Uncoupling of Brefeldin A-Mediated Coatomer ProteinComplex-I Dissociation from Golgi Redistribution

Eran Barzilay1, Nathalie Ben-Califa1, KoretHirschberg2,a and Drorit Neumann1,a,*

1Department of Cell and Developmental Biology, SacklerFaculty of Medicine, Tel-Aviv University, Ramat-Aviv69978, Israel2Department of Pathology, Sackler Faculty of Medicine,Tel-Aviv University, Ramat-Aviv 69978, Israel*Corresponding author: Drorit Neumann,histo6@post.tau.ac.il

The Golgi complex functions in transport of moleculesfrom the endoplasmic reticulum (ER) to the plasma mem-brane and other distal organelles as well as in retrogradetransport to the ER. The fungal metabolite brefeldin A(BFA) promotes dissociation of ADP-ribosylation-factor-1(ARF1) and the coatomer protein complex-I (COP-I) fromGolgi membranes, followed by Golgi tubulation andfusion with the ER. Here we demonstrate that the catio-nic ionophore monensin inhibited the BFA-mediatedGolgi redistribution to the ER without interfering withARF1 and COP-I dissociation. Preservation of a peri-nuclear Golgi despite COP-I and ARF1 dissociationenables addressing the involvement of these proteins inanterograde ER to Golgi transport. The thermo-reversiblefolding mutant of vesicular stomatitis virus G protein(VSVGtsO45) was retained in the ER in the presence ofboth monensin and BFA, thus supporting ARF1/COP-Iparticipation in ER-exit processes. Live-cell imagingrevealed that BFA-induced Golgi tubulation persistedlonger in the presence of monensin, suggesting thatmonensin inhibits tubule fusion with the ER. Moreover,monensin also augmented Golgi-derived tubules thatcontained the ER-Golgi-intermediate compartmentmarker, p58, in the absence of BFA, signifying the gen-erality of this effect. Taken together, we propose thatmonensin inhibits membrane fusion processes in thepresence or absence of BFA.

Key words: brefeldin A, COP-I, Golgi, intracellular traffick-ing, monensin

Received16February2005, revisedandaccepted forpublica-tion 13 June 2005, published on-line 28 July 2005

Golgi-associated retrograde and anterograde trafficking

processes involve pinching-off of vesicles (1) and tubule

formation (2,3). The coatomer protein complex-I (COP-I), a

key component of the intracellular transport machinery, is

recruited to membranes by ADP-ribosylation-factor-1

(ARF1) and takes part in intra-Golgi transport (4). The

participation of COP-I in endoplasmic reticulum (ER) exit

was also suggested (5,6). Central pathways underlying

molecular membrane trafficking have been identified by

the advent of pharmacological agents (7). The macrocyclic

lactone brefeldin A (BFA) has been extensively used for

unraveling the dynamics of the transport machinery and

was paramount for delineating the role of COP-I in intra-

cellular trafficking and maintenance of Golgi structure. The

hallmark of BFA treatment is the rapid collapse of the

Golgi into the ER (2). Brefeldin A binds to the sec7 domain

of ARF guanine-nucleotide-exchange factor (ARF-GEF) and

stabilizes the ARF-GDP-ARF-GEF protein complex,

thereby inhibiting the activity of ARF1 (8) and leading to

sequential dissociation of ARF1 and COP-I from the Golgi

membranes. Golgi redistribution is perceived to be a direct

result of this event (4) and has been segregated into at

least two steps, namely, Golgi tubulation and subsequent

fusion of Golgi membranes with the ER (2,9).

Retrograde Golgi to ER transport was suggested to occur

through the formation of Golgi-derived tubules that fuse

with the ER (2). In the presence of BFA, accentuated

tubule formation and their subsequent fusion with the

ER were observed (2). The cationic ionophore monensin

promotes Hþ/Naþ exchange, thereby raising the pH of the

Golgi complex (10). This agent blocks forward trafficking

from the Golgi to the plasma membrane and other distal

organelles (7). Because it is perceived that the effects of

monensin on intracellular trafficking are mediated by ion

exchange through the Golgi membrane, rather than by

inhibition of a specific molecular target (7), we questioned

whether monensin might also perturb Golgi to ER retro-

grade trafficking. In this study, we demonstrate for the

first time that monensin inhibits BFA-mediated redistribu-

tion of the Golgi to the ER, despite dissociation of ARF1

and COP-I from the Golgi membranes. Under combined

BFA and monensin treatment, ER to Golgi transport is still

inhibited, despite the presence of a perinuclear Golgi.

Monensin treatment augmented Golgi-derived tubules

that were evident both in the presence or absence of

BFA. Taken together, our data suggest that monensin

inhibits membrane fusion processes.

Results

Monensin inhibits BFA-mediated redistribution of the

Golgi into the ER

To examine whether monensin perturbs BFA-mediated

redistribution of the Golgi into the ER, CHO-K1 cells

were subjected to monensin and BFA treatment and sub-

sequently immunostained for the integral Golgi enzymeaCo-senior authors.

Traffic 2005; 6: 794–802Copyright # Blackwell Munksgaard 2005

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2005.00317.x

794

mannosidase-II (MannII). Figure 1 demonstrates that the

redistribution of MannII, apparent upon incubation of the

cells with 5 mg/mL BFA for 1 h, was blocked when mon-

ensin (100 nM) was applied together with BFA.

Monensin does not interfere with BFA-mediated

dissociation of ARF1 and eCOP

We raised the question as to whether the inhibition of

BFA-mediated redistribution of the Golgi complex by mon-

ensin reflects inhibition of BFA-conferred ARF1 and COP-I

dissociation. To explore this possibility, we used CHO-K1

cells expressing ARF1 fused to green fluorescent protein

(ARF1-GFP) and cells expressing the COP-I complex com-

ponent eCOP fused to GFP (eCOP-GFP) and subjected

them to monensin and BFA treatment. Figure 2 demon-

strates that incubation of cells with monensin together

with BFA did not inhibit BFA-induced dissociation of

ARF1-GFP and eCOP-GFP from the Golgi complex. Thus,

although BFA still promotes dissociation of ARF1 and

COP-I from the Golgi membranes in the presence of mon-

ensin, this does not culminate in collapse of the Golgi. This

could suggest that in addition to ARF1 and COP-I, there

are also other factors that participate in the collapse of the

Golgi. This concept is supported by reports on the role of

BFA-induced ADP ribosylation in the collapse of the Golgi

(11–13). Moreover, it has been demonstrated that com-

pounds that inhibit ADP ribosylation in vitro were also able

to inhibit BFA-mediated redistribution of the Golgi

apparatus (13). As monensin did not inhibit ADP-ribosyla-

tion in vitro (13), we propose that the inhibition of BFA-

mediated Golgi collapse by monensin is not a result of

blocking BFA-induced ADP ribosylation. Taken together,

Control

+B

FA

Monensin

Figure 1: Monensin inhibits brefeldin A (BFA)-mediated

redistribution of mannosidase-II (MannII). CHO-K1 cells were

incubated with or without monensin (100 nM) in the presence or

absence of BFA (5 mg/mL) for 1 h prior to fixation with 3% para-

formaldehyde and immunostaining with anti-MannII antibodies.

Control non-treated cells are depicted for comparison. Images

were obtained using a Zeiss LSM PASCAL laser scanning con-

focal microscope. Bar: 10 mm.

ControlA

B+

BF

A

Monensin

Control+

BF

AMonensin

Figure 2: Monensin does not interfere with brefeldin A

(BFA)-induced ADP-ribosylation-factor-1 (ARF1) and e-coato-mer protein complex (eCOP) dissociation. CHO-K1 cells tran-

siently expressing ARF1-GFP (A), or eCOP-GFP (B), were

incubated with monensin (100 nM) and/or BFA (5 mg/mL) for 1 h.

Cells were fixed with 3% paraformaldehyde, and images were

obtained using a Zeiss LSM PASCAL laser scanning confocal

microscope. Control non-treated cells are depicted for compari-

son. Bar: 10 mm.

Monensin Blocks BFA-Induced Golgi Collapse

Traffic 2005; 6: 794–802 795

monensin may confer stability to the Golgi cisternae by

inhibiting downstream processes that regulate BFA-

mediated Golgi collapse.

ER to Golgi transport is blocked under combined BFA

and monensin treatment

Monensin inhibits forward trafficking of cargo molecules

from the Golgi apparatus, thus promoting their accumula-

tion in this organelle (7). Brefeldin A inhibits intracellular

trafficking by inducing Golgi tubulation and subsequent

fusion with the ER (2). In addition, it was suggested that

BFA inhibits ER exit by promoting the dissociation of ARF1

from ER-exit sites (5,6). Based on our finding that monensin

inhibits BFA-mediated collapse of the Golgi apparatus with-

out affecting the dissociation of ARF1 and COP-I, we were

able to examine ER to Golgi anterograde transport in the

absence of Golgi membrane-bound COP-I/ARF1. Our strat-

egy was to use the thermo-reversible folding mutant of

vesicular stomatitis virus G protein VSVGtsO45 (3) in

order to examine ER to Golgi transport under combined

monensin and BFA treatment. At 40 �C, VSVGtsO45 is

misfolded and accumulates within the ER. Upon tempera-

ture shift to 32 �C, VSVGtsO45 is refolded, released from

the ER and directed to the plasma membrane (14). Cells

coexpressing VSVGtsO45 fused to yellow fluorescent pro-

tein (VSVGtsO45-YFP) and the Golgi marker galactosyl-

transferase fused to cyan fluorescent protein (GalT-CFP)

were thus incubated for 20 h at 40 �C and subjected to

BFA and/or to monensin 15 min prior to temperature shift

to 32 �C for 2 h. The results depicted in Figure 3

VSVG-YFP

40 °C

Control

Monensin

BFA

BFA + monensin

GalT-CFP Merge

Figure 3: VSVGtsO45 is retained

in the endoplasmic reticulum

(ER) under combined brefeldin A

(BFA) and monensin treatment.

CHO-K1 cells transiently expressing

VSVGtsO45-YFP and GalT-CFP

were incubated at 40 �C for 20 h.

Brefeldin A (5 mg/mL) and/or mon-

ensin (100 nM) were added 15 min

prior to temperature shift to 32 �Cfor 2 h. Cells were fixed with 3%

paraformaldehyde, and images

were obtained using a Zeiss LSM

PASCAL laser scanning confocal

microscope. Cells fixed prior to

temperature shift to 32 �C (40 �C)and control non-treated cells (con-

trol) are shown for comparison.

Bar: 10 mm.

Barzilay et al.

796 Traffic 2005; 6: 794–802

demonstrate that monensin promotes accumulation of

VSVGtsO45-YFP in the Golgi apparatus, whereas incuba-

tion with BFA alone mediated redistribution of GalT-CFP as

well as accumulation of VSVGtsO45-YFP within the ER.

Addition of monensin together with BFA displayed a differ-

ential effect; while redistribution of GalT-CFP to the ER was

blocked, VSVGtsO45-YFP remained in the ER. These data

suggest that BFA elicits two distinct processes, namely,

Golgi collapse and ER-exit block, that are differentially

affected by monensin. Inhibition of ER to Golgi transport

in the absence of membrane-bound COP-I conforms to

previous reports demonstrating a role for COP-I in ER to

Golgi anterograde transport (5,6). However, one cannot

exclude the possibility that inhibition of ER to Golgi

BFA

0

10

17

Zoom

Tim

e (m

in)

25

26

BFA + mon

Figure 4: Monensin augments

brefeldin A (BFA)-induced

tubulation of the Golgi mem-

branes and inhibits fusion.

CHO-K1 cells stably expressing

GalT-YFP were cultured on cham-

bered coverglasses. Images were

taken at time intervals of 30

seconds, beginning at the time

of BFA addition (5 mg/mL) in the

presence or absence monensin

(100 nM) using the Zeiss LSM

PASCAL laser scanning confocal

microscope. Arrows point at

Golgi-derived tubules. Bar:

10 mm.

Monensin Blocks BFA-Induced Golgi Collapse

Traffic 2005; 6: 794–802 797

transport may reflect a disfunction of the Golgi apparatus

and not a direct block of ER-exit processes.

Monensin inhibits fusion of Golgi-derived tubules

with the ER

The dynamics of BFA-mediated collapse of the Golgi to

the ER were previously described (2). Following dissocia-

tion of ARF1, BFA promotes the formation of tubules from

the Golgi membranes. These tubules persist for several

minutes prior to fusion with ER membranes. To determine

which step of the BFA-induced collapse of the Golgi into

the ER is inhibited by monensin, we used time-lapse

imaging in order to monitor GalT-YFP in CHO-K1 cells

during treatment with BFA and monensin. The results

depicted in Figure 4 (see also QuickTime movie in the

supplementary material available online at http://

www.traffic.dk/suppmat/6_9.asp) demonstrate that BFA-

induced tubulation of the Golgi was not inhibited by mon-

ensin. In fact, Golgi tubulation appeared to persist for

longer periods (up to 20–30 min) in cells subjected to

combined BFA and monensin treatment, compared with

cells that were treated with BFA alone. Hence, monensin

did not block BFA-induced tubulation of the Golgi, but

rather impeded their fusion and absorption into the ER. It

thus appears that the persistence of tubulation in cells

treated with monensin together with BFA is a direct result

of inhibition of tubule fusion with the ER.

Monensin inhibits BFA-induced N-glycan processing

of an ER-localized protein

Brefeldin A-induced relocalization of the Golgi to the ER

allows Golgi enzymes that process N-glycans to access

ER-localized glycoproteins and to catalyse their processing

within these mixed ER-Golgi membranes (15). We thus

assumed that monensin-induced inhibition of BFA-

mediated Golgi fusion with the ER should also be reflected

on N-glycan processing of ER-localized proteins. To

evaluate the inhibition of BFA-induced N-glycan proces-

sing by monensin, we utilized a truncated erythropoietin

receptor that lacks the cytosolic and transmembrane

domains (sEPO-R). This EPO-R derivative is localized pri-

marily in the ER and possesses one N-linked glycosylation

site which is endoglycosidase H (EndoH)-sensitive in most

molecules at steady state (16). Acquisition of resistance to

EndoH by sEPO-R can thus be used as biochemical read-

out for the BFA-induced merge of the Golgi and ER. Our

results show that sEPO-R gains resistance to EndoH upon

addition of BFA (Figure 5; see also supplemental figure 1

available online at http://www.traffic.dk/suppmat/

6_9.asp). Although BFA-induced processing of sEPO-R is

clearly observed after 6 h of incubation with BFA (Figure 5

lane 4, solid arrow), the majority of sEPO-R molecules

EndoH

1

Control mon

BF

A

BF

A +

mon

BF

A

BF

A +

mon

2 3 4 5 6

+–

Figure 5: Monensin inhibits brefeldin A (BFA)-mediated

N-glycan processing. COS-7 cells expressing sEPO-R were incu-

bated with or without monensin in the presence or absence of

BFA. The sample in lane 6 was exposed to BFA 10 min prior to

the addition of monensin. Lysates of control non-treated cells are

depicted with or without endoglycosidase H (EndoH) digestion for

comparison. Solid and empty arrowheads represent EndoH-resist-

ant and EndoH-sensitive forms of sEPO-R, respectively.

+BFA

Control

Monensin

Nigericin

Bafilomycin

Figure 6: Nigericin, but not bafilomycin A1, inhibits brefeldin

A (BFA)-mediated redistribution of the Golgi. CHO-K1 cells

were incubated with monensin (100 nM), nigericin (100 nM) or

bafilomycin A1 (50 nM) in the presence or absence of BFA(5 mg/mL) for 1 h prior to fixation with 3% paraformaldehyde and immuno-

staining with anti-MannII antibodies. Control non-treated cells are

depicted for comparison. Images were obtained using a Zeiss

LSM PASCAL laser scanning confocal microscope. Bar: 10 mm.

Barzilay et al.

798 Traffic 2005; 6: 794–802

remain EndoH-sensitive (Figure 5 lane 4, empty arrow).

The inefficiency of BFA-mediated N-glycan processing

may be ascribed to the change in lumenal pH and/or to

the reduced concentration of enzymes and substrates in

the mixed ER-Golgi membranes. Addition of monensin

inhibited the BFA-mediated processing of sEPO-R (Figure

5, lane 5). However, processing of sEPO-R was not inhib-

ited when monensin was added 10 min after BFA

(Figure 5, lane 6), a period when BFA-induced glycan pro-

cessing is not evident (supplemental figure 1 available

online at http://www.traffic.dk/suppmat/6_9.asp). We

thus conclude that monensin inhibits the redistribution of

Golgi enzymes to the ER and not their enzymatic activity.

Control MonensinA

Control Monensin

21

16

9.5

5

B

Figure 7: Monensin-induced augmentation of Golgi tubula-

tion is not limited to brefeldin A (BFA)-treated cells. COS-7

cells expressing p58-YFP were cultured on chambered cover-

glasses. Images were taken at time intervals of 30 seconds

using the Zeiss LSM PASCAL laser scanning confocal micro-

scope. Monensin (100 nM) was added 21 min after the first

image was acquired. A) Selected images of the same cell before

and after monensin addition. The time after monensin addition is

depicted in minutes, the matching control image represents the

same cell prior to the addition of monensin at a time point 21 min

earlier. Arrows point at Golgi-derived tubules. Bar: 10 mm. B)

Overlay of all images taken 21 min before and after monensin

addition (43 images each at time intervals of 30 seconds).

cargoGolgi residentARF-1COP-1

Untreated

BFA

Monensin + BFA

Figure 8: A scheme depicting the effect of brefeldin A (BFA)

and monensin treatment on the secretory pathway. Brefeldin

A treatment induces ADP-ribosylation-factor-1 (ARF1) and coat-

omer protein complex-I (COP-I) dissociation and subsequently

fusion of the Golgi membranes with the ER. Hence, both cargo

proteins and Golgi-resident proteins accumulate in the ER. When

monensin and BFA are added concomitantly, both anterograde

and retrograde transport processes are inhibited, thus preventing

Golgi fusion with the ER. Golgi-resident enzymes will hence

remain within the Golgi compartment without affecting ER-

localized proteins, and newly synthesized cargo molecules will

fail to reach the Golgi.

Monensin Blocks BFA-Induced Golgi Collapse

Traffic 2005; 6: 794–802 799

Nigericin but not bafilomycin A1 inhibits BFA-

mediated Golgi redistribution

As the hallmark of monensin activity is the elevation of

Golgi lumen pH (7), we examined whether other com-

pounds that elevate Golgi lumen pH might inhibit

the BFA-induced redistribution of the Golgi into the ER.

We compared the effect of monensin on BFA-mediated

Golgi redistribution with that of nigericin and bafilomycin

A1. Nigericin, similarly to monensin, elevates Golgi-lumen

pH by ion exchange. Whereas monensin promotes

Naþ/Hþ exchange, nigericin promotes Kþ/Hþ exchange

(7). While nigericin inhibited BFA-mediated Golgi redistri-

bution in a similar manner to monensin (Figure 6),

bafilomycin A1, which elevates Golgi lumen pH by inhibit-

ing the vacuolar-type, proton-translocating ATPase (17),

did not inhibit BFA-mediated Golgi redistribution. These

data suggest that elevated Golgi lumen pH is not

the sole factor responsible for inhibition of Golgi redistri-

bution. The inability of bafilomycin A1 to block BFA-

mediated Golgi collapse was observed at a wide

range of concentrations (data not shown). Nevertheless,

we cannot rule out that bafilomycin A1 and monensin

were not equipotent in their ability to change Golgi

lumen pH.

Monensin-induced augmentation of Golgi tubulation

is not limited to BFA-treated cells

The observation on the inhibitory effect of monensin on

BFA-mediated collapse of the Golgi to the ER led to the

question of the generality of monensin’s effect on retro-

grade trafficking. To determine whether constitutive retro-

grade trafficking is affected by monensin, we chose to use

time-lapse imaging in order to follow the cellular distribu-

tion of the ER-Golgi intermediate compartment (ERGIC)

marker p58. The results depicted in Figure 7 reveal that

Golgi-derived tubules are more prominent after addition of

monensin in comparison with the same cell prior to mon-

ensin treatment. It should be noted that augmented tubu-

lations were not discerned when individual images were

compared (Figure 7A). Yet, by comparing the composite

overlay of images (43 images each at time intervals of

30 seconds) captured before and after monensin addition,

the monensin-induced increase of Golgi-derived tubules is

clearly noticed (Figure 7B). It thus appears that augmenta-

tion of Golgi-derived tubules by monensin is not limited to

BFA-treated cells.

Discussion

Here we demonstrate for the first time that monensin

inhibits BFA-induced Golgi redistribution to the ER,

despite ARF1 and COP-I dissociation. Under combined

BFA and monensin treatment, VSVGtsO45-YFP fails to

reach the Golgi apparatus and is retained in the ER.

Inhibition of ER to Golgi transport could be ascribed

either to a direct interference with ER-exit processes or

to defective Golgi function under combined monensin and

BFA treatment. Hence, whereas monensin blocks BFA-

mediated collapse of the Golgi, it does not release BFA-

induced inhibition of ER to Golgi transport (Figure 8). This

lends support to the notion that monensin inhibits BFA

activity indirectly, possibly by inhibiting the actual Golgi

redistribution process. In view of the fact that Golgi tubu-

lation was not inhibited by monensin, and the observation

that BFA-induced Golgi-derived tubules actually persisted

longer, it appears that monensin inhibits the fusion of

Golgi-derived tubules with ER membranes. The finding

that monensin increased p58-YFP-containing tubules

could be attributed to inhibition of tubule fusion with the

ER, leading to increased persistence of the tubules. As the

augmented Golgi tubulation induced by monensin is not

limited to BFA-mediated Golgi tubulation, we suggest that

monensin may inhibit downstream fusion processes that

regulate both BFA-mediated Golgi collapse and constitu-

tive Golgi to ER retrograde transport.

Our data point to a modulatory role of electro-chemical

potential in membrane fusion processes. The hallmark of

monensin activity is the elevation of Golgi lumen pH (7);

however, as an ionophore, the impact of monensin on

electro-chemical potential is broader and includes changes

in Naþ and Hþ composition both in the Golgi lumen and in

the cytosol (18,19). The related compound nigericin, which

promotes Kþ/Hþ exchange, was similarly active in inhibit-

ing BFA-induced Golgi collapse. In contrast, bafilomycin

A1, another blocker of secretion that elevates Golgi lumen

pH by inhibiting the vacuolar-type, proton-translocating

ATPase (17), did not inhibit BFA-mediated redistribution

of the Golgi. This might indicate that changes in Golgi

lumen pH are not the sole cause of the inhibitory effect

of monensin on BFA-mediated Golgi redistribution. It is

thus possible that the monensin-sensitive component in

the fusion process of Golgi-derived tubules with the ER

resides in the cytosol. Unraveling the differential activities

of bafilomycin A1 and monensin may provide the key for

deciphering the mechanisms, whereby monensin and

nigericin confer inhibition of BFA-mediated Golgi collapse.

We further speculate that the same mechanisms respon-

sible for inhibition of BFA-mediated Golgi redistribution

demonstrated here may also account for inhibition of ante-

rograde transport by monensin and nigericin. As intra-

Golgi trafficking was suggested to occur via formation of

intercisternal tubules (20), the inhibition of anterograde

trafficking and accumulation of cargo within the Golgi

may be attributed to inhibition of intra-Golgi fusion pro-

cesses. Inhibition of intra-Golgi fusion may also explain

the segregation of Golgi elements previously demon-

strated to occur upon monensin treatment (21,22). Taken

together, we propose that the inhibition of both antero-

grade and retrograde transport processes by monensin is

attributed to a block in membrane fusion processes due to

a disturbance in the electro-chemical potential of the Golgi

membranes. Efforts to identify the monensin-sensitive

fusion components are currently underway and may pro-

vide novel targets for the control of intracellular trafficking.

Barzilay et al.

800 Traffic 2005; 6: 794–802

Materials and Methods

MaterialsMaterials were obtained from previously listed sources (23,24). Monensin

and BFA were obtained from Sigma (St. Louis, MO, USA).

AntibodiesRabbit polyclonal antibodies directed against the extracellular domain of

the erythropoietin receptor (EPO-R) were used at a 1:1000 dilution for

immunoblotting, as described (23). Rabbit polyclonal antibodies directed

against MannII (kindly provided by K. Moremen, Athens, GA, USA) were

used at a dilution of 1:1000 for immunofluorescence.

PlasmidsA plasmid containing a truncated erythropoietin receptor that lacks the

cytosolic and transmembrane domains (sEPO-R) was previously described

(16). Plasmids containing galactosyl transferase fused to cyan fluorescent

protein (GalT-CFP), GalT fused to yellow fluorescent protein (GalT-YFP) and

the thermo-reversible folding mutant of vesicular stomatitis virus G protein

fused to YFP (VSVGtsO45-YFP) were employed (25). Plasmids containing

ARF1 fused to green fluorescent protein (ARF-GFP) and eCOP fused to

GFP (eCOP-GFP) were kindly provided by J. Lippincott-Schwartz, NIH,

Bethesda, MD, USA (4). A plasmid encoding p58-YFP, a tagged full-length

transmembrane protein homologous to ERGIC53, was employed (6).

Cell culture and transfectionCHO-K1 and COS 7 cells were maintained in Dulbecco’s-modified Eagle’s

medium supplemented with 10% (v/v) fetal calf serum. Cells cultured to

60% confluence were transiently transfected using lipofectamine

(Invitrogene, Carlsbad, CA, USA) according to manufacturer’s instructions.

For generating stably transfected cells, CHO-K1 cells were subjected to

selection with 750 mg/mL G418 24 h after transfection and were main-

tained in this medium henceforth.

Cell lysis and EndoH digestionCells (105) were lysed in 30 mL phosphate-buffered saline containing 1%

(v/v) Triton X-100, 0.5% (w/v) deoxycholate and 5 mM ethylenediaminete-

traacetic acid supplemented with protease inhibitors (Complete Protease

Inhibitors, Roche Diagnostics, Basel Switzerland), denatured in 0.5% (w/v)

SDS for 5 min at 100 �C prior to addition of 3 mL 0.5 M sodium citrate and

incubation with or without 500 U EndoH (Endo Hf, New England Biolabs,

Inc., Ipswich, MA, USA) for 1 h at 37 �C. Samples were resolved on 7.5%

SDS-PAGE, prior to Western blot analysis with anti-EPO-R antibodies.

VSVG traffickingCHO cells transiently expressing VSVGtsO45-YFP and GalT-CFP, cultured

on glass coverslips, were incubated for 20 h at 40 �C. Brefeldin A and

monensin (5 mg/mL and 100 nM, respectively) were added 15 min prior to

the temperature shift to 32 �C for 2 h. Slides were fixed in 3% (w/v)

paraformaldehyde before the temperature shift and 2 h thereafter.

Confocal microscopy and time-lapse imagingImages were captured using the Zeiss LSM PASCAL laser scanning con-

focal microscope with 458, 488, 514 and 543 nm laser lines. For time-lapse

imaging, cells expressing GalT-YFP or p58-YFP were cultured on cham-

bered coverglasses. Images were taken at time intervals of 30 seconds,

beginning at the time of BFA addition (5 mg/mL) in the presence or absence

of monensin (100 nM) (GalT-YFP), or before and after addition of monensin

(100 nM) (p58-YFP).

Acknowledgments

We thank R. Sagi-Eisenberg, G. Z. Lederkremer and Z. Elazar for critically

reviewing the manuscript. This work was supported by grants from the

Israel Science Foundation administered by the Israel Academy of Sciences

and Humanities (574/99 18.2) and (650/02) to Drorit Neumann and Koret

Hirschberg, respectively, and by the German Israeli Foundation Grant

I-666–79.2/2000 to Drorit Neumann. This work was carried out in partial

fulfillment of the requirements for the PhD degree of Eran Barzilay from

the Sackler Faculty of Medicine, Tel.Aviv University, Israel.

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