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,[email protected]
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.
References
1. Rothman JE, Wieland FT. Protein sorting by transport vesicles.
Science 1996;272:227–234.
2. Sciaky N, Presley J, Smith C, Zaal KJ, Cole N, Moreira JE, Terasaki M,
Siggia E, Lippincott-Schwartz J. Golgi tubule traffic and the effects of
brefeldin A visualized in living cells. J Cell Biol 1997;139:1137–1155.
3. Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD,
Lippincott-Schwartz J. Kinetic analysis of secretory protein traffic and
characterization of Golgi to plasma membrane transport intermediates
in living cells. J Cell Biol 1998;143:1485–1503.
4. Presley JF, Ward TH, Pfeifer AC, Siggia ED, Phair RD, Lippincott-
Schwartz J. Dissection of COPI and Arf1 dynamics in vivo and role in
Golgi membrane transport. Nature 2002;417:187–193.
5. Scales SJ, Pepperkok R, Kreis TE. Visualization of ER-to-Golgi transport
in living cells reveals a sequential mode of action for COPII and COPI.
Cell 1997;90:1137–1148.
6. Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J.
Maintenance of Golgi structure and function depends on the integrity
of ER export. J Cell Biol 2001;155:557–570.
7. Dinter A, Berger EG. Golgi-disturbing agents. Histochem Cell Biol
1998;109:571–590.
8. Peyroche A, Antonny B, Robineau S, Acker J, Cherfils J, Jackson CL.
Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein
complex: involvement of specific residues of the Sec7 domain. Mol
Cell 1999;3:275–285.
9. Kano F, Sako Y, Tagaya M, Yanagida T, Murata M. Reconstitution of
brefeldin A-induced golgi tubulation and fusion with the endoplasmic
reticulum in semi-intact chinese hamster ovary cells. Mol Biol Cell
2000;11:3073–3087.
10. Nachliel E, Finkelstein Y, Gutman M. The mechanism of monensin-
mediated cation exchange based on real time measurements. Biochim
Biophys Acta 1996;1285:131–145.
11. Mironov A, Colanzi A, Silletta MG, Fiucci G, Flati S, Fusella A,
Polishchuk R, Mironov A Jr, Di Tullio G, Weigert R, Malhotra V,
Corda D, De Matteis MA, Luini A. Role of NADþ and ADP-ribosylation
in the maintenance of the Golgi structure. J Cell Biol 1997;139:
1109–1118.
12. Spano S, Silletta MG, Colanzi A, Alberti S, Fiucci G, Valente C, Fusella A,
Salmona M, Mironov A, Luini A, Corda D, Spanfo S. Molecular cloning
and functional characterization of brefeldin A-ADP-ribosylated sub-
strate. A novel protein involved in the maintenance of the Golgi struc-
ture. J Biol Chem 1999;274:17705–17710.
13. Weigert R, Colanzi A, Mironov A, Buccione R, Cericola C, Sciulli MG,
Santini G, Flati S, Fusella A, Donaldson JG, Di Girolamo M, Corda D,
De Matteis MA, Luini A. Characterization of chemical inhibitors of
brefeldin A-activated mono-ADP-ribosylation. J Biol Chem
1997;272:14200–14207.
14. Bergmann JE. Using temperature-sensitive mutants of VSV to study
membrane protein biogenesis. Methods Cell Biol 1989;32:85–110.
15. Daull P, Home W, Boileau G, LeBel D. Brefeldin A-induced prosoma-
tostatin N-glycosylation in AtT20 cells. Biochem Biophys Res Commun
2002;296:618–624.
16. Neumann D, Yuk MH, Lodish HF, Lederkremer GZ. Blocking
intracellular degradation of the erythropoietin and asialoglycoprotein
Monensin Blocks BFA-Induced Golgi Collapse
Traffic 2005; 6: 794–802 801
receptors by calpain inhibitors does not result in the same increase in
the levels of their membrane and secreted forms. Biochem J
1996;313:391–399.
17. Drose S, Altendorf K. Bafilomycins and concanamycins as inhibitors of
V-ATPases and P-ATPases. J Exp Biol 1997;200:1–8.
18. Tapper H, Sundler R. Role of lysosomal and cytosolic pH in the regula-
tion of macrophage lysosomal enzyme secretion. Biochem J
1990;272:407–414.
19. Smallridge RC, Gist ID, Kiang JG. Naþ-Hþ antiport and monensin
effects on cytosolic pH and iodide transport in FRTL-5 rat thyroid
cells. Am J Physiol 1992;262:E834–E839.
20. Trucco A, Polishchuk RS, Martella O, Di Pentima A, Fusella A, Di
Giandomenico D, San Pietro E, Beznoussenko GV, Polishchuk EV,
Baldassarre M, Buccione R, Geerts WJ, Koster AJ, Burger KN,
Mironov AA et al. Secretory traffic triggers the formation of tubular
continuities across Golgi sub-compartments. Nat Cell Biol
2004;6:1071–1081.
21. Griffiths G, Quinn P, Warren G. Dissection of the Golgi complex. I.
Monensin inhibits the transport of viral membrane proteins from med-
ial to trans Golgi cisternae in baby hamster kidney cells infected with
Semliki Forest virus. J Cell Biol 1983;96:835–850.
22. Quinn P, Griffiths G, Warren G. Dissection of the Golgi complex. II.
Density separation of specific Golgi functions in virally infected cells
treated with monensin. J Cell Biol 1983;96:851–856.
23. Neumann D, Wikstrom L, Watowich SS, Lodish HF. Intermediates in
degradation of the erythropoietin receptor accumulate and are
degraded in lysosomes. J Biol Chem 1993;268:13639–13649.
24. Supino-Rosin L, Yoshimura A, Yarden Y, Elazar Z, Neumann D.
Intracellular retention and degradation of the epidermal growth factor
receptor, two distinct processes mediated by benzoquinone ansamy-
cins. J Biol Chem 2000;275:21850–21855.
25. NicholsBJ, KenworthyAK, PolishchukRS, LodgeR,Roberts TH,HirschbergK,
Phair RD, Lippincott-Schwartz J. Rapid cycling of lipid raftmarkers between the
cell surface and Golgi complex. J Cell Biol 2001;153:529–541.
Barzilay et al.
802 Traffic 2005; 6: 794–802
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