INTRO

Duringcompleinput aendoploutput vesicle

of a Gopincottthere is great interest in the retrograde pathway because it isassumed to fulfill the role of returning escaped ER residentproteins, which eventually reach the Golgi complex (Lewis andPelham, 1992; Pelham, 1988) as well as misfolded proteins thatare not suitable to proceed along the exocytic pathway(Hammond and Helenius, 1994). On the basis of observationsmade on cells treated with the fungal metabolite brefeldin A(BFA), the retrograde pathway has been described to occur bythe microtubule-dependent extension of tubule processeswhich emanate from the Golgi cisternae and fuse with the ER(Lippincott-Schwartz et al., 1990). By contrast, anterogrademembrane transport is thought to be mediated by carriervesicles which transport both proteins and lipids from the ERto the cis-Golgi and between the different Golgi compartments(Rothman and Orci, 1992; Takizawa and Malhotra, 1993).Conditions that alter the normal balance existing betweenanterograde and retrograde membrane pathways lead to dis-ruption of the Golgi organization. Thus, treatment with BFA

i aonut o

i r-ri19x

et r

process that influences both Golgi structural maintenance andfunction.

The mechanism underlying anterograde transfer betweenmembrane compartments of the exocytic pathway has beenextensively investigated using both biochemical and geneticapproaches (Rothman and Orci, 1992). By contrast, littleattention has been given to the factors that regulate retrogradetransport. Studies using BFA have indicated that redistributionof Golgi membranes to the ER is inhibited by both the non-hydrolyzable analog of GTP, GTP

γS, and the activator oftrimeric G proteins, AlF(3-5), suggesting that GTP-bindingproteins might be involved (Donaldson et al., 1991b; Tan etal., 1992). However, both GTPγS and AlF(3-5) primarilyinterfere with the association/disassociation cycle of the coatproteins to Golgi membranes and therefore they also inhibitanterograde transport as well (Donaldson et al., 1991a;Melançon et al., 1987). Part of the difficulty in understandingthe basic mechanism of retrograde transport is due to the lim-

Streptconcengeneramic reII, galfluoresER ofproteinimmunATP amerizaGolgi This s

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*Author f

Journal oPrinted in

nterograde transport but not the retro-sequently the Golgi complex is disas-es into the ER (Klausner et al., 1992).bserved in two other situations: over-eceptor for ER lumenal proteins (Hsubosylation of rho, a small GTP-binding92a,b). In addition, cell mutants have

hibit a BFA-like phenotype (Kao andal., 1991). Taken together these studiesetrograde transport is a highly regulated

1805

ts, and mastoparan, indicating thec G proteins. At a later stage, vesiclesfuse with the ER. This part of ther in cells incubated at either 15°C orethylmaleimide. In cells treated withussis toxin Golgi redistribution intoold lower requirement for cytosolicted cells. These data suggest a regu-s and αi trimeric G proteins in therograde transport taking place in

x, retrograde transport, G protein

ribution of Golgi

/n, 41012-Seville, Spain

olysin O-permeabilized cells incubated with a high transducin βγ subu

ARY

eric G proteins regulate the cytosol-induced redi

mes into the endoplasmic reticulum

na Hidalgo, Manuel Muñiz and Angel Velasco*

ment of Cell Biology, Faculty of Biology, University of Seville, Avd. Reina Mercede

or correspondence

f Cell Science 108, 1805-1815 (1995) Great Britain © The Company of Biologists Limited 1995

DUCTION

interphase the cisternal organization of the Golgix is maintained by a controlled balance of membranend output. The input pathway consists of anterogradeasmic reticulum (ER)-Golgi membrane transport. Thedepends on both the production of sorting and exocytics from the trans-Golgi network (TGN) and the existencelgi-ER retrograde pathway (Klausner et al., 1992; Lip-

-Schwartz, 1993; Rothman and Orci, 1992). Currently

inhibits the ER-Golggrade pathway and csembled and redistribThese effects are alsoexpression of a Golget al., 1992) and ADPprotein (Sugai et al., been described that eDraper, 1992; Zuber suggest that Golgi-ER

tration (5-10 mg/ml) of cytosolic proteins and ATP-ting system exhibit redistribution into the endoplas-ticulum (ER) of Golgi integral proteins (mannosidaseactosyltransferase, TGN 38), detected by immuno-cence. In addition, mannosidase II is detected in the cells exposed to a high concentration of cytosolics and processed for immunolectron microscopy byoperoxidase. The redistribution process requiresnd is not affected by previous microtubule depoly-tion. Ultrastructural observations indicate thatdisassembly occurs by budding of coated vesicles.tage of the process is inhibited by GTPγS, AlF(3-5),

involvement of trimelose their coats andprocess does not occ20°C, or exposed to either cholera or pethe ER shows a 50factors than in untrlatory role for bothnormal Golgi-ER rintact cells.

Key words: Golgi comp

1806

itations itubule pr(Lippinc1992; WGolgi-ERmediatedGolgi ciabsence supply (Cthese tubin vivo relevancesembly oStreptolyconcentrrequires regulated

MATER

MaterialsTissue cuburg, M(Beckenh3,3′-diamof the ATLouis, MOland). BacCA). AntsyltransfeFarquhar versité deBiology LZürich, Srespectivesecondaryfrom TAGkindly proIL) (Jaffe

Cytosol Cytosol wbovine bra500 mM Ktrifuged adialysed aMg(AcO)remove pliquid nitr

Cell cultNormal rglucose Dµg/ml strcoated glaunit/ml SOMg(AcO)this buffethe cells KOH, pHmM CaCendogenodehydroge

J

mposed by the use of BFA, a drug that also inducesocesses from the TGN, endosomes, and lysosomes

ott-Schwartz et al., 1991; Tooze and Hollinshead,ood et al., 1991). Thus there is no direct evidence that retrograde transport in the absence of BFA is tubule- (Pelham, 1991). Recently tubule formation fromsternae has been reported to occur in vitro in theof BFA and under conditions of low ATP or cytosol

luett et al., 1993; Weidman et al., 1993). Althoughule processes have been assimilated to those formed

during retrograde transport their physiological is uncertain. In the present study we show disas-f the Golgi complex and redistribution into the ER insin O (SO)-permeabilized cells incubated with a highation (5-10 mg/ml) of cytosolic proteins. This processATP, is independent of tubule formation, and is by trimeric G proteins.

. Hidalgo, M. Muñiz and A. Velasco

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at 37°C in transport buffer containing bovine brain cytosol (0.1-10 mg

Fig. 1. Redistribution of Golgi α-mannosidase II in SO-permeabilized cells incubated with a high concentration of cytosolicproteins. NRK cells grown on coverslips were SO-permeabilized andincubated for 1 hour at 37°C in transport buffer containing ATP-generating system plus cytosol at either 0.1 mg/ml (A,A′) or 5 mg/ml(B,B′) protein concentration. Cells were fixed and processed forindirect immunofluorescence with anti-mannosidase II antibodies.Arrows in B′ indicate the localization of possible tubulovesicularnetworks seen in Fig. 5C. Bar, 20 µm.

ALS AND METHODS

ture reagents were obtained from GIBCO BRL (Gaithers-). SO was purchased from Wellcome Diagnostics

m, UK). N-ethylmaleimide (NEM), GTPγS, nocodazole,nobenzidine tetrahydrochloride, saponin, and componentsP-generating system were from Sigma Chemical Co (St). Mastoparan was from Fluka Chemika (Buchs, Switzer-

terial toxins were purchased from Calbiochem (San Diego,bodies against α-mannosidase II, β-COP, KDEL, galacto-ase, and TGN38 were kindly provided by Drs M. G.University of California San Diego, CA), T. Kreis (Uni- Genève, Switzerland), S. Fuller (European Molecularaboratory, Heidelberg, Germany), E. Berger (Universitätitzerland), and G. Banting (University of Bristol, UK),

ly. FITC-, TRITC-, and peroxidase-conjugated goat antibodies against rabbit and mouse IgG were purchasedO (Burlingame, CA). Purified transducin βγ subunits werevided by Dr Y. K. Ho (University of Illinois at Chicago,et al., 1993).

reparationas prepared as described (Taylor et al., 1992). Briefly,

ins were homogenized at 4°C in 25 mM Tris-HCl (pH 8.0),Cl, 250 mM sucrose, 1 mM DTT, 1 mM PMSF and cen-

t 125,000 g for 90 minutes. Supernatant was extensivelygainst 25 mM Hepes-KOH (pH 7.2), 50 mM KAcO, 1 mM and then centrifugated at 10,000 g for 20 minutes toecipitates. Aliquots (15-20 mg protein/ml) were frozen inogen and stored at −70°C.

re, permeabilization, and incubation conditionst kidney (NRK) and HeLa cells were cultured in highME containing 2 mM glutamine, 50 U/ml penicillin, 50ptomycin, and 10% FCS. Cells grown on poly-L-lysine-ss coverslips were incubated on ice for 5 minutes with 1 in 20 mM Hepes-KOH (pH 7.2), 110 mM KOAc, 2 mM, 1 mM DTT. Excess SO was removed by washing with at 0°C. Permeabilization was then achieved by incubatingt 37°C for 5 minutes in transport buffer (25 mM Hepes-7.2, 75 mM KOAc, 2.5 mM Mg(AcO)2, 5 mM EGTA, 1.82). Cells were thoroughly rinsed in this buffer to releases cytosolic proteins (i.e. more than 50% of the total lactatenase activity was usually lost). They were then incubated

protein/ml) and ATP-generating system (1 mM ATP, 5 mM creatinephosphate, 0.2 i.u. rabbit muscle creatine phosphokinase).

ImmunofluorescenceCells were fixed for 30 minutes in 3% paraformaldehyde in phosphatebuffer, pH 7.4, and then incubated for 10 minutes with 0.05% saponinand 0.5% BSA in PBS. Cells to be processed for β-COP staining werepermeabilized with 0.2% Triton X-100 plus 0.5% SDS. Incubationwith antibodies was performed at room temperature for 1-2 hours.Coverslips were mounted in 10% PBS/90% glycerol.

Immunoelectron microscopyConventional electron microscopy was performed on 2.5% glu-taraldehyde fixed cells as described (Hidalgo et al., 1992). Forimmunoperoxidase, cells were fixed in periodate-lysine-paraformaldehyde fixative for 4 hours and permeabilized with 0.005%saponin in PBS/0.5% BSA. They were incubated overnight at 4°Cwith anti-mannosidase II antibody and 2 hours at room temperaturewith HRP-conjugated secondary antibody. Cells were then processedas described (Velasco et al., 1993).

Cytosol-induced G

RESULTS

Redistribution of the Golgi complex into the ERoccurs in SO-permeabilized cells incubated with ahigh concentration of cytosolic proteinsSO-permeabilized NRK cells incubated with transport buffercontaining 0.05-0.1 mg/ml cytosolic proteins and ATP-gener-ating system maintained the organization of the Golgi complexas viewed by indirect immunofluorescence. Thus, under theseconditions α-mannosidase II, a mid-Golgi resident in this celltype (Velasco et al., 1993), was detected in the perinuclearregion (Fig. 1A,A′). In contrast, a reticular staining pattern,reminiscent of the ER, was observed in cells similarlyincubated with a high concentation (5-10 mg/ml) of cytosolicproteins (Fig. 1B,B′). We studied the time-course of thisprocess by performing double immunofluorescence staining ofthe cells with both anti-mannosidase II and an antibody rec-ognizing the KDEL sequence, here used as an ER marker.Golgi disruption began after 10-15 minutes incubation andafter 40 minutes or longer extensive immunofluorescence colo-calization of both Golgi and ER antigens occurred (Fig. 2).This effect was not observed when normal cytosol wasreplaced by heat-inactivated cytosol or bovine serum albumin(not shown).

These results suggested that cytosolic factors induced thefusion of Golgi membranes with the ER. Indeed, we detected

by electron microscopy mannosidstaining in the ER and nuclear envel5 mg/ml cytosolic proteins (Figcytosolic factors induce Golgi disasER.

Golgi disruption induced by cyinvolves vesicle buddingAt the ultrastructural level the Golcells was found to be composed of number of associated vesicles (Fig. ization was maintained in SO-permwith a low concentration (0.1 mg(Fig. 4B). In contrast, Golgi structuin permeabilized cells exposed tproteins. Disassembly seemed to oclation of the Golgi cisternae (Figincrease in the number of small, 50comitant decrease in the number ofthe Golgi area during the first minuteconcentration of cytosolic proteinproduced were coated (Fig. 5B, inwere also present in the Golgi aobtained that they were derived froconnections between convoluted envelope or ER cisternae were note

Figmagalredandcovper37°mecytgenwerminproimmantgalof Hwitmaseqcas

1807olgi redistribution

ase II immunoperoxidaseope of cells incubated with. 3). We concluded thatsembly and fusion with the

tosolic factors

gi complex in intact NRK4-6 stacked cisternae and a4A). In general this organ-eabilized cells incubated

/ml) of cytosolic proteinsre was dramatically alteredo 5 mg/ml of cytosoliccur by progressive vesicu-. 5A,B). Thus, significant-60 nm, vesicles and con-

m

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cisternae was observed ins of incubation with a highs. Many of the vesiclessert). Convoluted tubules

rea but no evidence was Golgi cisternae; instead,

tubules and the nucleard (Fig. 5C). Following 40-

. 2. Time-course of Golgi α-nosidase II and

ctosyltransferasestributions into the ER. NRK HeLa cells grown onerslips were SO-

eabilized and incubated atC in complete incubationium containing 5 mg/mlsolic proteins and ATP-

erating system. Coverslipse fixed at 5, 20 and 40utes of incubation andcessed for

unofluorescence withbodies againstctosyltransferase in the caseeLa cells, or double-stained antibodies against α-nosidase II and the KDEL

uence (ER marker) in the of NRK cells. Bar, 20 µm.

1808

60 minutsupposedtubuloveiniscent 1992). Scells. Thinvolvedtreatmen1992; Hstained w(not showrespond inuclear fluoresce

CytosodifferenIn additiorescencinto the E(Roth anresident substanticytosolichowevernever ca

We alCOP in concentrwas primaddition,cytoplasβ-COP rnosidasespots of

J

peroxidase detectionse II in the cytoplasmilized NRK cells5 mg/ml cytosolic

. Hidalgo, M. Muñiz and A. Velasco

Fig. 3. Immunoof α-mannosidaof SO-permeabincubated with

es incubation the number of vesicles decreased as theyly fused with the ER (Fig. 5C). At this time extensivesicular networks were also seen. The latter were rem-of those observed in BFA-treated cells (Hidalgo et al.,imilar structures were not detected in normal, intactey have been proposed to represent Golgi remnants in Golgi reorganization during recovery from BFAt (De Lemos-Chiarandini et al., 1992; Hendricks et al.,idalgo et al., 1992). The tubulovesicular networkseakly with anti-mannosidase II by immunoperoxidasen). In addition, membrane structures that might cor-

to tubulovesicular networks were often seen in the per-region of cells stained with this antibody by immuno-nce (Fig. 1B′).

lic factors induce the redistribution oft Golgi compartments into the ERon to mannosidase II we also studied by immunoflu-e the redistribution of other Golgi integral proteinsR. Both the trans-Golgi enzyme galactosyltransferase

d Berger, 1982) (Fig. 2) and the trans-Golgi networkprotein TGN38 (Luzio et al., 1990) (Fig. 6) showedal relocation after incubation with an excess of proteins. Redistribution of these two proteins,, occurred slower than that of mannosidase II and itme to completion (Figs 2,6). so examined the redistribution of the coat protein β-SO-permeabilized NRK cells incubated with a highation of cytosolic proteins. As shown in Fig. 6 β-COParily found associated with Golgi membranes and, in in punctate accumulations distributed throughout them. During redistribution of mannosidase II into the ERemained associated with Golgi fragments. Once man- II redistribution was completed β-COP localized indifferent sizes. At no time did we observe diffuse,

cytosolic staining for β-COP du isorganization of theGolgi.

prot uter membrane of thenucl e (arrowheads) andthe E were stained.Imm ase reaction productwas alized at the ERmem ws) although it alsofille of some cisternae(aste 0.2 µm.

Fig. 4. Golgi complex organizationNRK cells. (A) Golgi complex in a(B) Structure of the Golgi complexincubation for 1 hour at 37°C with ATP-generating system. Bar, 0.2 µ

ring the d

eins. The oear envelopR cistenaeunoperoxid

mainly locbrane (arro

d the lumenrisk). Bar,

in intact and SO-permeabilized normal, intact NRK cell. after cell permeabilization and0. 1 mg/ml cytosolic proteins andm.

1809Cytosol-induced Golgi redistribution

Fig. 5. Ultrastructural visualization of Golgi disassembly induced by cytosolic proteins. SO-permeabilized NRK cells were incubated at 37°Cin complete incubation medium containing 5 mg/ml cytosolic proteins and ATP-generating system for 5 (A), 20 (B), and 60 minutes (C) beforefixation and processing for electron microscopy. (A,B) Non-clathrin coated vesicles (arrowheads) are originated from Golgi cisternae duringthe initial disassembly of the Golgi complex. (C) After Golgi redistribution to the ER is completed extensive tubulovesicular networks (NT)persisted in the perinuclear region. Arrows indicate connections between convoluted tubules and ER elements. Bar, 0.2 µm.

1810

We therefore concluded that Golgi rcytosolic factors affects different Goloccurs without previous β-COP dimembranes.

Requirements for Golgi redistribcytosolGolgi redistribution into the ER requoccur when ATP was omitted from (Fig. 7A). It was also inhibited at low15°C (Fig. 7B) or 20°C (not showappeared partially fragmented as judgrescence detection of mannosidase IIfailed to redistribute into the ER at th

Golgi redistribution did not require iwas shown in cells which were treateto permeabilization and incubation w

J. Hidalgo, M. Muñiz and A.

Fig. 7. Requirements for Golgi redistributiproteins. NRK cells were permeabilized ancytosolic proteins as indicated in Fig. 1 excomitted from the incubation medium or inc15°C. Cells were also treated at 37°C for 1nocodazole before permeabilization with Saddition, nocodazole-treated, SO-permeabat 37°C for 1 hour with ATP-generating sycytosolic proteins in the presence of nocodlocalization of α-mannosidase II is shown.

Velasco

edistribution induced bygi compartments and itssociation from Golgi

ution induced by

ired ATP as it did notthe incubation medium temperature. At eithern) the Golgi complexed by the immunofluo-; this protein, however,ese temperatures. ntact microtubules. Thisd with nocodazole priorith cytosol. Microtubule

Fig. 6. Redistribution of β-COP andTGN38. SO-permeabilized NRKcells were incubated with 5 mg/mlcytosolic proteins and ATP-generating system. At 15, 30, and60 minutes the cells were fixed anddouble-stained with antibodiesagainst β-COP and α-mannosidaseII or, alternatively, single-stainedwith an antibody against TGN38.Bar, 20 µm.

on induced by cytosolicd incubated with 5 mg/mlept that either ATP wasubation was performed at hour with 5 µMO (NCDZ 0h). Inilized cells were incubatedstem and 5 mg/mlazole (NCDZ 1h). The Bar, 20 µm.

Cytosol-induced Golg

depolymerization by nocodazole is known to induce Golgifragmentation into individual stacks (Turner and Tartakoff,1989). Thus, when nocodazole treated cells were permeabi-lized and processed for indirect immunofluorescence with anti-mannosidase II antibody the Golgi complex appeared frag-mented into spots distributed throughtout the cytoplasm (Fig.7C). Staining of these cells with anti-tubulin antibody showedthat microtubules were indeed depolymerized (not shown).Nocodazole-treated cells were also permeabilized andincubated with a high concentration of cytosolic proteins andin the continuous presence of nocodazole to prevent micro-tubule reassembly. In these cells mannosidase II stainingshowed an ER-like reticular pattern (Fig. 7D). This resultindicated that Golgi redistribution into the ER induced bycytosolic factors did not depend upon an intact microtubulesystem for it to take place.

Inhibition by GTPγS and NEMThe above ultrastructural results provided evidence that redis-tribution of Golgi enzymes into the ER induced by cytosolicfactors might be vesicle-mediated. Both GTPγS and NEM arepotent inhibitors of the vesicular anterograde transport (Balchet al., 1984; Melançon et al., 1987). We determined byimmunofluorescence the effects of these two agents on Golgiredistribution (Fig. 8). Permeabilized cells were incubated with

a high concentration of cytosolic protNEM was added at different time continued for 1 hour in the presence completely blocked Golgi redistributiowas only effective if GTPγS was addeminutes) of incubation. Addition of Gmedium after this time did not inhibit that GTP-binding proteins are requirebreakdown. In contrast, NEM was induring the first 15-20 minutes of incuprocess became insensitive to this agentconcluded that both GTPγS and NEM ithe Golgi redistribution process.

We exploited these differences in ordmediates in the route by electron microscells exposed for 1 hour to 5 mg/ml GTPγS we detected the presence of coaintact Golgi cisternae (Fig. 9A). We trthese coated vesicles. Following 10 micytosolic proteins and no inhibitor, NEbation continued for 1 hour. This aprocess to occur during the preincubatdisappearance of the Golgi complex instead of coated vesicles different kiwere seen in these cells (Fig. 9B). We

Fig. 8. Time-course of GTPγS and NEM inhibitions on Golgi redistribution induced by cytosolic proteins. SO-permeaincubated with 5 mg/ml cytosolic proteins and ATP-generating system at 37°C. Either 100 µM GTPγS or 1 mM NEMand 25 minutes and the incubation continued for 1 hour. Cells were then fixed and processed for immunofluorescence II antibody. Bar, 20 µm.

1811i redistribution

eins. Either GTPγS orpoints and incubationof each agent. GTPγSn. However, inhibitiond at the begining (3-5

TPγS to the incubationthe process, suggestingd at the time of Golgihibitory only if addedbation, after which the (Fig. 8). Therefore, wenhibit different steps of

er to characterize inter-copy. In permeabilizedcytosolic proteins andted vesicles attached toied to assess the fate of

nutes of incubation withM was added and incu-

llowed for the buddingion period giving rise to(Fig. 9B). Noteworthy,

nds of uncoated vesiclesconcluded that the redis-

bilized NRK cells were was added at 2, 6, 15,with anti-α-mannosidase

1812

tributioncytosolicGolgi ciER.

TrimeridisasseInhibitioGTP-binGolgi rmonometrimeric then testin Fig. 1the ER.proteinssubunitsseparate

Additproteins(Bomselmimics ai.e. activgarten etif Golgiseveral Gthis hyp

J

Fig. 9. Chincubatedincubatiohour. Cel

. Hidalgo, M. Muñiz and A. Velasco

s

e

0

li

o

bacterial toxins known to carry out ADP-icular Gα subunits and hence affect their987). Pretreatment of cells with eitherussis toxin stimulated Golgi redistribution.Golgi disassembly and redistribution intoa low concentration of cytosolic proteins

was indicative of the involvement of boths, the former being irreversibly activated

thway. (A) Cells were SO-permeabilized andM GTPγS. (B) Following a 10 minute

was added and incubation continued for 1of uncoated vesicles. Bar, 0.2 µm.

of the Golgi complex into the ER induced by factors is mediated by coated vesicles that bud fromternae and then lose their coats before fusion with the

c G proteins are involved in Golgimblyn by GTPγS was indicative of the involvement of

bating the cells withribosylation of partactivity (Gilman, 1cholera toxin or pertThus, in both cases the ER occurred at (Fig. 10D-F). This αs and αi G protein

aracterization of vesicular intermediates in the cytosol-induced Golgi-ER retrograde pa for 1 hour at 37°C with 5 mg/ml cytosolic proteins, ATP-generating system, and 100 µn with 5 mg/ml cytosolic proteins, ATP-generating system, and no inhibitor, 1 mM NEMls were fixed and processed for electron microscopy. Arrowheads indicate the presence

ding proteins in an early step of the cytosol-induceddistribution into the ER. GTPγS activates both

ric and trimeric GTP-binding proteins whereas onlyG proteins are activated by AlF(3-5) (Kahn, 1991). Weed the effect of adding AlF(3-5) to our assay. As shownB AlF(3-5) inhibited mannosidase II redistribution intoFurther support for the involvement of trimeric Gin the process was obtained by adding transducin βγ (Fig. 10C) and mastoparan (not shown), both of whichy inhibited Golgi redistribution. on of βγ subunits should give rise to inactivation of Gas the βγ subunits complex with the free α subunits and Mostov, 1992). In contrast, mastoparan, whichn activated receptor, should render the opposite effect,ation of G proteins (Higashijima et al., 1990; Wein- al., 1990). Inhibition by both agents can be explained redistribution into the ER would be controlled by

proteins exerting opposite regulatory roles. To testthesis we examined the effects resulting from incu-

by cholera toxin treatment and αi being inhibited by pertussistoxin. The emerging idea was that Golgi redistribution into theER was stimulated by either αs activation or αi inhibition.

DISCUSSION

In this paper we have described the redistribution of Golgicomponents into the ER originated by the incubation of SO-permeabilized cells with an excess of cytosolic proteins. Asimilar phenomenon occurs in BFA-treated cells that alsoundergo Golgi disassembly and fusion with the ER (Klausneret al., 1992; Lippincott-Schwartz et al., 1989). However,important differences between both processes were noted.First, we did not observe by immunofluorescence tubuleprocesses emerging from the Golgi cisternae during their redis-tribution into the ER. Instead, our ultrastructural observationsindicate that the cytosol-induced Golgi redistribution occurs bybudding of coated vesicles from the Golgi cisternae. In

1813Cytosol-induced Golgi redistribution

additiocytosoit appaGolgi cells inintact into thGolgi bition resultsmay astill un

Role oredistCytosoresult dpathwacomplmisfoltively,norma

r

c

ed l

eems unlikely since the fragmented Golgi membranesfically fuse with the ER and morphological changes in organelles were not observed during incubation of SO-eabilized cells with cytosolic proteins.

Fig. 10. Involvement of trimeric Gproteins in Golgi disassembly.NRK cells were treated or not with1 µg/ml cholera toxin (CT), 0.5µg/ml pertussis toxin (PT) for 16hours at 37°C. Following SOpermeabilization the cells wereincubated at 37°C for 1 hour incomplete incubation mediumcontaining low (0.1 mg/ml) orhigh (5 mg/ml) concentration ofcytosolic proteins plus ATP-generating system. Effectsresulting from adding either 50µM AlCl3 plus 30 mM NaF (B) or5 µM of transducin βγ subunits(C) on Golgi redistribution areshown. Cells were fixed andstained for immunofluorescencewith anti-α-mannosidase IIantibody. Bar, 20 µm.

n, the mechanism of Golgi disruption induced bylic factors is different from that activated by BFA sincerently occurs without previous β-COP dissociation frommembranes. Furthermore, in the case of permeabilized

ity sspeciotherperm

cubated with a high concentration of cytosolic proteinsmicrotubules are not required for Golgi redistributione ER. Finally, unlike the BFA effect, cytosol-inducededistribution does not seem to be a consequence of inhi-of the ER-Golgi anterograde transport. Instead, our

suggest the presence in the cells of cytosolic factors thattively promote the Golgi-ER retrograde pathway by adetermined mechanism.

f cytosolic factors in promoting Golgiributionl-induced Golgi redistribution into the ER may be theerived from an exaggeration of the Golgi-ER retrogradey taking place in vivo during retrieval from the Golgix of either ER resident molecules (Pelham, 1988) ored proteins (Hammond and Helenius, 1994). Alterna-this redistribution process may be unrelated with the Golgi-ER retrograde pathway. However, this possibil-

The Golgi-ER retrograde pathway has been shown to beregulated by both the amount of ER proteins to be recycledfrom the Golgi and the number of specific receptors availablefor them. For instance, a BFA-like effect has been describedin cells overexpressing ELP-1, a putative receptor for lumenalER proteins bearing the KDEL sequence (Hsu et al., 1992). Inaddition, a striking redistribution of the KDEL receptor fromthe Golgi region to the ER occurs in cells overexpressingproteins with this retention sequence (Lewis and Pelham,1992). The conclusion derived from these studies is that theretrograde pathway is somehow activated by the formation ofligand-receptor complexes in the Golgi. In this regard, involve-ment of cytosolic factors could introduce additional controls inthe pathway. For instance, by influencing the activity of par-ticular trimeric G proteins cytosolic factors might modulate theassociation of coat proteins with membranes. This, in turn,could be responsible for maintaining a controlled balancebetween anterograde and retrograde membrane routes. It has

1814

been recto Golgivesicles receptor1993). Itoxin hacAMP (Smessengmembra

Steps iThe usethe GoAccordibuddingThis prothereafteindepend

VesicleGolgi dcytosoliprocessewith theal., 1992tubule pSO-permobservatmassivecisternaeprocess for this Treatmeand/or Therefomediateroute coin studie1993; Lvesicle pthe Golhinderedsuch assdepletioIndeed membrarograde coat proproteinsproposedsignals Letourne

The membratulated inherentproteinscontrast,previousthe absetubules a

J

ently reported that both the association of coat proteins membranes and the constitutive release of secretoryare stimulated by the activation of cell surface

s and by protein kinase C activators (De Matteis et al.,n addition, Golgi-ER retrograde transport of shigas been shown to be stimulated by both butyric acid and

andvig et al., 1994). Therefore it is likely that seconders and other cytosolic factors might regulatene traffic.

n Golgi redistribution of a permeabilized cell system allowed us to dissectlgi redistribution process into different stages.ng to our data Golgi disassembly is achieved by of non-clathrin coated vesicles from Golgi cisternae.cess is regulated by trimeric G proteins. The vesiclesr lose their coat and fuse to the ER in a microtubule-

Other evidence indicates that tubule processes are normallyformed in vivo. For instance, tubules have been described tooccur in cells incubated at low (16°C) temperature (Lippincott-Schwartz et al., 1990; Tang et al., 1993). In this case, however,mid- and trans-Golgi proteins do not enter the tubules whilep53, a protein localized at the intermediate compartmentsituated between the ER and the Golgi, does (Lippincott-Schwartz et al., 1990). Therefore, whereas it is evident thattubule processes can be originated from the Golgi region undercertain conditions, it remains to be determined if they reallyaccount for the recycling pathway existing between the Golgiand the ER.

Regulation of Golgi redistribution by trimeric GproteinsOur data indicate that the Golgi-ER retrograde transportstudied in SO-permeabilized cells is modulated by both αs and

. Hidalgo, M. Muñiz and A. Velasco

ent, temperature- and NEM-sensitive manner. αi. These two Gα proteins, however, should have opposite reg-

vs tubule-mediated retrograde transportisassembly and redistribution into the ER induced byc proteins does not occur by the extension of tubules from Golgi cisternae. This is a significant difference redistribution caused by BFA treatment (Klausner et). Thus we could not observe by immunofluorescence

rocesses emerging from the Golgi during incubation ofeabilized cells with cytosol. Instead, ultrastructural

ions indicated that Golgi disassembly was achieved by budding of 50-60 nm coated vesicles from Golgi. GTPγS was an efficient inhibitor of this budding

consistent with recent data indicating an inhibitory roleagent in vesicle formation (Weidman et al., 1993).nt with NEM, on the other hand, inhibited targetingfusion of these vesicles with the ER membranes.re these results suggest the existence of a vesicle-d Golgi-ER retrograde pathway. In intact cells thisuld coexist with others mediated by tubules and showns with BFA or in cells depleted of ATP (Cluett et al.,ippincott-Schwartz et al., 1990). Alternatively, theathway could be the normal route operating between

gi and the ER in cells whose coat proteins are not from becoming associated with membranes; lack of

ulatory roles, since either αs activation (by cholera toxintreatment) or αi inhibition (by pertussis toxin treatment) stim-ulates Golgi redistribution into the ER. Similar findings havebeen described for the production of secretory vesicles fromthe TGN (Leyte et al., 1992). This suggests the existence of abasic, G protein-regulated mechanism common to the twoprocesses.

We thank Dr Marilyn G. Farquhar for critical reading of the man-uscript and R. Garcia and F. J. Fuentes for their technical work. Thisresearch was supported by grant 93/0824 from FISs (to J. Hidalgo)and grant PB92-0674 from Spanish DGICYT (to A. Velasco). M.Muñiz was supported by a predoctoral fellowship fromUNICAJA/Junta de Andalucia.

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