Department of Cell Biology, Sciences III, University of Geneva, Geneva, Switzerland, and * Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Germany
Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer
from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of -COP, inhibit
BFA-induced redistribution of
-COP in vivo and
block transfer of resident proteins of the Golgi complex
to the ER; tubulo-vesicular clusters accumulate and
Golgi membrane proteins concentrate in cytoplasmic
patches containing
-COP. These patches are devoid of
marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where
it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of
stacks of Golgi cisternae following BFA wash-out is inhibited to only ~50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at
least as efficiently as GTP
S. Taken together these results suggest that enhanced binding of coatomer to
membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER,
probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of
the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde
membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993
), and corroborate that
COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.
VECTORIAL protein and membrane transport in the
secretory pathway is mediated by vesicular carriers.
Despite the enormous membrane traffic through
this pathway, each compartment maintains a unique and
defined membrane composition; in addition a balance between forward membrane transport and recycling must be
kept. Two basic mechanisms operating in the regulation of
this membrane flow are sorting and recycling. Coat proteins (COPs)1 have been implicated in sorting of cargo
into coated buds and formation of vesicular transport intermediates (Robinson, 1994 The organization of membrane traffic in tissue culture
cells is disrupted by BFA. BFA interferes with the function of a membrane bound GTP exchange factor and thus
prevents association of ARFs, small GTPases (Boman and
Kahn, 1995 Microinjection of antibodies into living cells has proven
a powerful alternative approach to studies with cell-free
systems and genetic analyses in yeast for characterizing
functions of COPs in membrane traffic in mammalian cells
in vivo. In this study we have used specific polyclonal antibodies raised against peptides of Cell Culture, Microinjection, and Microscopy
Vero cells (African green monkey kidney cells, Amer. Type Culture Collection, Rockville, MD; CCL81) were maintained and infected with tsO45VSV (Indiana serotype) as described earlier (Kreis and Lodish, 1986 Images of immunolabeled cells were recorded on a Zeiss inverted fluorescence microscope (Axiovert TV135) equipped with a cooled CCD
camera (Photometrics CH250, 1317X1035 pixels, Tucson, AZ), controlled
by a Power Macintosh 8100/100 and the software package IPLab spectrum
V2.7 (Signals Analytics Corp., Vienna, VA). Images were further processed using Adobe Photoshop V3.0 before printing with a Xanté laser
press 1800 (Conware Informatik AG, Baar, Switzerland).
Quantitative electron microscopy (EM) on microinjected cells was performed as described (Pepperkok et al., 1993 Metabolic Labeling and Biochemical Analyses of
Maturation of ts-O45-G
500-1,000 cells, grown on a glass coverslip (Pepperkok et al., 1993 Preparation of Rat Liver Cytosol and Enriched
IC/Golgi Membranes
To prepare rat liver cytosol 30 g of frozen rat liver was thawed at 4°C,
minced in 60 ml of homogenization buffer (20 mM Hepes-KOH [pH 7.0],
100 mM KCl, 2.5 mM MgCl2, 1 mM PMSF, 0.5 mM 1:10 phenanthroline, 2 mM pepstatin A, 2 mg/ml aprotinin, 0.5 µg/ml leupeptin) and homogenized with a Polytron using three 30-s bursts on setting 4. The resulting
homogenate was centrifuged at 12,000 g for 15 min in a 18.50 rotor (Heraeus SA, Carouge-Genève, Switzerland). The supernatant was collected
and centrifuged at 100,000 g for 1 h in a TST 41.14 rotor (Koutron Instruments, Schlieren, Switzerland).AU: Please
give name of
of manufacturer, city
and state The resulting supernatant was collected
and centrifuged for a further 2 h at 200,000 g in the TST 41.14 rotor. The
200,000-g supernatant (rat liver cytosol) was frozen in liquid nitrogen and
stored at Quantifying Binding of Coatomer to
Membranes In Vitro
Incubations (120 µl total volume) were carried out for 10 min at 37°C in
the presence of 14 µg Golgi/IC membranes, 25 mM Hepes-KOH (pH 7.0),
25 mM KCl, 2.5 mM MgCl2, 50 µM ATP, 2 mM creatine phosphate, 12.5 U/ml creatine kinase, and 160 µg rat liver cytosol. Further additions were
made as indicated in the figure legends. Reactions were layered on top of
200 µl of 15% sucrose (in 25 mM Hepes-KOH [pH 7.0], 25 mM KCl) and
centrifuged at 4°C in a microcentrifuge for 30 min at 15,000 rpm. The supernatants were discarded and the membrane pellets resuspended in 15 µl
SDS sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Microinjection of Anti-EAGE Inhibits BFA-induced
Transfer of Golgi Enzymes to the ER
Anti-EAGE, when microinjected into cells, inhibit anterograde transport of ts-O45-G from the ER/IC to the Golgi
complex (Pepperkok et al., 1993 To analyze a possible recycling of Golgi enzymes through
the ER/IC, Vero cells were infected with tsO45-VSV and
kept at nonpermissive temperature (39.5°C, predominantly ER accumulation), or 15°C (IC accumulation). Under these conditions, ts-O45-G arrested in the ER or IC
may serve as a substrate for oligosaccharide-processing Golgi enzymes, and transport of these enzymes into the
ER/IC can be studied by biochemical analyses of modifications of the oligosaccharide side chains of the viral glycoprotein (Doms et al., 1989
Under normal conditions, ts-O45-G is not modified by
Golgi enzymes at 39.5°C and therefore remains sensitive
to digestion with endo H for at least 6 h (Fig. 1 A, n.i. 6h).
Treatment of cells with BFA relocates Golgi enzymes to
the ER within 15-30 min (Domes et al., 1989; LippincottSchwartz et al., 1989) and renders ER accumulated ts-O45-G
endo H resistant at 39.5°C (Fig. 1 B, n.i. 2.5h). However,
ts-O45-G remains completely endo H sensitive at this
temperature in cells, microinjected with anti-EAGE or its
Fab-fragments, which have subsequently been treated
with BFA (Fig. 1 B, EAGE). Thus, microinjection of antiEAGE blocks the transfer of Golgi enzymes to the ER
where ts-O45-G is accumulated. This inhibition is specific
for anti-EAGE, since microinjected anti-110-12 have no
effect on BFA induced acquisition of endo H resistance of
ts-O45-G (Fig. 1 B, 110-12).
Distribution of Golgi, IC, and ER Proteins in
BFA-treated Cells Microinjected with Antibodies
against In control noninjected cells ts-O45-G accumulates in the
ER at 39.5°C and colocalizes with PDI or calnexin (data not
shown). Microinjection of anti-EAGE has no effect on the
localization of ts-O45-G in the ER at nonpermissive temperature (e.g., Fig. 3, a and b). The lack of BFA-induced
acquisition of endo H resistance of ts-O45-G in these cells
must therefore be due to inhibition of delivery of Golgi
enzymes to the ER. To analyze this transport block morphologically, we colocalized microinjected antibodies and
several marker proteins for the ER, IC, and Golgi complex by immunofluorescence microscopy.
The resident Golgi membrane proteins NAGT I (Fig. 2 e)
and Man II (not shown), as well as p58, a protein associated with the cytoplasmic surface of Golgi membranes
(Bloom and Brashear, 1989
Interestingly, upon addition of BFA to noninjected virusinfected control cells kept at nonpermissive temperature, a
significant fraction of ts-O45-G also accumulates in patchy
structures (Fig. 5; see also Fig. 3, c and e). These patches
show no enhanced staining with antibodies against PDI or
calnexin (not shown) which retain their typical ER distribution (Fig. 5 b). However, they are enriched in ERGIC53
(Fig. 5 d), p58 (Fig. 5 h), and contain
Quantitative EM analysis of thin sections revealed that
virtually no identifiable Golgi stacks remained after a 1-h
treatment with BFA at 39.5°C in control (anti-110-12) or
anti-EAGE-injected cells (Fig. 6 a). Thus, the scattered
patches of Golgi membranes positive for
Microinjected Anti-EAGE Inhibits Reformation
of Golgi Stacks after BFA Wash-Out
Anti-EAGE inhibit protein transport in the anterograde
(Pepperkok et al., 1993 Virus-infected Vero cells were treated with BFA for 1 h,
microinjected with antibodies in the presence of BFA at
39.5°C, and subsequently transferred to medium at permissive temperature lacking the drug. Initially, ts-O45-G is
accumulated in ERGIC53 positive patches (Fig. 8, a and b).
In uninjected cells or cells injected with anti-110-12, tsO45-G is transported normally to the plasma membrane
through the reforming Golgi apparatus (data not shown). However, in cells microinjected with anti-EAGE, ts-O45-G
is arrested in tubular structures (Fig. 8 c) positive for
ERGIC53 (Fig. 8 d), suggesting that under these conditions anterograde protein transport is blocked in the IC,
apparently in identical fashion to injected cells shifted to
permissive temperature that had not been treated with BFA
(Pepperkok et al., 1993
Golgi complex reassembly during BFA wash-out was
further studied by quantitative EM on thin sections of microinjected cells and determination of the volume density
of Golgi membranes was performed. Golgi stacks were absent in BFA-treated cells before the wash-out of the drug
(not shown). In uninjected control cells or cells injected
with anti-110-12, Golgi stacks reappear rapidly after washout of BFA (Fig. 6 a). The percent cell volume of reformed Golgi stacks was however inhibited by ~50% in cells microinjected with anti-EAGE (Fig. 6 a) and in contrast to the
control cells, many of the BFA-induced tubulo-vesicular
clusters remained (Fig. 6 b). No dense spherical aggregates
have, however, been detected in cells injected with either
of the two antibodies after BFA wash-out (Fig. 6 c). We
thus conclude that reassembly of the Golgi complex is only
partly inhibited by the injected anti-EAGE. Interestingly, biochemical analyses also revealed only ~50% inhibition
of acquisition of endo H resistance of ts-O45-G, although
virtually no viral glycoprotein reaches the cell surface under these conditions (Pepperkok et al., 1993 Anti-EAGE Stabilizes Membrane Binding of Relocation of coatomer is a rapid and early event of BFA
action (Donaldson et al., 1990 A small fraction of
We microinjected specific antibodies against Anti-EAGE stabilizes in vitro binding of coatomer to
membranes at least as efficiently as GTP In normal cells at steady state, membrane-bound coatomer
visualized with antibodies against Interestingly, injected anti-EAGE neither leads to accumulation of KDEL receptor in the Golgi complex in normal
cells, nor inhibit BFA-dependent relocation of KDEL receptor to the IC. It has been reported that at steady state
most of the KDEL receptor is localized to the cis-Golgi
(Lewis and Pelham, 1992 We consider it likely that anti-EAGE inhibits the budding of COPI-coated vesicles in vivo. Indeed, the number
of COPI-coated vesicles decreases significantly in microinjected cells (Pepperkok et al., 1993 Anti-EAGE inhibits reformation of Golgi stacks upon
BFA wash-out to only ~50%. This indicates that two mechanisms may lead to Golgi complex reassembly, one COPI
dependent and one COPI independent. This is consistent
with previous observations that not all Golgi membranes
partition into the ER upon treatment of cells with BFA (Oprins et al., 1993 Microinjected anti-EAGE appear to inhibit membrane
traffic from the IC to the cis-Golgi by a different mechanism from that by which they inhibit BFA-induced relocation of Golgi enzymes to the ER. Only ~50% of the normal transport of newly synthesized ts-O45-G to the Golgi
is inhibited by the injected antibodies (Pepperkok et al.,
1993 Given the recent evidence for a role of a coat immunologically related to COPI in the endocytic pathway (Whitney et al., 1995; Rothman, 1994
; Kreis et al.,
1995
; Schekman and Orci, 1996
): COPII at the ER (Barlowe et al., 1994
; Aridor et al., 1995
), COPI at the ER/IC
(Pepperkok et al., 1993
; Griffiths et al., 1995
) and Golgi
complex (Malhotra et al., 1989
), clathrin and AP1 (Robinson, 1994
), as well as so far poorly characterized COPs at
the trans-Golgi network (TGN) the exit site of the Golgi
complex (Griffiths et al., 1985
; Ladinsky et al., 1994
;
Narula and Stow, 1995
). Immunolocalization of COPs in
mammalian cells is consistent with their sites of action (Oprins et al., 1993
; Griffiths et al., 1995
; Shaywitz et al., 1995
). In yeast COPI and COPII appear both involved in
the formation of distinct anterogradely directed transport
intermediates from ER membranes (Bednarek et al.,
1995
). Missorted ER proteins and components of the targeting and fusion machinery bearing specific signals are
retrieved from the IC and the Golgi complex by specific receptors, like the KDEL receptor for example (Pelham,
1995
; see also Miesenbock and Rothman, 1995
), and genetic evidence in yeast indicates that retrograde transport
of proteins with an ER retrieval signal KKXX depends on
COPI (Letourneur et al., 1994
; Cosson et al., 1996
; see
however also Duden et al., 1994
; Wuestehube et al., 1996
).
), with membranes (Donaldson et al., 1992
; Helms
and Rothman, 1992
; Stamnes and Rothman, 1993
). As a
consequence, binding of COPI (and most likely some other related COPs), as well as the TGN, but not the cell surface, clathrin adaptors, to membranes is inhibited (Donaldson et al., 1990
; Robinson and Kreis, 1992
). The final
result is a dramatic reorganization of endocellular membranes; membranes of compartments connected in the
secretory and endocytic pathways appear to fuse with one another in a nonregulated manner and sorting of cargo is
impaired (Hunziker et al., 1991
; Lippincott-Schwartz et al.,
1991
; Orci et al., 1991
; Wood et al., 1991
). Most significantly, membranes of the Golgi complex relocate into the
ER leading to the rapid disappearance of morphologically
distinct stacks of Golgi cisternae (Misumi et al., 1986
; Oda
et al., 1987
; Lippincott-Schwartz et al., 1989
; see also Ulmer and Palade, 1991
). This BFA induced relocation has
given valuable insights into components and mechanisms that might be involved in the regulation of transport in the
early secretory pathway (Klausner et al., 1992
). Yet, it remains to be shown how closely BFA-induced relocation of
Golgi components into the ER resembles the normal retrograde pathway(s) involved in recycling material from
the Golgi complex to the IC/ER, and the role of coatomer
in these processes is not yet fully understood (Pelham, 1991
).
The BFA-induced transfer of Golgi components to the ER
may be due to uncovering a fusion machinery following
dissociation of coatomer (Orci et al., 1991
). This process
could then lead to a nonregulated fusion of related membranes that normally do not fuse, a consequence of uncoupling the sequential events of budding, uncoating, and fusion of transport intermediates (Elazar et al., 1994
; Rothman
and Warren, 1994
). Alternatively, and not mutually exclusive, exposure of receptors for microtubule-based molecular motors may induce the movement of cis-Golgi membranes to sites adjacent to ER membranes and somehow
facilitate their fusion (Pelham, 1991
; Lippincott-Schwartz
et al., 1995).
-COP, a subunit of
coatomer (Duden et al., 1991
), for further characterizing
the role of COPI in membrane traffic between the ER/IC
and Golgi complex. Microinjected antibodies against the EAGE peptide inhibit transport of newly synthesized temperature sensitive membrane glycoprotein of vesicular stomatitis virus (ts-O45-G) between the ER/IC and the Golgi
complex (Pepperkok et al., 1993
). We show here that these
antibodies also interfere with the BFA-induced transport
of Golgi proteins into the ER. Biochemical and morphological analyses of these inhibitory effects suggest that
anti-EAGE interfere with dissociation of coatomer from
membranes leading to the inhibition of the subsequent fusion of membranes of the Golgi complex with those of the
ER. The inhibitory effects of anti-EAGE on anterograde
transport from the IC to the Golgi complex and on BFAinduced retrograde transport appear to be different, suggesting that the function of COPI may be regulated differently in these two pathways.
Materials and Methods
). Microinjection was performed on a computer automated microinjection system
(AIS, Zeiss, Zürich, Switzerland) as described (Pepperkok et al., 1993
). Vero
cells stably expressing myc-tagged NAGT1 were obtained from Dr. Brian
Storrie (Blacksburg). Immunofluorescence was performed as described
(Pepperkok et al., 1993
) with antibodies against calnexin (Hammond and
Helenius, 1994
), PDI (Vaux et al., 1990
), ERGIC53 (Schweizer et al.,
1988
), KDEL receptor (Tang et al., 1995
), p58 (Bloom and Brashear,
1989
),
-COP (Pepperkok et al., 1993
),
-COP (Lowe and Kreis, 1995
),
and VSV-G (Kreis, 1986
). The Golgi complex was visualized in Vero cells
expressing myc-tagged NAGT1 with a monoclonal antibody against the
myc-epitope (Evan et al., 1985
) as described (Pierre et al., 1994
).
), and the volume density of
Golgi membranes per cell determined by point counting (Weibel, 1979
).
), were
infected with tsO45-VSV and incubated for 1.5 h in culture medium followed by 10 min incubation in labeling medium (culture medium lacking
methionine and cysteine). Cells were metabolically labeled with 2.8 mCi
[35S]methionine (Amersham Corp., Arlington Heights, IL) per ml labeling medium for 20 min. Cells were washed and placed into low carbonate culture medium containing 100 µg/ml cycloheximide for microinjection (all
incubations up to this step and the microinjection were done at 39.5°C).
Cells were further chased (in culture medium containing 100 µg/ml cycloheximide) as indicated in the figure legends. Preparation of cell lysates,
immunoprecipitation of ts-O45-G, endoglycosidase H (endo H) digestion,
gel electrophoresis, and fluorography were performed as described (Pepperkok et al., 1993
).
80°C. Golgi/IC enriched membranes were prepared from rat
liver using the method of Malhotra et al. (1989)
.
-COP was detected using
the monoclonal antibody M3A5 (Allan and Kreis, 1986
) at a 1:1,000 dilution followed by horseradish peroxidase-conjugated goat anti-mouse IgG
(Cappel, Malvern, PA) at a 1:2,000 dilution. Peroxidase labeling was detected using the ECL kit (Amersham) and quantified by densitometry.
Results
). These antibodies may
directly inhibit this transport by interfering with the formation or function of transport vesicles, or the inhibition
may be indirect by preventing recycling of essential factors
for anterograde transport. In an attempt to further clarify this issue we analyzed the effect of injected anti-EAGE on
the putative recycling of Golgi enzymes between the Golgi
complex and the ER/IC, and on BFA-induced relocation
of Golgi proteins to the ER/IC. If continuous recycling of
Golgi enzymes through the ER/IC normally occurred, one
should expect an accumulation of these enzymes in the
ER/IC when anterograde transport between the ER/IC and
Golgi complex is selectively blocked. As a consequence, Golgi specific trimming of the viral glycoprotein accumulated in the ER/IC should be detected.
). Even after accumulation of
ts-O45-G in the ER or IC for up to 6 h no trace of endo
H-resistant glycoprotein could be detected in cells injected
with anti-EAGE at the respective temperature (as in noninjected control cells; Fig. 1 A). Moreover, when cells were
injected with anti-EAGE at 39.5°C and kept for three additional hours at the nonpermissive temperature and then
for 3 h at 15°C with ts-O45-G finally accumulated in the IC
(anti-EAGE does not inhibit ER to IC transport) no endo
H-resistant fraction of the viral glycoprotein could be detected (not shown). Thus, either the recycling of (even a
small fraction of) Golgi enzymes through the ER/IC is
inhibited by the injected antibodies, or these enzymes,
once located at their proper site of function within the
Golgi complex, are retained. To investigate directly the
role of coatomer in transport from the Golgi complex to
the ER/IC in vivo, we used BFA-induced transfer of Golgi
enzymes into the ER combined with microinjection of specific antibodies raised against synthetic peptides of
-COP
(anti-EAGE and anti-110-12; Pepperkok et al., 1993
) and
-COP (Lowe and Kreis, 1995
).
Fig. 1.
Biochemical analysis of transfer of Golgi enzymes to
the ER. Transfer of Golgi enzymes to the ER was determined
biochemically by analyzing acquisition of endo H resistance of tsO45-G arrested in the ER. 500-1000 Vero cells infected with
tsO45-VSV were incubated for 1.5 h at 39.5°C, metabolically labeled for 10 min with 35S-met and microinjected at 39.5°C or 15°C
with Fab-fragments of anti-EAGE (EAGE), divalent anti-110-12
(110-12), or kept as noninjected controls (n.i.). (A) Noninjected
control cells were lysed directly after the pulse with 35S-met and
ts-O45-G analyzed for endo H sensitivity (0 h). 39.5°C: control or
injected cells were lysed and ts-O45-G analyzed for endo H sensitivity after a chase of 6 h at 39.5°C; 15°C: temperature was shifted
for 2 h to 15°C before injection at 15°C, and the cells were kept
for an additional 4 h at 15°C before lysis and analysis of endo H
resistance of ts-O45-G. (B) Control and injected cells were
chased for 0 h or 2.5 h at 39.5°C in the presence of 5 µg/ml BFA
before lysis and analysis of endo H resistance of ts-O45-G. Microinjected anti-EAGE, but not anti-110-12 block BFA induced acquisition of endo H resistance of ts-O45-G.
[View Larger Version of this Image (38K GIF file)]
-COP
Fig. 3.
Effect of microinjected
anti-EAGE and BFA on the distribution of ts-O45-G. ts-O45 VSV-infected
Vero cells, kept for 2 h at 39.5°C,
were microinjected at nonpermissive
temperature with Fab-fragments of
anti-EAGE (a-d) or with anti-110-12
(e and f). After injection control cells
were incubated for further 6 h at
39.5°C (a and b), or for 2.5 h at 39.5°C
with 5 µg/ml BFA (c-f). Cells were then fixed and labeled for injected
antibodies (b, d, and f) and ts-O45-G
(a, c, and e). Injected anti-EAGE has
no effect on ts-O45-G in the ER in
the control cells kept at 39.5°C (a and
b). BFA treatment, however, induces accumulation of ts-O45-G in
cytosolic patches (arrows in c and e)
which are distinct from the patches
where anti-EAGE accumulate (arrowheads in d). Anti-110-12 maintains its diffuse cytosolic distribution
after BFA treatment (f). Bar, 15 µm.
[View Larger Version of this Image (99K GIF file)]
), accumulate in
-COP positive patches (Fig. 2 f) when virus-infected cells, microinjected with anti-EAGE, are treated with BFA while continuously kept at 39.5°C. These patches do not contain the "IC marker lectin" ERGIC53 (Schweizer et al., 1988
; Schindler et al., 1993
; Arar et al., 1995
; Fig. 2, c and d), and the
ER marker proteins, PDI and calnexin (not shown), maintain their reticular distributions and are not enriched in
these structures either (Fig. 2, a and b). Furthermore, no
ts-O45-G accumulates in these patches under nonpermissive conditions (Fig. 3, c and d). Interestingly, the normally
predominantly cis-Golgi localized KDEL receptor accumulates in these BFA-treated cells in the patches containing ts-O45-G (Fig. 4, d and e) and ERGIC53, and not in those positive for Golgi enzymes or injected anti-EAGE
(Fig. 4 c). This result indicates that injected anti-EAGE
does not interfere with the BFA-induced relocation of
KDEL receptor to the IC. Furthermore, the distribution
of KDEL receptor (Fig. 4 a) appears not significantly changed in control cells microinjected with anti-EAGE
(Fig. 4 b); occasionally, formation of tubular structures can
be observed. The formation of these aggregates is specific
for injected anti-EAGE, since neither microinjected anti110-12 nor anti-
-COP (not shown; these antibodies when
injected into cells bind to coatomer in vivo) lead to an accumulation of coat proteins in patches under these conditions (Fig. 3, e and f), and no significant number of the
-COP and Golgi protein containing aggregates form in
the absence of BFA at nonpermissive temperature (Fig. 3,
a and b). Furthermore, the effect of injected anti-EAGE is
not dominant over the effect of BFA, since if the antibodies were injected after BFA treatment of infected cells, no
patches could be seen (not shown). It is also an early effect
of BFA action, since patches containing Golgi proteins
and
-COP can already be observed ~5 min after addition of the drug (data not shown). These results corroborate our biochemical data which suggest that ts-O45-G does not meet Golgi enzymes in BFA-treated cells which have
been injected with anti-EAGE. Thus, injected anti-EAGE
inhibits the BFA-induced transfer of resident Golgi proteins
to the ER and leads to a rapid accumulation of Golgi proteins in COPI containing cytoplasmic aggregates distinct
from the ER/IC.
Fig. 2.
Effect of microinjected anti-EAGE on BFA induced relocation of Golgi enzymes. Vero cells were microinjected with Fabfragments of anti-EAGE. 30 min after injection 5 µg/ml BFA was added to the medium and cells were incubated for further 2.5 h at
37°C. Cells were then fixed and double stained for injected antibodies (b, d, and f) and marker proteins for (a) the ER (PDI), (c) the intermediate compartment (ERGIC 53), and (e) the Golgi complex (NAGT1). Microinjected antibodies accumulate in patches (arrowheads in d; arrows in f) which colocalize with NAGT1 (arrows in e) but not ERGIC 53 and PDI. The injected anti-EAGE thus affect the
BFA-induced relocalization of Golgi proteins to the ER. Bar, 10 µm.
[View Larger Version of this Image (137K GIF file)]
Fig. 4.
Effect of microinjected
anti-EAGE on BFA induced relocation of KDEL receptor. ts-O45VSV-infected Vero cells were microinjected at 39.5°C with Fab-fragments of anti-EAGE (asterisks). 30 min after injection, cells were transferred into medium containing 5 µg/ml BFA (c-e) or kept in normal
medium (a and b). Cells were fixed
after 2 h and double labeled with a
murine monoclonal antibody recognizing KDEL receptor (a) and rabbit anti-EAGE (b), or triple labeled
for injected anti-EAGE (c), KDEL
receptor (d), and ts-O45-G (e).
Anti-KDEL receptor was visualized with cy3-labeled antibodies against mouse IgG and anti-EAGE
Fab-fragments with fluorescein antibodies against rabbit IgG. Cells
were then incubated with 5 mg/ml
mouse IgG, after this first incubation with secondary antibodies, to
saturate free binding sites for
mouse IgG on cy3-anti-mouse. Finally, ts-O45-G was labeled with
cy5-conjugated P5D4. Arrows indicate aggregates containing antiEAGE; note that these patches do
not contain KDEL receptor or
ts-O45-G. Arrowheads indicate patches containing KDEL receptor
and ts-O45-G note that these patches do not contain antiEAGE. Bar, 10 µm.
[View Larger Version of this Image (150K GIF file)]
-COP (Fig. 5 f).
These patches are usually not observed in noninfected
cells. These results suggest that in these BFA-treated cells
newly synthesized membrane proteins destined to the cell
surface, as well as Golgi proteins and proteins recycling
between the ER/IC and the Golgi complex, either accumulate in a subdomain of the ER which probably corresponds to the IC (see also Hammond and Helenius, 1994
),
or are transported in a BFA/COPI independent way from
the ER to the IC.
Fig. 5.
Effect of BFA on
the distribution of ER, IC,
and Golgi complex-associated
proteins in VSV-infected cells.
ts-O45 VSV-infected Vero
cells were incubated with 5 µg/ml BFA for 2.5 h at 39.5°C and then fixed and double
stained for ts-O45-G (a, c, e,
and g) and the ER protein
PDI (b), the IC marker protein ERGIC 53 (d), -COP
(f), and Golgi membrane associated p58 (h). ts-O45-G
accumulates in patches enriched in ERGIC53,
COP,
and p58; PDI maintains its
typical ER distribution and is
not affected by BFA. Bar,
10 µm.
[View Larger Version of this Image (112K GIF file)]
-COP seen by
immunofluorescence microscopy in cells injected with antiEAGE and after BFA treatment are unlikely to represent
Golgi mini-stacks. In contrast, numerous dense spherical aggregates (Fig. 7 c) were found in the BFA-treated cells
microinjected with anti-EAGE but not anti-110-12 (Fig. 6 c).
The number of these spherical aggregates appears to correspond roughly with the number of
-COP-positive patches
expected from light microscopy observations. Similar structures shown to contain
-COP and membranes have previously been observed in different cell types under various
conditions of treatment (Oprins et al., 1993
; Orci et al.,
1993
; Pepperkok et al., 1993
; Hendricks et al., 1993
). Furthermore, a significant fraction of tubulo-vesicular structures (Fig. 7, a, d, and e) appear in the injected cells; although more of these clusters are found in cells injected
with anti-EAGE, a significant number is also present in
control injected cells (Fig. 6 b). Thus, it is possible that in
cells injected with anti-EAGE, BFA-induced relocation of
resident Golgi proteins is arrested in these spherical aggregates. Since the tubulo-vesicular clusters are present in cells
injected with either antibodies, they probably represent the structures where ts-O45-G, ERGIC53, and Golgi
markers accumulate in BFA-treated cells, i.e., the IC.
Fig. 6.
Quantitative EM analysis of the effect of injected antibodies against -COP on the BFA-induced reorganization of the
Golgi complex. ts-O45 VSV-infected (2 h) Vero cells were microinjected with antibodies against
-COP before or after treatment
with 5 µg/ml BFA for 1 h. Thin sections of these cells were analyzed by EM and the volume density of Golgi stacks, tubulo-
vesicular clusters, and dense spherical aggregates determined
(Weibel, 1979
) in at least 10 different fields (see Fig. 6 for examples of these structures). Injected antibodies have no effect on the
disappearance of distinct Golgi stacks. Injected Fab-fragments of
anti-EAGE, but not anti-110-12, inhibit, however, the reassembly
of Golgi stacks by ~50% after BFA wash-out for 1 h and numerous tubulo-vesicular clusters remain which are abundant after
treatment of cells with BFA. Also, electron dense spherical aggregates form in anti-EAGE-injected BFA-treated cells, and
they readily disappear upon removal of the drug. Error bars represent the standard mean error.
[View Larger Version of this Image (18K GIF file)]
Fig. 7.
Morphological EM analysis of the effect of injected antibodies against -COP on the BFA-induced reorganization of the
Golgi complex. Typical examples for the structures identified and quantified in Fig. 5 in BFA-treated ts-O45 VSV-infected cells injected
with Fab-fragments of anti-EAGE (a-c and f) or with anti-110-12 (d, e, and g) are shown. Cells in a-e have been treated for 1 h with 5 µg/ml BFA 30 min after injection with antibodies, while BFA has been washed-out for 1 h from cells shown in f and g. a shows a low magnification and b a higher magnification micrograph of tubulo-vesicular clusters that typically accumulate in BFA-treated cells injected with
anti-EAGE; electron dense spherical aggregates are illustrated in c. In d and e tubulo-vesicular clusters are shown at low and high magnification in control cells injected with anti-110-12. Reassembled stacks of Golgi cisternae are shown in cells injected with anti-EAGE (f)
or anti-110-12 (g). C, cisterna; CE, centriole pair; G, stacks of Golgi cisternae; N, nucleus; RER, rough ER; asterisks in a and d indicate
tubulo-vesicular clusters, and in c, dense spherical aggregates. Arrowheads indicate possible COP-coated buds or vesicles. Bars: (b, c,
and e) 100 nm; (a, d, f, and g) 200 nm.
[View Larger Version of this Image (144K GIF file)]
), as well as in the BFA-induced
retrograde (this report) direction in vivo. While mixing of
Golgi and ER membranes is induced by BFA, sorting of
Golgi- from ER-specific components and subsequent transfer of these components to the location where the new
Golgi complex will reassemble must occur upon removal
of the drug. These processes may depend on the machinery regulating anterograde transport or reflect an intrinsic
property of Golgi specific components to self assemble. If
coatomer is essential for anterograde membrane traffic,
and Golgi reformation after wash-out of BFA followed
that pathway, then microinjected anti-EAGE should inhibit Golgi reassembly.
). Similarly to the anti-EAGE-
injected cells not treated with BFA,
-COP accumulates in
patches which were often found on or at the ends of tubules containing ERGIC53 and ts-O45-G (Fig. 8, e and f).
Interestingly, NAGT1 (myc-tagged and stably expressed
in Vero cells) and p58 (not shown) resumed an apparently
normal Golgi distribution in about half of the cells injected
with anti-EAGE within 1 h after removal of BFA (Fig. 9, c
and d); in the other cells, NAGT1 remained in perinuclear
clusters (e.g., lower injected cell in Fig. 9 c). No significant
effect on Golgi reassembly was found in cells injected with
anti-110-12 (Fig. 9, a and b). It appears that coatomer does not accumulate in a vesicular pattern at the periphery of
the Golgi complex demarcated by a medial-Golgi resident
protein (NAGT1) in cells injected with anti-EAGE (Fig. 9,
c and d), as is typically observed in control cells or cells injected with anti-110-12 (see arrows in Fig. 9, a, b, e, and f).
Fig. 8.
Effect of microinjected antiEAGE on transport of ts-O45-G following BFA wash-out. ts-O45 VSVinfected Vero cells were treated for 1 h
at 39.5°C with 5 µg/ml BFA. These
cells were then microinjected at nonpermissive temperature with Fab-fragments of anti-EAGE and coumarin
conjugated BSA as an injection indicator. Cells were incubated for further 30 min at 39.5°C in the presence of BFA after microinjection and then fixed (a and
b) or incubated in medium without
BFA for one more hour at 31°C to
wash-out the drug before fixation (c-f).
Cells were immunolabeled for ERGIC
53 (b and d), ts-O45-G (a, c, and e), or
injected anti-EAGE (f). Injected cells
were identified in a-d by the co-injected
coumarine BSA (not shown). ts-O45-G
is arrested in tubular structures colocalizing with ERGIC 53 (arrowheads
in c-e). Arrows in e and f indicate ts-O45-G positive dots which are labeled with the injected antibodies against -COP. Bar, 10 µm.
[View Larger Version of this Image (121K GIF file)]
Fig. 9.
Effect of injected antibodies against COP on the localization of NAGT1 after BFA wash-out in VSV-infected cells. ts-O45 VSV-infected Vero cells were incubated for 1 h at 39.5°C with 5 µg/ml BFA and subsequently microinjected at 39.5°C with Fab-fragments of anti-EAGE or with anti-110-12 in the presence of BFA. Cells were transferred into normal medium immediately after injection and BFA washed-out at 31°C for 1 h. Cells were then fixed and stained for injected antibodies (b and d) and NAGT1 (a and c). Control
cells were double labeled for NAGT1 (e) and
-COP (f) for comparison. In many cells anti-EAGE interfere with the proper reassembly
of a compact Golgi complex in a juxtanuclear region (e.g., lower injected cells in d). Injected anti-EAGE also appears to interfere with the
distribution of coatomer;
-COP and NAGT1 are more tightly colocalized in these injected cells (c and d) than in cells injected with anti110-12 or in control cells where
-COP is "wrapped-around" the NAGT1 positive membranes (arrows in a, b, e, and f). Bar, 10 µm.
[View Larger Version of this Image (153K GIF file)]
).
-COP
). In cells injected with antiEAGE, however,
-COP remains associated with patches
containing Golgi membrane proteins for at least 2 h after
addition of the drug (Fig. 3, e and f). This is an effect specific for microinjected anti-EAGE. Analyses of early time
points (1-20 min) after addition of the drug to cells microinjected with these antibodies gave no evidence for an intermittent dissociation of
-COP from membranes, indicating that
-COP remains associated with the membranes
during their redistribution into patches. Thus anti-EAGE
appears to inhibit the dissociation of coatomer from membranes in vivo. Since interference with the dynamics of membrane interactions of
-COP could be the mechanism by
which anti-EAGE affects membrane traffic, we characterized the membrane binding of
-COP in a cell-free system.
-COP remains associated in vitro
with membranes enriched in Golgi and IC after incubation
with rat liver cytosol (Fig. 10). This binding is significantly
increased (up to threefold) in the presence of GTP
S, in
agreement with previous reports (Donaldson et al., 1991
).
A 2-3-fold enhanced binding of coatomer to Golgi membranes, similar to the levels obtained with GTP
S, can also
be observed in the presence of anti-EAGE (Fig. 10). This
increased binding occurs also with monovalent Fab-fragments, can be completely competed by pre-incubation of
anti-EAGE with the EAGE peptide, and is specific since
it does not occur with control antibodies anti-110-12 (Fig.
10). Thus, anti-EAGE significantly increases the fraction
of membrane-bound coatomer. These results and the block
of BFA's activity to remove
-COP from membranes in
living cells microinjected with anti-EAGE suggest that
anti-EAGE interferes with the regulation of dissociation
of
-COP from membranes, most likely by locking it together with the other subunits of coatomer, in its membrane-bound form. We assume that this inhibition of
membrane dissociation of coatomer by anti-EAGE interferes with membrane traffic between the ER/IC and the
Golgi complex.
Fig. 10.
Anti-EAGE increases binding of COP to membranes in vitro. 14 µg of membranes enriched in IC and Golgi were
incubated for 10 min at 37°C with 160 µg rat liver cytosol in a reaction mixture containing either no additions (cytosol), 25 µM
GTP
S (GTP
S), 10 µg of anti-EAGE (EAGE), 10 µg Fab-fragments of anti-EAGE (EAGE-Fabs), 10 µg anti-110-12 (110-12),
anti-EAGE preincubated with the EAGE peptide (EAGE + Peptide), or 30 µg EAGE peptide (Peptide). To block anti-EAGE
antibodies 10 µg IgG was incubated with 30 µg EAGE peptide in
PBS for 30 min on ice immediately before addition to the reaction. Membrane bound
COP was detected by immunoblotting with M3A5 as described in Materials and Methods. Shown is a
quantitation of the immunoblots from two duplicate experiments
(top) and a corresponding autoradiogram (bottom). Error bars
represent the standard mean error.
[View Larger Version of this Image (23K GIF file)]
Discussion
-COP to
further dissect the role of coatomer in membrane traffic in
living mammalian cells. We show here that microinjected
antibodies directed against the EAGE-peptide of
-COP
block BFA-induced transfer of resident Golgi enzymes to
the ER, presumably by inhibiting the fusion of Golgi with
ER membranes. Interestingly, relocation of KDEL receptor, a membrane protein cycling between the cis-Golgi and
the IC, is not affected in these cells. Together with our previous results, which show inhibition of anterograde protein
transport from the ER/IC to the Golgi complex by the
same antibodies (Pepperkok et al., 1993
), these results indicate that COPI is involved in the regulation of both anterograde (IC to Golgi) and retrograde (Golgi to ER)
membrane traffic in the early exocytic pathway.
S, and in vivo, a
significant fraction of coatomer remains bound to aggregates of membranes in cells treated with BFA. Out of two
dozen antibodies raised against peptides along the sequence of
-COP only two (anti-EAGE, anti-110-12) bound
to coatomer when injected into cells and only anti-EAGE
interferes with coatomer function (Pepperkok et al., 1993
).
The other epitopes must thus be buried inside a protein fold,
or are inaccessible within the stable hetero-oligomeric protein complex. Indeed, upon reversible disassembly of
coatomer into its monomeric subunits these "hidden"
epitopes of
-COP become accessible to the respective peptide antibodies (Lowe and Kreis, 1995
). Thus, the domain
around the EAGE-epitope appears to be a major site for
heterologous liaisons involved in regulating membrane
binding of coatomer. Anti-EAGE may either mask the
site where a factor binds
-COP that regulates dissociation
of coatomer from its membrane receptor(s), or interfere
with a conformational change of
-COP essential for dissociation of coatomer from membranes. While strong evidence suggests that a subcomplex of coatomer composed of
-,
-, and
-COP binds to membrane proteins with an
ER-retrieval motif (Cosson and Letourneur, 1994
; Lowe
and Kreis, 1995
), and that another coatomer subcomplex
(containing
- and
-COP) may interact with members of
the p24 membrane proteins with a putative "phenylalanine"
anterograde transport motif (Fiedler et al., 1996
; Harter
et al., 1996
), our data indicate that
-COP must be intimately involved in the regulation of membrane binding of
the coatomer complex. Interestingly, binding of the
-,
-,
and
-COP subcomplex to membranes in vitro is insensitive to GTP
S (Lowe and Kreis, 1995
). It is possible that
-COP, perhaps together with
-COP with which it interacts directly in the complex (Lowe and Kreis, 1995
), may
be involved in conferring ARF-dependent, GTP sensitivity to coatomer-membrane interaction.
-COP is on vesicular
structures scattered throughout the cytoplasm or closely
attached to, and surrounding, the Golgi complex (Duden
et al., 1991
; Kreis et al., 1995
). Interestingly, while about
half of the
-COP coated vesicular structures that can be
identified immediately upon shifting tsO45-VSV-infected cells to the permissive temperature (after accumulation of
ts-O45-G in the IC at 15°C) colocalize with ts-O45-G, the
other half appears not to contain viral glycoprotein and
may thus be recycling vesicles (Griffiths et al., 1995
). This
observation is consistent with the hypothesis that
coatomer is involved in both anterograde and retrograde
membrane transport. When injected anti-EAGE interfere with transport from the IC to the Golgi complex, cargo
(e.g., ts-O45-G) is found in tubular structures containing
ERGIC53 and
-COP (Pepperkok et al., 1993
). However,
when anti-EAGE interferes with BFA induced relocation
of resident Golgi membrane proteins to the ER, virtually
no overlap of
-COP with ERGIC-53 and KDEL receptor
is observed, yet coatomer closely colocalizes with "Golgiderived cargo." In both situations no obvious vesicular
structures accumulate, but numerous aggregates can be
seen with which
-COP is associated (see also Pepperkok
et al., 1993
; Oprins et al., 1993
). Since these aggregates are
significantly more abundant in injected, BFA-treated cells
and contain Golgi resident membrane proteins, but not recycling KDEL receptor, ERGIC53, or ER proteins, they are most likely Golgi derived. These results are thus consistent with genetic data from yeast suggesting that
coatomer is involved in regulating membrane transport
from the Golgi complex to the ER.
; Tang et al., 1993
). Since the
BFA-induced relocation of Golgi resident membrane proteins to the ER/IC aggregates is inhibited by injected antiEAGE, direct retrograde transport of KDEL receptor to
the IC and BFA-induced transfer of Golgi resident proteins to the ER must follow independent routes. The simplest explanation for this finding is that the bulk of the
KDEL receptor cycles at, or between, the interfaces between the ER (IC) and the Golgi complex (cis-Golgi network; see also Griffiths et al., 1994
; Tang et al., 1995
), and
that in this cycle, recycling is independent of COPI that is
recognized by anti-EAGE. Anti-EAGE will only affect relocation to the ER of resident Golgi proteins. This observation in fact further supports a role for COPI in anterograde early secretory membrane traffic. If recycling of an
essential factor were inhibited by injected anti-EAGE,
then KDEL receptor retrieval should also be affected. It is
possible that in normal cells anti-EAGE affects anterograde COPI-dependent membrane traffic more effectively, because it binds to this form of coatomer with higher affinity. This possibility is fully consistent with the hypothesis
that coatomer is involved in more than one transport step,
and that for each distinct transport step, coatomer has different conformation or composition (i.e., different posttranslational modification of subunits, different subunit
isoforms, etc.; see Whitney et al., 1995
; Scheff et al., 1996;
Fiedler et al., 1996
; Lowe and Kreis, 1996
).
). This is in contrast
to the action of GTP
S, which also stabilizes binding of
coatomer to membranes and inhibits BFA-induced relocation of resident Golgi proteins to the ER in permeabilized
cells (Donaldson et al., 1991
), but does not interfere with
the formation of COPI-coated vesicles in vitro (Melançon et al., 1987
). The nature of the accumulating tubulo-vesicular clusters in BFA-treated cells injected with anti-EAGE
(many of which remain after wash-out of the drug) is unclear; they probably represent ER/IC membranes that accumulate when early exocytic membrane traffic is inhibited by the injected antibodies and membranes pile up as
COPI-coated vesicles cannot bud (see also Pepperkok et
al., 1993
). On the other hand, the aggregates (containing
resident Golgi transmembrane proteins) that form in the
BFA-treated cells injected with anti-EAGE are most likely
remnants of Golgi complex derived membranes which cannot fuse with ER membranes as a consequence of the antibody induced stabilization of membrane bound coatomer. It is tempting to speculate that components of the membrane fusion machinery (e.g., v-SNAREs) are present in
these aggregates and that they are inactive while covered
by coatomer.
; Orci et al., 1993
; Hendricks et al., 1993
). In addition, two functionally different domains have been
predicted within Golgi cisternae based on two different
pathways leading to mitotic Golgi disassembly (Misteli and
Warren, 1995
), and postmitotic reassembly of the Golgi
complex is regulated by two distinct fusion events depending on NSF-SNAPs-p115 and p97 (Rabouille et al., 1995
;
Acharya et al., 1995
). Interestingly however, although
coatomer is stably bound to membranes of the Golgi complex in cells injected with anti-EAGE, and coatomerdependent budding of vesicles appears inhibited, BFA still
leads to the complete disappearance of stacked Golgi cisternae. This result suggests that the BFA induced morphological changes of the Golgi complex cannot be attributed
alone to dissociation of COPI from Golgi membranes.
Other factors (e.g., ARF), closer in the cascade of events
to the action of BFA, may be more directly responsible for
this process.
), whereas virtually no BFA-induced transfer of Golgi
glycosidases to the ER can be measured in cells injected with anti-EAGE. While the precise reason for this major
difference remains unclear, several possibilities can be discussed. It could for example be argued that coatomer alone
is essential for all retrograde transport, but to some extent
redundant (with COPII) for transport in the anterograde
direction. It could also be speculated that significantly
more binding sites for coatomer reside on membranes of
the Golgi complex (consistent with the predominant localization of COPI to the region of the Golgi complex in normal cells) and that as a consequence inhibition of BFA-
induced Golgi to ER transport by injected anti-EAGE is
more efficient than is transport from the IC to the cisGolgi. Anti-EAGE may also more efficiently stabilize Golgi
membrane bound coatomer due to different conformations of coatomer subcomplexes (Lowe and Kreis, 1995
,
1996
; Fiedler et al., 1996
) primed to produce retrograde or
anterograde directed transport vesicles. Clearly, further
experiments will be required to resolve this conundrum.
; Aniento et al., 1996
) and the presence of
COPI subunit isoforms generated by differential phosphorylation (Sheff et al., 1996
), it would not be surprising if
different forms of coatomer mediated anterograde and
retrograde transport between the ER/IC and the Golgi
complex. Alternatively, COPI-coated vesicles may provide a "paternoster" continuously recycling immature cargo, its
receptors, as well as membrane proteins of the vesicle
docking and fusion machinery between the ER/IC and the
Golgi complex. One signal for release of cargo into the
Golgi complex would then be its dissociation from chaperones which are part of the sequential quality control machinery (Hammond and Helenius, 1995
). Simultaneous visualization of movement of fluorescently modified cargo
and coat proteins in living cells may provide further insight
into the mechanisms of regulation of early secretory membrane traffic.
The present address of J. Scheel is the Department of Physical Biology, Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
Received for publication 26 September 1996 and in revised form 24 January 1997.
R. Pepperkok was a recipient of a European Molecular Biology Organization (EMBO) longterm postdoctoral fellowship and M. Lowe was supported by a Traveling Research Fellowship from the Welcome Trust. T.E. Kreis was supported by grants from the Fonds Nationale Suisse, the Canton de Genève, and the International Human Frontier Science Program.We thank Jean Gruenberg and Andy Whitney for helpful comments on the manuscript, Brian Storrie for the NAGT1-Vero cells, and George Bloom, Steve Fuller, Hans-Peter Hauri, Ari Helenius, Wanjin Hong, and Bor Luen Tang for antibodies; we appreciated Heinz Horstmann's help with electron microscopy.
BFA, brefeldin A; COP, coat protein; EM, electron microscopy; endo H, endoglycosidase H; IC, intermediate compartment.