We have investigated the effect of
nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the
intracellular protein transport and the structure of the Golgi complex.
Pulse-chase experiments and immunoelectron microscopy showed that NDGA
strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of
secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that
oligosaccharide-processing enzymes are redistributed from the Golgi to
the ER. Morphological observations further revealed that
-mannosidase II (a cis/medial-Golgi marker),
but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the
disappearance of the characteristic Golgi structure. Upon removal of
the drug, the Golgi complex was reassembled into the normal structure
as judged by perinuclear staining of
-mannosidase II and by
restoration of the secretion. These effects of NDGA are quite similar
to those of brefeldin A. However, unlike brefeldin A, NDGA did not
cause a dissociation of
-coatomer protein, a subunit of coatomer,
from the Golgi membrane. On the contrary, NDGA exerted the stabilizing
effect on
-coatomer protein/membrane interaction against the
dissociation caused by brefeldin A and ATP depletion. Taken together,
these results indicate that NDGA is a potent agent disrupting the
structure and function of the Golgi complex with a mechanism different
from those known for other drugs reported so far.
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INTRODUCTION |
Newly synthesized secretory proteins are transported from the
endoplasmic reticulum (ER)1
to the cell surface via the Golgi complex, which is comprised of
structurally and functionally distinct subcompartments including the
cis-Golgi network, the Golgi stack, and the
trans-Golgi network (TGN) (1). In the secretory pathway, the
Golgi complex plays a key role in the sorting and modification of
proteins. Resident ER proteins are sorted at the cis-Golgi
network and recycled back to the ER (2), and lysosomal proteins are
sorted at the TGN (3). Modifications such as oligosaccharide processing
(4), proteolytic processing (5), and sulfation (6) are carried out by
enzymes with specific localizations in the Golgi subcompartments. Each
transport step along the secretory pathway is mediated by small
vesicles that bud from a donor compartment and fuse with a target
compartment membrane. Biochemical and genetic studies have identified a
number of proteins involved in the vesicular transport (1, 7-9). These
include coat protein complexes (COPI and COPII) and ADP-ribosylation
factor (ARF) or Sar1p for vesicle budding, and
N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion attachment proteins, and
soluble N-ethylmaleimide-sensitive fusion attachment protein
receptors (SNAREs) for vesicle docking and fusion. It is postulated
that the specificity of vesicle targeting is generated by complexes formed between membrane proteins on the transport vesicles (v-SNAREs) and those on the target compartments (t-SNAREs) (10).
The use of drugs affecting the secretory process at distinct sites in
the cell may prove valuable for more detailed studies of specific steps
in secretion and may lead to the understanding of the molecular basis
of the mechanisms involved in intracellular transport. One of the most
useful and characterized drugs is the fungal metabolite brefeldin A
(BFA). BFA strongly blocks secretion by apparently inhibiting protein
transport from the ER to the Golgi (11, 12) and causes redistribution
of resident Golgi proteins into the ER (12-15). The primary action of
the drug is now believed to be to inhibit Golgi membrane-catalyzed
GDP/GTP exchange of ARF (16, 17) that is required for assembly of ARF
and COPI onto the Golgi membrane (18), resulting in lack of formation
of transport vesicles. The redistribution of resident Golgi proteins to
the ER in the presence of BFA is microtubule-dependent (19)
and suggests the existence of a retrograde transport pathway without
involving COPI vesicles. This is in contrast to the recent evidence
that resident ER proteins are retrieved from the cis-Golgi network by COPI-dependent transport vesicles (2), which
also raises a question as to how COPI proteins are involved in both the
anterograde and retrograde transport from the cis-Golgi
network (9, 20). Thus, more data are required to elucidate the details of the vesicular transport mechanism.
Nordihydroguaiaretic acid (NDGA), a polyhydroxyphenolic antioxidant, is
known to exert the inhibitory effect on lipoxygenase pathways of
arachidonic acid metabolism (21, 22). It was recently demonstrated that
NDGA also inhibits the secretion of prolactin from GH3 cells (23) and
the intracellular transport of vesicular stomatitis virus G protein
(24). The drug is likely to inhibit protein transport from the ER to
the Golgi and also from the TGN to the cell surface. In the present
study we examined the effect of NDGA on secretion and intracellular
processing of secretory proteins, confirming that the drug indeed
blocks the protein transport from the ER to the Golgi complex. In
addition, we have found that NDGA rapidly disrupts the cisternal
organization of the Golgi complex and causes the redistribution of
resident Golgi proteins into the ER without dissociation of COPI from
the membrane in contrast to the effects of BFA.
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EXPERIMENTAL PROCEDURES |
Materials--
NDGA was obtained from Sigma. BFA was from Wako
Chemicals (Osaka, Japan). Antibodies to
-COP (25) and
dipeptidyl-peptidase IV (DPP IV) (26) were raised in rabbits as
described. Anti-TGN38 antibody was generously provided by Dr. G. Banting (University of Bristol, U.K.) and monoclonal
anti-
-mannosidase II (Man II) was from Berkeley Antibody Co.
(Richmond, CA). Antibodies to albumin, third component of complement
(C3), and
1-protease inhibitor (
1-PI)
were purchased from Organon Teknika (Durham, NC). Fluorescein isothiocyanate-conjugated anti-rabbit IgG and tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG were from Dakopatts (Glostrup, Denmark). Peroxidase-conjugated goat anti-rabbit IgG was from Biosys
(Compiegne, France). Biotinylated anti-mouse IgG and
peroxidase-conjugated streptavidin were from Vector Laboratories
(Burlingame, CA).
Cell Culture and Transfection--
HepG2 and H35P15 cells were
cultured in Eagle's minimum essential medium containing 10% fetal
calf serum. NRK and COS-1 cells were cultured in Dulbecco's modified
Eagle's minimum essential medium with 10% fetal calf serum.
Transfection experiments were carried out as described (27, 28), for
which a plasmid encoding a mutant DPP IV (mDPP IV) with substitution of
Gly633
Arg was constructed. The plasmid (20 µg) was
transfected into COS-1 cells with the Lipofectin Reagent. The
transfected cells were cultured for 2 days before use.
Labeling of Cells and Immunoprecipitation--
Cells in 60-mm
plastic dishes were pulse-labeled for 10 or 20 min with
[35S]methionine (50 µCi/dish) in methionine-free
Eagle's minimum essential medium and chased in the complete Eagle's
minimum essential medium in the presence or absence of NDGA (30 µM) or BFA (5 µg/ml) (11). Cycloheximide (50 µM) was added to the chase medium. At the indicated
times, the cells were separated from the medium, and cell lysates were
prepared (29). 35S-Labeled proteins of cell lysates and
medium were immunoprecipitated with the indicated antibodies,
extensively washed, and subjected to enzyme digestion when indicated
(29).
Enzyme Digestion--
Samples were incubated with endo H (0.2 unit/ml) in 50 mM acetate buffer (pH 5.5) at 37 °C for
16 h, or with neuraminidase (0.2 unit/ml) in 50 mM
acetate buffer (pH 5.0) for 20 h.
Polyacrylamide Gel Electrophoresis--
Proalbumin (pI 6.0) and
albumin (pI 5.8) were separated by electrofocusing on 5%
polyacrylamide gels (pH range from 5 to 8) as described previously
(11). SDS-PAGE was carried out on 7.5% gels (for C3) and on 10% gels
(for
1-PI) according to Laemmli (30), and the gels were
processed for fluorography (29).
Determination of Cellular ATP Level--
HepG2 cells (8 × 106 cells/150-mm dish), before or after the NDGA treatment,
were washed and suspended in phosphate-buffered saline (PBS). An equal
volume of 0.6 M perchloric acid was added to the cell
suspension and mixed well, followed by centrifugation at 32,000 × g for 5 min. The resulting supernatant was neutralized with
5 M KOH and used for the determination of ATP as described (11).
Cell Fractionation and Immunoblotting--
NRK cells were
suspended in 0.2 ml of a buffer containing 25 mM Hepes-KOH
(pH 7.2), 115 mM KCl, 2.5 mM magnesium acetate, 1 mM dithiothreitol and 0.2 M sucrose (buffer
A) and homogenized by passing 15 times through a 25-gauge needle.
Nuclei and cell debris were removed by centrifugation at 600 × g for 5 min. The postnuclear supernatant was separated into
cytosol and membrane fractions by centrifugation at 125,000 × g for 1 h. Proteins of each sample were separated by
SDS-PAGE (8% gels) and transferred to a polyvinylidene difluoride
membrane (Millipore). The membrane was incubated with rabbit
anti-
-COP IgG (25 µg/ml) for 1 h, followed by incubation with
peroxidase-conjugated anti-rabbit IgG for 1 h. The immunoreaction
was visualized by an enhanced chemiluminescence detection system
(Sigma).
Immunofluorescence Microscopy--
Cells were grown on glass
coverslips. After the indicated treatments, cells were briefly washed
with PBS and fixed with 3% paraformaldehyde in PBS for 15 min at room
temperature. The fixed cells were washed, permeabilized with 0.1%
saponin in PBS and incubated with the indicated primary antibodies for
15 min, followed by incubation for 15 min with fluorescein
isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated
secondary antibodies. To see the membrane association of
-COP, cells
on a coverslip were perforated according to Simons and Vitra (31) and
washed before fixation and immunostaining. Briefly, a HATF filter
(0.45-µm pore size, Millipore) presoaked in buffer A was placed on a
cell monolayer for 1 min. The filter was gently peeled off the cell
monolayer on the coverslip, and the perforated cells were washed with
buffer A for 5 min at room temperature. Drugs, when indicated, were
present throughout the perforation and washing steps. The cells were
then washed with PBS, fixed, and stained for
-COP as above.
Immunoelectron Microscopy--
HepG2 cells (for albumin) and
H35P15 cells (for Man II) were fixed for 2 h with the
paraformaldehyde/lysine/periodate fixative (32) and permeabilized with
0.05% saponin (12). The HepG2 cells were incubated for 2 h with
anti-albumin antibodies, followed by incubation for 1 h with
peroxidase-conjugated goat anti-rabbit IgG. The H35P15 cells were
incubated with anti-Man II antibodies and then with biotinylated
anti-mouse IgG and peroxidase-conjugated streptavidin for 1 h
each. After the peroxidase reaction, the cells were processed for
electron microscopy as described previously (12).
 |
RESULTS |
Inhibition of Secretion and Accumulation of Secretory Proteins in
the ER--
HepG2 cells synthesize various plasma proteins including
albumin, the C3, and
1-PI. In addition, albumin and C3
are initially synthesized as proforms, which are converted into mature
forms at the TGN (5, 11). In control cells, newly synthesized albumin was rapidly converted to the mature form and secreted into the medium
(Fig. 1A). The presence of a
small amount of proalbumin in the medium may be due to the relatively
low activity of the converting enzyme in HepG2 cells as compared with
that of hepatocytes (33). In the presence of 30 µM NDGA,
however, the processing and secretion of albumin were strongly blocked
(Fig. 1B). Upon removal of the drug, the labeled albumin was
normally processed and secreted into the medium (Fig. 1C),
indicating the reversibility of the drug effect. Since protein
synthesis was found to be significantly inhibited at higher
concentrations of the drug, we used NDGA at 30 µM
throughout the following experiments. Essentially the same results were
obtained for the processing and secretion of pro-C3. In the control
cells, pro-C3 synthesized as a single polypeptide of 180 kDa and was
processed into the
(115 kDa) and
(65 kDa) subunits, which were
secreted into the medium, although a considerable amount of the proform
was also secreted (Fig. 1D). The processing and secretion of
pro-C3 were completely blocked by NDGA (Fig. 1E), and this
inhibitory effect was reversible (Fig. 1F). These results
suggest that NDGA blocks secretion by inhibiting the intracellular transport of proteins before the site where the proforms of albumin and
C3 are processed into the mature forms.

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Fig. 1.
Effect of NDGA on proteolytic processing of
proalbumin and pro-C3. HepG2 cells were pulse-labeled with
[35S]methionine (50 µCi/dish) for 10 min and chased for
the indicated periods in the absence (A and D) or
presence (B and E) of 30 µM NDGA.
In C and F, the drug was removed from the medium
at 30 min of chase, and the cells were further chased in the medium
without the drug. Immunoprecipitates of albumin
(A-C) and C3 (D-F) were prepared from cell lysates (lanes 1-5) and media
(lane 6) and analyzed by gel electrofocusing (pH 5-8) for
albumin or by SDS-PAGE (7.5% gels) for C3, followed by fluorography.
Lane 1, no chase; lane 2, chase 30 min;
lane 3, 1 h; lane 4, 2 h; lanes
5 and 6, 3 h. PA and SA
(A-C) indicate proalbumin and serum-type albumin, respectively. P, , and (D-F)
denote the proform and the and subunits, respectively, of
C3.
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Immunofluorescence microscopy showed that albumin was markedly
concentrated in the juxtanuclear region in the control cells (Fig.
2A), whereas the protein was
distributed in reticular network structures extending throughout the
cytoplasm in the NDGA-treated cells (Fig. 2B). The
alteration in localization of albumin was examined in more detail by
immunoelectron microscopy. In the control cells, albumin was most
heavily stained in the Golgi complex, although also detectable in the
ER and nuclear envelope (Fig. 2C). In the cells treated with
the drug for 2 h (Fig. 2D), however, we could not
identify the characteristic Golgi stack structure where albumin had
been concentrated. The immunoreaction product was detected exclusively
in the ER and nuclear envelope. Although some mitochondria appeared to
be slightly swollen, structures of other organelles were not
significantly changed by treatment with the drug. These results suggest
that NDGA primarily blocks the protein transport from the ER, resulting
in the accumulation of secretory proteins in the ER.

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Fig. 2.
Changes in intracellular localization of
albumin caused by NDGA. HepG2 cells were incubated at 37 °C for
2 h in the absence (A and C) or presence
(B and D) of 30 µM NDGA. The cells were fixed and stained for albumin by immunofluorescence microscopy (A and B) or by immunoperoxidase electron
microscopy (C and D). ER, endoplasmic
reticulum; GA, Golgi complex; M, mitochondria; N, nucleus. Bars represent 5 µm (A
and B) and 1 µm (C and D).
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Effect of NDGA on Cellular ATP Levels--
Since it is known that
intracellular protein transport is critically dependent on cellular ATP
levels, we examined the effect of NDGA on the ATP level in HepG2 cells.
The ATP content in control cells was measured to be about 17 nmol/mg
protein. When cells were treated with the drug, the cellular ATP level
was rapidly decreased to about 40% of the control, a level that was
maintained throughout an incubation time of up to 3 h with the
drug (Fig. 3). However, removal of the
drug from the medium at 30 min of incubation did not allow the ATP
level to be recovered during the following incubation times without the
drug. This is in contrast to the reversible effect of NDGA on secretion
and proteolytic processing (Fig. 1). These results suggest that the
inhibitory effect of NDGA on protein transport may not be directly
coupled with the reduction in the cellular ATP levels caused by the
drug.

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Fig. 3.
Effect of NDGA on cellular ATP levels.
HepG2 cells were incubated at 37 °C in the absence ( ) or presence
( ) of 30 µM NDGA. In one experiment (X),
the drug was removed from the medium at 30 min of incubation ( ) and
further incubated in a fresh medium without the drug. At the indicated
times of incubation, cells were scraped from the dishes, deproteinized,
and subjected to the determination of ATP contents as described under
"Experimental Procedures." Values are expressed as percentages of
the ATP content in nontreated cells (means of three separate
experiments).
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Effect of NDGA on Oligosaccharide Processing--
If NDGA blocks
the transport of secretory glycoproteins out of the ER, it is expected
that their oligosaccharides will remain as high mannose type sensitive
to endo H digestion. To test this, we examined the biosynthesis and
processing of the secretory glycoprotein
1-PI in HepG2
cells. In control cells,
1-PI was initially synthesized as a 51-kDa form and subsequently converted to a 56-kDa form, which was
secreted into the medium (Fig.
4A, lanes 1-6).
The 51-kDa form was sensitive to endo H digestion, whereas the 56-kDa
form was resistant to endo H (Fig. 4A, lanes
7-11). In addition, the latter form decreased the molecular size
to 51 kDa when treated with neuraminidase (Fig. 4A,
lane 12). In contrast, in NDGA-treated cells, the newly
synthesized 51-kDa form was neither converted into the mature 56-kDa
form nor secreted into the medium (Fig. 4B, lanes
1-6). The 51-kDa form in the treated cells, however, showed
different responses to endo H depending on the chase times. Upon
digestion with endo H, the protein was initially converted to the
completely sensitive 41-kDa form, then to partially sensitive forms,
and finally to a single resistant 51-kDa form (Fig. 4B, lanes 7-11). The final 51-kDa form obtained at 3-h chase
was insensitive to neuraminidase treatment (Fig. 4B,
lane 13) and had the same molecular size as that of the
neuraminidase-treated form in the control cells (Fig. 4A,
lane 12). The results indicate that the 51-kDa form contains
complex type oligosaccharides without sialylation. Immunofluorescence
microscopy confirmed that
1-PI was retained in the
ER-like structures in the treated cells (Fig. 4D).

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Fig. 4.
Effect of NDGA on oligosaccharide processing
of 1-PI. HepG2 cells were pulse-labeled and chased
in the absence (A) or presence (B) of 30 µM NDGA as in Fig. 1. Immunoprecipitates of
1-PI were prepared from cell lysates (lanes
1-5, 7-11, and 13) and media (lanes
6 and 12). The samples before (lanes 1-6) and after endo H (lanes 7-11) or neuraminidase (lanes
12 and 13) digestion were analyzed by SDS-PAGE (10%
gels), followed by fluorography. Lanes 1 and 7,
no chase; lanes 2 and 8, chase 30 min;
lanes 3 and 9, 1 h; lanes 4 and
10, 2 h; lanes 5, 6,
11, 12, and 13, 3 h. Molecular
masses (kDa) are indicated at the left. In C and D, cells were incubated at 37 °C for 3 h in the
absence (C) or presence (D) of NDGA. The cells
were fixed, permeabilized, and stained with goat
anti- 1-PI in combination with tetramethylrhodamine isothiocyanate-conjugated anti-goat IgG. Bar = 5 µm.
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The finding that the oligosaccharides of
1-PI retained
in the ER are converted to the complex type might suggest that the processing enzymes localized in the Golgi complex are redistributed into the ER in the presence of NDGA. This possibility was confirmed by
another set of experiments. DPP IV is an ectoenzyme with
N-linked oligosaccharide chains (26). We found that a mutant
with substitution of Gly633
Arg (mDPP IV) is retained
in the ER and degraded there without being transported to the Golgi
complex (27, 28). When mDPP IV was expressed in COS-1 cells by
transfection, the protein was retained in the ER (Fig.
5D) and remained completely
sensitive to endo H even after 4-h chase in control cells (Fig.
5A). In the presence of NDGA, however, the protein acquired
the resistance to endo H after the chase (Fig. 5B) as
observed in BFA-treated cells (Fig. 5C). There was no
significant difference in distribution of mDPP IV between the control
and treated cells, demonstrating its retention in the ER (Fig. 5,
D and E). These results suggest that NDGA causes
the redistribution of the Golgi-resident processing enzymes to the ER
as effectively as BFA.

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Fig. 5.
Effect of NDGA on oligosaccharide processing
of ER-retained mutant DPP IV. COS-1 cells transfected with mDPP IV
cDNA were pulse-labeled for 20 min with
[35S]methionine and chased for 4 h in the absence
(A) or presence of 30 µM NDGA (B)
or BFA (5 µg/ml) (C). The cells were lysed and immunoprecipitated with anti-DPP IV. The immunoprecipitates before (lanes 1 and 2) and after (lanes 3 and
4) treatment with endo H were analyzed by SDS-PAGE and
fluorography. Lanes 1 and 3, no chase;
lanes 2 and 4, chase 4 h. Molecular mass
markers used are the (115 kDa) and (65 kDa) subunits of C3 and
transferrin (78 kDa). Localization of mDPP IV was observed by
immunofluorescence microscopy after cells were incubated in the absence
(D) or presence (E) of NDGA at 37 °C for
4 h. Bar = 10 µm.
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Redistribution of Resident Golgi Proteins to the ER--
Man II
has been used as a cis/medial-Golgi marker (4,
14), whereas TGN38 is a membrane protein localized in the TGN (34). Changes in localizations of Man II and TGN38 were examined in NRK cells
as a function of time after exposure to NDGA. Man II localized in
perinuclear regions in control cells (Fig.
6A) still remained in the same
regions with slightly fragmented and dispersed structures in cells
treated with the drug for 5 min (Fig. 6C). After 30 min of
incubation, Man II was distributed on punctate or reticular structures
over the cytoplasm, and no significant perinuclear staining was
detected (Fig. 6E). A more intense staining pattern for Man
II on the reticular structures was observed in cells exposed for 2 h (Fig. 6G). This effect of NDGA was found to be reversible.
The reticular distribution of Man II in cells treated with the drug for
1 h (Fig. 7A) completely
reversed to a perinuclear localization after 2 h of incubation
without the drug (Fig. 7D) through intermediate stepwise
changes (Fig. 7, B and C) reciprocal to those
observed in the presence of the drug (Fig. 6, C and
E). In contrast to Man II, the localization of TGN38 was not
so significantly changed by the drug (Fig. 6, B, D, F, and H), indicating that NDGA has
little effect, if any, on the TGN structure.

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Fig. 6.
Effect of NDGA on localization of resident
Golgi proteins. NRK cells were incubated at 37 °C with NDGA for
0 min (A and B), 5 min (C and
D), 30 min (E and F), and 2 h
(G and H). The cells were fixed, permeabilized,
and incubated with antibodies to Man II (A, C,
E, and G) or TGN38 (B, D,
F, and H), followed by incubation with
tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG.
Bar = 10 µm.
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Fig. 7.
Reversibility of the NDGA effect on the
localization of Man II. NRK cells were pretreated with NDGA for
1 h at 37 °C. The cells were then incubated in a fresh medium
without the drug for 0 min (A), 30 min (B),
1 h (C), and 2 h (D), fixed,
permeabilized, and stained for Man II by immunofluorescence as in Fig.
6. Bar = 10 µm.
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The change in localization of Man II was examined by immunoelectron
microscopy, for which rat hepatoma H35P15 cells were used since Man II
was more intensely stained in these cells than in the NRK cells.
Immunofluorescence microscopy confirmed the same staining profiles of
Man II in H35P15 cells (Fig. 8,
A and B) as in NRK cells (Fig. 6, A
and G). When observed by immunoelectron microscopy, Man II
was detected only in the Golgi cisternae in control cells (Fig.
8C). In contrast, no immunostainable Golgi structures could
be observed in cells treated with NDGA for 3 h. Instead, Man II
was clearly detected in the ER and weakly detected in the nuclear
envelope (Fig. 8D). Taken together, these results indicate
that the cis/medial-Golgi protein, but not the
TGN marker, redistribute into the ER in the presence of NDGA.

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Fig. 8.
Immunoelectron microscopy for localization of
Man II. Rat hepatoma H35P15 cells were incubated at 37 °C for
3 h in the absence (A and C) or presence
(B and D) of NDGA. In A and B, Man II was stained by immunofluorescence as in Fig. 6.
Bar = 10 µm. In C and D, cells
were fixed, permeabilized, and incubated with anti-Man II. The cells
were processed for immunoperoxidase electron microscopy using a
biotin-streptavidin reaction system. Thin sections were observed
without counterstaining. ER, endoplasmic reticulum;
GA, Golgi complex; N, nucleus.
Bar = 1 µm.
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Effects of NDGA on the Coatomer/Membrane Interaction--
The data
presented here show that the effects of NDGA are quite similar to those
caused by BFA. BFA is known to prevent the attachment of ARF and
coatomer (COPI) to the Golgi membrane, resulting in their rapid
dissociation from the membrane (16, 17, 35). To see whether NDGA also
has the same effect, we examined the response of
-COP, a subunit of
COPI, upon NDGA treatment.
-COP was colocalized with Man II in the
Golgi complex in control cells (Fig. 9,
panel A, a and b). In cells treated
with NDGA for 10 min,
-COP was detected in large vesicular
structures scattered in the cytoplasm and colocalized with Man II (Fig.
9, panel A, c and d). After 30 min of
incubation,
-COP and Man II were dispersed into the cytoplasm and
showed essentially the same staining profile (Fig. 9, panel
A, e and f). Immunoelectron microscopy
confirmed that
-COP was localized to the ER and nuclear envelope in
the treated cells (data not shown) as observed for Man II (Fig.
8D). When cells were perforated and washed,
-COP was
completely removed from cells that had been treated with BFA (Fig. 9,
panel B, c and d), whereas the
dispersed
-COP remained in the NDGA-treated cells (Fig. 9,
panel B, a and b). In addition, an
immunoblot analysis revealed that
-COP remained associated with
membranes in the NDGA-treated cells even after prolonged incubation
periods (Fig. 9, panel C, lanes 1-6) in contrast
to its rapid dissociation from the membranes in the BFA-treated cells
(lanes 7 and 8).

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Fig. 9.
Effect of NDGA on the distribution of
-COP. In panel A, NRK cells were treated at 37 °C
with 30 µM NDGA for 0 (a and b), 10 (c and d), or 30 (e and f)
min. The cells were fixed, permeabilized, and double-stained for
-COP (a, c, and e) and Man II
(b, d, and f). Bar = 10 µm. In panel B, cells were incubated with 30 µM NDGA (a and b) or 5 µg/ml BFA
(c and d) at 37 °C for 30 min. The cells were
immediately fixed (a and c) or fixed after being
perforated and washed (b and d), and stained for
-COP by immunofluorescence. Bar = 10 µm. In
panel C, membrane (lanes 1, 3,
5, and 7) and cytosol (lanes 2,
4, 6, and 8) fractions were prepared
from NRK cells that had been incubated with 30 µM NDGA
for 0 (lanes 1 and 2), 30 (lanes 3 and
4), or 60 (lanes 5 and 6) min or with
5 µg/ml BFA for 30 min (lanes 7 and 8). The
samples (25 µg of protein/lane) were analyzed by SDS-PAGE (8% gel)
and immunoblotting with anti- -COP IgG.
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When cells were treated with NDGA for 5 min and then incubated with BFA
and NDGA for 2 min,
-COP was colocalized with Man II in the
perinuclear Golgi (Fig. 10,
A and B). In contrast, when cells were first
treated with BFA for 2 min followed by incubation together with NDGA
for 5 min,
-COP was completely dissociated from the Golgi (Fig.
10C), whereas Man II was still associated with the Golgi
(Fig. 10D). Depletion of cytosolic ATP is also known to
cause the dissociation of the coatomer from the Golgi membrane (35).
When cells were treated for 10 min with 2-deoxyglucose and sodium
azide,
-COP was completely dissociated into the cytoplasm while the
Golgi structure with Man II remained unaffected (Fig. 10, E
and F). In contrast, pretreatment with NDGA prevented the dissociation of
-COP from the membrane caused by the ATP depletion (Fig. 10, G and H). Once COPI was dissociated
from the membrane by prior ATP depletion treatment, however, the
addition of NDGA exerted no effect (Fig. 10, I and
J). Thus, it is likely that NDGA does not primarily
dissociate the coatomer from the Golgi membrane but rather exerts the
stabilizing effect, even against the dissociation caused by BFA and by
ATP depletion.

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Fig. 10.
Effect of NDGA on the dissociation of
-COP from the Golgi caused by BFA or by ATP depletion. In
A and B, NRK cells were treated at 37 °C first
with NDGA for 5 min and then together with BFA (2 µg/ml) for 2 min.
In C and D, cells were incubated with BFA for 2 min and then together with NDGA for 5 min. In E and
F, cells were incubated with 50 mM
2-deoxyglucose and 0.05% sodium azide for 10 min. In G and
H, cells were treated first with NDGA for 5 min and then
together with 2-deoxyglucose and sodium azide for 10 min. In
I and J, cells were treated with 2-deoxyglucose and sodium azide for 10 min and then together with NDGA for 5 min. The
cells were fixed, permeabilized, and double-stained for -COP
(A, C, E, G, and
I) and Man II (B, D, F,
H, and J). Bar = 10 µm.
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 |
DISCUSSION |
In this study we have demonstrated that NDGA strongly blocks the
secretion and causes the redistribution of resident Golgi proteins into
the ER. It is important to use about 30 µM NDGA because
higher concentrations such as 100 µM of the drug
completely inhibits protein synthesis and causes irreversible effects
on the Golgi structure and protein transport. There are several reports that demonstrate the inhibitory effect of NDGA on protein secretion and
suggest the involvement of arachidonate metabolites in the secretory
mechanism (23, 24, 36-38). It is, however, unlikely that this effect
of the drug is due to its well known inhibitory effect on lipoxygenase
pathways of arachidonic acid metabolism, since other selective
inhibitors against the pathways including caffeic acid, circumin, and
esculetin did not affect protein secretion even at considerably high
concentrations (0.1-0.2 mM) (data not shown). NDGA also
caused the reduction of cellular ATP levels, as was previously shown by
experiments in vitro (39, 40) and in vivo (41).
Although intracellular protein transport is critically dependent on the
cellular ATP level (42, 43), we assume that the reduction of the
cellular ATP level by NDGA observed here is not directly related to the
inhibition of protein transport. This is based on the results that the
intracellular transport and secretion of proteins were rapidly
recovered to the control level after removal of the drug from the
medium, whereas the ATP level was not recovered. Thus, it is likely
that the reduction of cellular ATP level to 40% of normal has little
effect, if any, on the transport from the ER to the Golgi. This is
supported by a previous finding that protein transport from the ER to
the Golgi is only slightly blocked at 30% of the normal ATP level,
although strongly blocked below 10% of the normal level (44).
In the presence of NDGA, the newly synthesized glycoproteins,
1-PI and the mutant DPP IV, despite accumulating in the
ER, acquired endo H resistance, indicating that their oligosaccharides are modified by processing enzymes localized in the
cis/medial Golgi (4). This is in contrast to a
previous finding that a glycoprotein accumulated in the ER by NDGA was
sensitive to endo H (24). The discrepancy may be explained by the
difference of incubation times with the drug; for 30 min (24) and up to
3 h (this study). On the other hand, no sialylation of the
oligosaccharides nor proteolytic processing of the proforms, which are
carried out by the TGN-localized enzymes (5, 45), took place. These results indicate that the early (the cis/medial-
and possibly trans-) Golgi enzymes are redistributed into
the ER, whereas the TGN enzymes are not. Supporting these biochemical
data, immunocytochemical analysis demonstrated that the early Golgi
marker Man II was rapidly redistributed into the ER, whereas the TGN
marker TGN38 was not. The observations confirm the proposal that the
TGN is a compartment distinct from the
cis/medial/trans-Golgi cisternae (46).
These effects of NDGA are quite similar to those of BFA; proteins
resident in the early Golgi subcompartments (cis-,
medial-, and trans-cisternae) are rapidly
redistributed into the ER in the presence of BFA (47), resulting in
disappearance of the characteristic Golgi stack structure (12-14). The
fate of the TGN after treatment with NDGA, however, is different from
that in the BFA-treated cells. Long tubular processes emanating from
the TGN in the BFA-treated cells (48, 49) could not be observed in the
NDGA-treated cells. Those tubular processes caused by BFA are supposed
to reflect the mixing of the TGN with early endosomes. Therefore, it is
unlikely that NDGA causes the fusion of TGN with endosomes. It was also
confirmed by electron microscopy that the intracellular distribution of horseradish peroxidase, a marker for fluid phase endocytosis, was not
significantly changed after treatment with NDGA (data not shown).
To elucidate the mechanism by which NDGA inhibits the protein transport
from the ER to the Golgi, one would focus on the formation of transport
vesicles. Two sets of non-clathrin coats, COPI and COPII, drive the
formation of vesicles that mediate the transport between the ER and the
Golgi (9, 50). The blockade of transport by BFA could be explained by
no assembly of ARF and COPI onto the membranes, which is due to the
inhibition of Golgi membrane-catalyzed exchange of GDP/GTP bound to ARF
(16, 17). Contrary to our expectation, and unlike BFA, NDGA did not
cause a dissociation of
-COP from the Golgi membrane. Instead,
-COP was stably associated with the membrane during incubation with
NDGA. In addition, NDGA was found to stabilize the binding of
-COP
to the membrane, preventing its dissociation from the membrane caused
by BFA or ATP depletion. It is thought that the active vesicular
transport is carried out by dynamic cycling of ARF and COPI between the
Golgi membrane and the cytosol (10). NDGA probably interrupts this
dynamic cycling by locking the COPI components onto the membrane
thereby preventing the functional formation and/or fusion of transport vesicles with the target membrane in the secretory pathway.
Recent studies revealed that COPI coatomer also plays an essential role
in retrograde transport of resident ER proteins with the COOH-terminal
KKXX motif from the Golgi to the ER (2), suggesting that
COPI is primarily involved in retrograde transport rather than in
anterograde transport (2, 20, 51). The apparent blockade of anterograde
transport by BFA (11-15) or by mutation of COPI subunits (52) may be
explained by no retrieval of cargo receptors to the ER. The coated
transport vesicles must be uncoated before fusing with the target
membrane (53). The blocking of the uncoating step for example with
GTP
S causes an accumulation of the coated vesicles that do not fuse
with the target membrane, resulting in no transport of cargo (1, 53,
54). In the presence of NDGA, COPI or at least
-COP was not
dissociated from the Golgi membranes. The Golgi membranes, however, was
initially converted into aggregate-like or fragmented structures and
finally fused with the ER, suggesting that the Golgi membranes without detachment of COPI are able to fuse with the ER membrane in the presence of NDGA. In the presence of BFA that prevents the association of COPI with the membranes, a direct membrane fusion also occurs between the ER and the Golgi in a microtubule-dependent
manner (19). These observations suggest that there would be another pathway for redistribution of the resident Golgi proteins into the ER,
independent from the COPI-dependent pathway for retrieval of resident ER proteins (2). The pathway would become evident only when
vesicular transport is blocked, regardless of the blocking sites at the
formation or fusion of transport vesicles.
Rab6, a ubiquitous Rab associated with the Golgi membranes, was found
to function in intra-Golgi transport (55). Most recently it has been
reported that overexpression of a GTP-bound mutant of Rab6 (Rab6 Q72L)
inhibits the anterograde transport (55) and induces the redistribution
of Golgi proteins into the ER (56). It was also pointed out that the
typical necklaces and tubular structures emanating from the Golgi of
BFA-treated cells (19) were never observed in cells overexpressing Rab6
Q72L even at early times of overexpression (56). Similarly our
immunocytochemical stainings failed to identify such structures in the
NDGA-treated cells. Although the NDGA treatment and overexpression of
Rab6 Q72L cause almost the same phenotypic effects as does the addition of BFA, it is unlikely that these effects arise via analogous mechanisms. Despite recent remarkable advances, details of the mechanisms for anterograde and retrograde transport in the secretory pathway still remain to be elucidated. NDGA could provide a useful tool
for investigating vesicular transport and its role in the formation and
maintenance of the Golgi complex.
During revision of this manuscript, Yamaguchi et al. (57)
also reported that NDGA caused disassembly of the Golgi complex and
suggested the possible involvement of heterotrimeric GTP-binding proteins in the organization of the Golgi complex.
We thank Drs. S. Ogata and K. Moriyama for
useful suggestions, Dr. G. Banting for providing anti-TGN38, and Dr. M. Tagaya for useful information prior to publication. We also thank Dr. J. L. Millan for critical reading of the manuscript.