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.