu amine, 50 U/ml penicillin, 50 µg/ml s ep omycin, and 10% FCS. Cells g own on poly-L-lysine- coa ed glass co e slips we e incuba ed on ice o 5 minu es wi h 1 uni /ml SO in 20 mM Hepes-KOH (pH 7.2), 110 mM KOAc, 2 mM Mg(AcO)2, 1 mM DTT. Excess SO was emo ed by washing wi h his bu e a 0°C. Pe meabiliza ion was hen achie ed by incuba ing he cells a 37°C o 5 minu es in anspo bu e (25 mM Hepes- KOH, pH 7.2, 75 mM KOAc, 2.5 mM Mg(AcO)2, 5 mM EGTA, 1.8 mM CaCl2). Cells we e ho oughly insed in his bu e o elease endogenous cy osolic p o eins (i.e. mo e han 50% o he o al lac a e dehyd ogenase ac i i y was usually los ). They we e hen incuba ed a 37°C in anspo bu e con aining bo ine b ain cy osol (0.1-10 mg p o ein/ml) and ATP-gene a ing sys em (1 mM ATP, 5 mM c ea ine phospha e, 0.2 i.u. abbi muscle c ea ine phosphokinase). Immuno luo escence Cells we e ixed o 30 minu es in 3% pa a o maldehyde in phospha e bu e , pH 7.4, and hen incuba ed o 10 minu es wi h 0.05% saponin and 0.5% BSA in PBS. Cells o be p ocessed o β-COP s aining we e pe meabilized wi h 0.2% T i on X-100 plus 0.5% SDS. Incuba ion wi h an ibodies was pe o med a oom empe a u e o 1-2 hou s. Co e slips we e moun ed in 10% PBS/90% glyce ol. Immunoelec on mic oscopy Con en ional elec on mic oscopy was pe o med on 2.5% glu- a aldehyde ixed cells as desc ibed (Hidalgo e al., 1992). Fo immunope oxidase, cells we e ixed in pe ioda e-lysine- pa a o maldehyde ixa i e o 4 hou s and pe meabilized wi h 0.005% saponin in PBS/0.5% BSA. They we e incuba ed o e nigh a 4°C wi h an i-mannosidase II an ibody and 2 hou s a oom empe a u e wi h HRP-conjuga ed seconda y an ibody. Cells we e hen p ocessed as desc ibed (Velasco e al., 1993). J. Hidalgo, M. Muñiz and A. Velasco Fig. 1. Redis ibu ion o Golgi α-mannosidase II in SO- pe meabilized cells incuba ed wi h a high concen a ion o cy osolic p o eins. NRK cells g own on co e slips we e SO-pe meabilized and incuba ed o 1 hou a 37°C in anspo bu e con aining ATP- gene a ing sys em plus cy osol a ei he 0.1 mg/ml (A,A′) o 5 mg/ml (B,B′) p o ein concen a ion. Cells we e ixed and p ocessed o indi ec immuno luo escence wi h an i-mannosidase II an ibodies. A ows in B′indica e he localiza ion o possible ubulo esicula ne wo ks seen in Fig. 5C. Ba , 20 µm. 1807Cy osol-induced Golgi edis ibu ion RESULTS Redis ibu ion o he Golgi complex in o he ER occu s in SO-pe meabilized cells incuba ed wi h a high concen a ion o cy osolic p o eins SO-pe meabilized NRK cells incuba ed wi h anspo bu e con aining 0.05-0.1 mg/ml cy osolic p o eins and ATP-gene - a ing sys em main ained he o ganiza ion o he Golgi complex as iewed by indi ec immuno luo escence. Thus, unde hese condi ions α-mannosidase II, a mid-Golgi esiden in his cell ype (Velasco e al., 1993), was de ec ed in he pe inuclea egion (Fig. 1A,A′). In con as , a e icula s aining pa e n, eminiscen o he ER, was obse ed in cells simila ly incuba ed wi h a high concen a ion (5-10 mg/ml) o cy osolic p o eins (Fig. 1B,B′). We s udied he ime-cou se o his p ocess by pe o ming double immuno luo escence s aining o he cells wi h bo h an i-mannosidase II and an an ibody ec- ognizing he KDEL sequence, he e used as an ER ma ke . Golgi dis up ion began a e 10-15 minu es incuba ion and a e 40 minu es o longe ex ensi e immuno luo escence colo- caliza ion o bo h Golgi and ER an igens occu ed (Fig. 2). This e ec was no obse ed when no mal cy osol was eplaced by hea -inac i a ed cy osol o bo ine se um albumin (no shown). These esul s sugges ed ha cy osolic ac o s induced he usion o Golgi memb anes wi h he ER. Indeed, we de ec ed by elec on mic oscopy mannosidase II immunope oxidase s aining in he ER and nuclea en elope o cells incuba ed wi h 5 mg/ml cy osolic p o eins (Fig. 3). We concluded ha cy osolic ac o s induce Golgi disassembly and usion wi h he ER. Golgi dis up ion induced by cy osolic ac o s in ol es esicle budding A he ul as uc u al le el he Golgi complex in in ac NRK cells was ound o be composed o 4-6 s acked cis e nae and a numbe o associa ed esicles (Fig. 4A). In gene al his o gan- iza ion was main ained in SO-pe meabilized cells incuba ed wi h a low concen a ion (0.1 mg/ml) o cy osolic p o eins (Fig. 4B). In con as , Golgi s uc u e was d ama ically al e ed in pe meabilized cells exposed o 5 mg/ml o cy osolic p o eins. Disassembly seemed o occu by p og essi e esicu- la ion o he Golgi cis e nae (Fig. 5A,B). Thus, signi ican inc ease in he numbe o small, 50-60 nm, esicles and con- comi an dec ease in he numbe o cis e nae was obse ed in he Golgi a ea du ing he i s minu es o incuba ion wi h a high concen a ion o cy osolic p o eins. Many o he esicles p oduced we e coa ed (Fig. 5B, inse ). Con olu ed ubules we e also p esen in he Golgi a ea bu no e idence was ob ained ha hey we e de i ed om Golgi cis e nae; ins ead, connec ions be ween con olu ed ubules and he nuclea en elope o ER cis e nae we e no ed (Fig. 5C). Following 40- Fig. 2. Time-cou se o Golgi α- mannosidase II and galac osyl ans e ase edis ibu ions in o he ER. NRK and HeLa cells g own on co e slips we e SO- pe meabilized and incuba ed a 37°C in comple e incuba ion medium con aining 5 mg/ml cy osolic p o eins and ATP- gene a ing sys em. Co e slips we e ixed a 5, 20 and 40 minu es o incuba ion and p ocessed o immuno luo escence wi h an ibodies agains galac osyl ans e ase in he case o HeLa cells, o double-s ained wi h an ibodies agains α- mannosidase II and he KDEL sequence (ER ma ke ) in he case o NRK cells. Ba , 20 µm. 1808 60 minu es incuba ion he numbe o esicles dec eased as hey supposedly used wi h he ER (Fig. 5C). A his ime ex ensi e ubulo esicula ne wo ks we e also seen. The la e we e em- iniscen o hose obse ed in BFA- ea ed cells (Hidalgo e al., 1992). Simila s uc u es we e no de ec ed in no mal, in ac cells. They ha e been p oposed o ep esen Golgi emnan s in ol ed in Golgi eo ganiza ion du ing eco e y om BFA ea men (De Lemos-Chia andini e al., 1992; Hend icks e al., 1992; Hidalgo e al., 1992). The ubulo esicula ne wo ks s ained weakly wi h an i-mannosidase II by immunope oxidase (no shown). In addi ion, memb ane s uc u es ha migh co - espond o ubulo esicula ne wo ks we e o en seen in he pe - inuclea egion o cells s ained wi h his an ibody by immuno- luo escence (Fig. 1B′). Cy osolic ac o s induce he edis ibu ion o di e en Golgi compa men s in o he ER In addi ion o mannosidase II we also s udied by immuno lu- o escence he edis ibu ion o o he Golgi in eg al p o eins in o he ER. Bo h he ans-Golgi enzyme galac osyl ans e ase (Ro h and Be ge , 1982) (Fig. 2) and he ans-Golgi ne wo k esiden p o ein TGN38 (Luzio e al., 1990) (Fig. 6) showed subs an ial eloca ion a e incuba ion wi h an excess o cy osolic p o eins. Redis ibu ion o hese wo p o eins, howe e , occu ed slowe han ha o mannosidase II and i ne e came o comple ion (Figs 2,6). We also examined he edis ibu ion o he coa p o ein β- COP in SO-pe meabilized NRK cells incuba ed wi h a high concen a ion o cy osolic p o eins. As shown in Fig. 6 β-COP was p ima ily ound associa ed wi h Golgi memb anes and, in addi ion, in punc a e accumula ions dis ibu ed h oughou he cy oplasm. Du ing edis ibu ion o mannosidase II in o he ER β-COP emained associa ed wi h Golgi agmen s. Once man- nosidase II edis ibu ion was comple ed β-COP localized in spo s o di e en sizes. A no ime did we obse e di use, cy osolic s aining o β-COP du ing he diso ganiza ion o he Golgi. J. Hidalgo, M. Muñiz and A. Velasco Fig. 3. Immunope oxidase de ec ion o α-mannosidase II in he cy oplasm o SO-pe meabilized NRK cells incuba ed wi h 5 mg/ml cy osolic p o eins. The ou e memb ane o he nuclea en elope (a owheads) and he ER cis enae we e s ained. Immunope oxidase eac ion p oduc was mainly localized a he ER memb ane (a ows) al hough i also illed he lumen o some cis e nae (as e isk). Ba , 0.2 µm. Fig. 4. Golgi complex o ganiza ion in in ac and SO-pe meabilized NRK cells. (A) Golgi complex in a no mal, in ac NRK cell. (B) S uc u e o he Golgi complex a e cell pe meabiliza ion and incuba ion o 1 hou a 37°C wi h 0. 1 mg/ml cy osolic p o eins and ATP-gene a ing sys em. Ba , 0.2 µm. 1809Cy osol-induced Golgi edis ibu ion Fig. 5. Ul as uc u al isualiza ion o Golgi disassembly induced by cy osolic p o eins. SO-pe meabilized NRK cells we e incuba ed a 37°C in comple e incuba ion medium con aining 5 mg/ml cy osolic p o eins and ATP-gene a ing sys em o 5 (A), 20 (B), and 60 minu es (C) be o e ixa ion and p ocessing o elec on mic oscopy. (A,B) Non-cla h in coa ed esicles (a owheads) a e o igina ed om Golgi cis e nae du ing he ini ial disassembly o he Golgi complex. (C) A e Golgi edis ibu ion o he ER is comple ed ex ensi e ubulo esicula ne wo ks (NT) pe sis ed in he pe inuclea egion. A ows indica e connec ions be ween con olu ed ubules and ER elemen s. Ba , 0.2 µm. 1810 We he e o e concluded ha Golgi edis ibu ion induced by cy osolic ac o s a ec s di e en Golgi compa men s and i occu s wi hou p e ious β-COP dissocia ion om Golgi memb anes. Requi emen s o Golgi edis ibu ion induced by cy osol Golgi edis ibu ion in o he ER equi ed ATP as i did no occu when ATP was omi ed om he incuba ion medium (Fig. 7A). I was also inhibi ed a low empe a u e. A ei he 15°C (Fig. 7B) o 20°C (no shown) he Golgi complex appea ed pa ially agmen ed as judged by he immuno luo- escence de ec ion o mannosidase II; his p o ein, howe e , ailed o edis ibu e in o he ER a hese empe a u es. Golgi edis ibu ion did no equi e in ac mic o ubules. This was shown in cells which we e ea ed wi h nocodazole p io o pe meabiliza ion and incuba ion wi h cy osol. Mic o ubule J. Hidalgo, M. Muñiz and A. Velasco Fig. 6. Redis ibu ion o β-COP and TGN38. SO-pe meabilized NRK cells we e incuba ed wi h 5 mg/ml cy osolic p o eins and ATP- gene a ing sys em. A 15, 30, and 60 minu es he cells we e ixed and double-s ained wi h an ibodies agains β-COP and α-mannosidase II o , al e na i ely, single-s ained wi h an an ibody agains TGN38. Ba , 20 µm. Fig. 7. Requi emen s o Golgi edis ibu ion induced by cy osolic p o eins. NRK cells we e pe meabilized and incuba ed wi h 5 mg/ml cy osolic p o eins as indica ed in Fig. 1 excep ha ei he ATP was omi ed om he incuba ion medium o incuba ion was pe o med a 15°C. Cells we e also ea ed a 37°C o 1 hou wi h 5 µM nocodazole be o e pe meabiliza ion wi h SO (NCDZ 0h). In addi ion, nocodazole- ea ed, SO-pe meabilized cells we e incuba ed a 37°C o 1 hou wi h ATP-gene a ing sys em and 5 mg/ml cy osolic p o eins in he p esence o nocodazole (NCDZ 1h). The localiza ion o α-mannosidase II is shown. Ba , 20 µm. 1811Cy osol-induced Golgi edis ibu ion depolyme iza ion by nocodazole is known o induce Golgi agmen a ion in o indi idual s acks (Tu ne and Ta ako , 1989). Thus, when nocodazole ea ed cells we e pe meabi- lized and p ocessed o indi ec immuno luo escence wi h an i- mannosidase II an ibody he Golgi complex appea ed ag- men ed in o spo s dis ibu ed h ough ou he cy oplasm (Fig. 7C). S aining o hese cells wi h an i- ubulin an ibody showed ha mic o ubules we e indeed depolyme ized (no shown). Nocodazole- ea ed cells we e also pe meabilized and incuba ed wi h a high concen a ion o cy osolic p o eins and in he con inuous p esence o nocodazole o p e en mic o- ubule eassembly. In hese cells mannosidase II s aining showed an ER-like e icula pa e n (Fig. 7D). This esul indica ed ha Golgi edis ibu ion in o he ER induced by cy osolic ac o s did no depend upon an in ac mic o ubule sys em o i o ake place. Inhibi ion by GTPγS and NEM The abo e ul as uc u al esul s p o ided e idence ha edis- ibu ion o Golgi enzymes in o he ER induced by cy osolic ac o s migh be esicle-media ed. Bo h GTPγS and NEM a e po en inhibi o s o he esicula an e og ade anspo (Balch e al., 1984; Melançon e al., 1987). We de e mined by immuno luo escence he e ec s o hese wo agen s on Golgi edis ibu ion (Fig. 8). Pe meabilized cells we e incuba ed wi h a high concen a ion o cy osolic p o eins. Ei he GTPγS o NEM was added a di e en ime poin s and incuba ion con inued o 1 hou in he p esence o each agen . GTPγS comple ely blocked Golgi edis ibu ion. Howe e , inhibi ion was only e ec i e i GTPγS was added a he begining (3-5 minu es) o incuba ion. Addi ion o GTPγS o he incuba ion medium a e his ime did no inhibi he p ocess, sugges ing ha GTP-binding p o eins a e equi ed a he ime o Golgi b eakdown. In con as , NEM was inhibi o y only i added du ing he i s 15-20 minu es o incuba ion, a e which he p ocess became insensi i e o his agen (Fig. 8). The e o e, we concluded ha bo h GTPγS and NEM inhibi di e en s eps o he Golgi edis ibu ion p ocess. We exploi ed hese di e ences in o de o cha ac e ize in e - media es in he ou e by elec on mic oscopy. In pe meabilized cells exposed o 1 hou o 5 mg/ml cy osolic p o eins and GTPγS we de ec ed he p esence o coa ed esicles a ached o in ac Golgi cis e nae (Fig. 9A). We ied o assess he a e o hese coa ed esicles. Following 10 minu es o incuba ion wi h cy osolic p o eins and no inhibi o , NEM was added and incu- ba ion con inued o 1 hou . This allowed o he budding p ocess o occu du ing he p eincuba ion pe iod gi ing ise o disappea ance o he Golgi complex (Fig. 9B). No ewo hy, ins ead o coa ed esicles di e en kinds o uncoa ed esicles we e seen in hese cells (Fig. 9B). We concluded ha he edis- Fig. 8. Time-cou se o GTPγS and NEM inhibi ions on Golgi edis ibu ion induced by cy osolic p o eins. SO-pe meabilized NRK cells we e incuba ed wi h 5 mg/ml cy osolic p o eins and ATP-gene a ing sys em a 37°C. Ei he 100 µM GTPγS o 1 mM NEM was added a 2, 6, 15, and 25 minu es and he incuba ion con inued o 1 hou . Cells we e hen ixed and p ocessed o immuno luo escence wi h an i-α-mannosidase II an ibody. Ba , 20 µm. 1812 ibu ion o he Golgi complex in o he ER induced by cy osolic ac o s is media ed by coa ed esicles ha bud om Golgi cis e nae and hen lose hei coa s be o e usion wi h he ER. T ime ic G p o eins a e in ol ed in Golgi disassembly Inhibi ion by GTPγS was indica i e o he in ol emen o GTP-binding p o eins in an ea ly s ep o he cy osol-induced Golgi edis ibu ion in o he ER. GTPγS ac i a es bo h monome ic and ime ic GTP-binding p o eins whe eas only ime ic G p o eins a e ac i a ed by AlF(3-5) (Kahn, 1991). We hen es ed he e ec o adding AlF(3-5) o ou assay. As shown in Fig. 10B AlF(3-5) inhibi ed mannosidase II edis ibu ion in o he ER. Fu he suppo o he in ol emen o ime ic G p o eins in he p ocess was ob ained by adding ansducin βγ subuni s (Fig. 10C) and mas opa an (no shown), bo h o which sepa a ely inhibi ed Golgi edis ibu ion. Addi ion o βγ subuni s should gi e ise o inac i a ion o G p o eins as he βγ subuni s complex wi h he ee α subuni s (Bomsel and Mos o , 1992). In con as , mas opa an, which mimics an ac i a ed ecep o , should ende he opposi e e ec , i.e. ac i a ion o G p o eins (Higashijima e al., 1990; Wein- ga en e al., 1990). Inhibi ion by bo h agen s can be explained i Golgi edis ibu ion in o he ER would be con olled by se e al G p o eins exe ing opposi e egula o y oles. To es his hypo hesis we examined he e ec s esul ing om incu- ba ing he cells wi h bac e ial oxins known o ca y ou ADP- ibosyla ion o pa icula Gα subuni s and hence a ec hei ac i i y (Gilman, 1987). P e ea men o cells wi h ei he chole a oxin o pe ussis oxin s imula ed Golgi edis ibu ion. Thus, in bo h cases Golgi disassembly and edis ibu ion in o he ER occu ed a a low concen a ion o cy osolic p o eins (Fig. 10D-F). This was indica i e o he in ol emen o bo h αsand αiG p o eins, he o me being i e e sibly ac i a ed by chole a oxin ea men and αi being inhibi ed by pe ussis oxin. The eme ging idea was ha Golgi edis ibu ion in o he ER was s imula ed by ei he αsac i a ion o αi inhibi ion. DISCUSSION In his pape we ha e desc ibed he edis ibu ion o Golgi componen s in o he ER o igina ed by he incuba ion o SO- pe meabilized cells wi h an excess o cy osolic p o eins. A simila phenomenon occu s in BFA- ea ed cells ha also unde go Golgi disassembly and usion wi h he ER (Klausne e al., 1992; Lippinco -Schwa z e al., 1989). Howe e , impo an di e ences be ween bo h p ocesses we e no ed. Fi s , we did no obse e by immuno luo escence ubule p ocesses eme ging om he Golgi cis e nae du ing hei edis- ibu ion in o he ER. Ins ead, ou ul as uc u al obse a ions indica e ha he cy osol-induced Golgi edis ibu ion occu s by budding o coa ed esicles om he Golgi cis e nae. In J. Hidalgo, M. Muñiz and A. Velasco Fig. 9. Cha ac e iza ion o esicula in e media es in he cy osol-induced Golgi-ER e og ade pa hway. (A) Cells we e SO-pe meabilized and incuba ed o 1 hou a 37°C wi h 5 mg/ml cy osolic p o eins, ATP-gene a ing sys em, and 100 µM GTPγS. (B) Following a 10 minu e incuba ion wi h 5 mg/ml cy osolic p o eins, ATP-gene a ing sys em, and no inhibi o , 1 mM NEM was added and incuba ion con inued o 1 hou . Cells we e ixed and p ocessed o elec on mic oscopy. A owheads indica e he p esence o uncoa ed esicles. Ba , 0.2 µm. 1813Cy osol-induced Golgi edis ibu ion addi ion, he mechanism o Golgi dis up ion induced by cy osolic ac o s is di e en om ha ac i a ed by BFA since i appa en ly occu s wi hou p e ious β-COP dissocia ion om Golgi memb anes. Fu he mo e, in he case o pe meabilized cells incuba ed wi h a high concen a ion o cy osolic p o eins in ac mic o ubules a e no equi ed o Golgi edis ibu ion in o he ER. Finally, unlike he BFA e ec , cy osol-induced Golgi edis ibu ion does no seem o be a consequence o inhi- bi ion o he ER-Golgi an e og ade anspo . Ins ead, ou esul s sugges he p esence in he cells o cy osolic ac o s ha may ac i ely p omo e he Golgi-ER e og ade pa hway by a s ill unde e mined mechanism. Role o cy osolic ac o s in p omo ing Golgi edis ibu ion Cy osol-induced Golgi edis ibu ion in o he ER may be he esul de i ed om an exagge a ion o he Golgi-ER e og ade pa hway aking place in i o du ing e ie al om he Golgi complex o ei he ER esiden molecules (Pelham, 1988) o mis olded p o eins (Hammond and Helenius, 1994). Al e na- i ely, his edis ibu ion p ocess may be un ela ed wi h he no mal Golgi-ER e og ade pa hway. Howe e , his possibil- i y seems unlikely since he agmen ed Golgi memb anes speci ically use wi h he ER and mo phological changes in o he o ganelles we e no obse ed du ing incuba ion o SO- pe meabilized cells wi h cy osolic p o eins. The Golgi-ER e og ade pa hway has been shown o be egula ed by bo h he amoun o ER p o eins o be ecycled om he Golgi and he numbe o speci ic ecep o s a ailable o hem. Fo ins ance, a BFA-like e ec has been desc ibed in cells o e exp essing ELP-1, a pu a i e ecep o o lumenal ER p o eins bea ing he KDEL sequence (Hsu e al., 1992). In addi ion, a s iking edis ibu ion o he KDEL ecep o om he Golgi egion o he ER occu s in cells o e exp essing p o eins wi h his e en ion sequence (Lewis and Pelham, 1992). The conclusion de i ed om hese s udies is ha he e og ade pa hway is somehow ac i a ed by he o ma ion o ligand- ecep o complexes in he Golgi. In his ega d, in ol e- men o cy osolic ac o s could in oduce addi ional con ols in he pa hway. Fo ins ance, by in luencing he ac i i y o pa - icula ime ic G p o eins cy osolic ac o s migh modula e he associa ion o coa p o eins wi h memb anes. This, in u n, could be esponsible o main aining a con olled balance be ween an e og ade and e og ade memb ane ou es. I has Fig. 10. In ol emen o ime ic G p o eins in Golgi disassembly. NRK cells we e ea ed o no wi h 1 µg/ml chole a oxin (CT), 0.5 µg/ml pe ussis oxin (PT) o 16 hou s a 37°C. Following SO pe meabiliza ion he cells we e incuba ed a 37°C o 1 hou in comple e incuba ion medium con aining low (0.1 mg/ml) o high (5 mg/ml) concen a ion o cy osolic p o eins plus ATP- gene a ing sys em. E ec s esul ing om adding ei he 50 µM AlCl3plus 30 mM NaF (B) o 5 µM o ansducin βγ subuni s (C) on Golgi edis ibu ion a e shown. Cells we e ixed and s ained o immuno luo escence wi h an i-α-mannosidase II an ibody. Ba , 20 µm.