* Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud,
66030 Santa Maria Imbaro (Chieti), Italy; and Department of Biology, University of California at San Diego, La Jolla,
California 92093
We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114-1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065-7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200-14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre-ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50-enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.
THE Golgi apparatus is a complex structure that can
be schematically viewed as composed of two basic
elements: flat disc-shaped cisternae and tubular-
reticular networks. Groups of three to eight cisternae piled
in stacks are in continuity with cisternae of adjacent stacks
through tubular-reticular elements. The overall tridimensional appearance of the Golgi complex is therefore ribbon-like, with alternating compact (stacked cisternae) and
noncompact (tubular-reticular) zones; the cis and trans
poles of the complex are made mostly of tubular networks
(Tanaka et al., 1986 A potentially important tool to approach the problem of
the Golgi structure is brefeldin A (BFA)1, a fungal macrocyclic lactone originally discovered as an antiviral agent and
well known for its ability to inhibit constitutive protein secretion (Takatsuki and Tamura, 1985 We have recently reported that BFA potently stimulates
an endogenous ADP-ribosylation reaction that selectively
modifies two cytosolic proteins of 38 kD (GAPDH), and
50 kD (De Matteis et al., 1994 Cell Culture
Rat basophilic leukemia (RBL)-2H3 cells were grown in DME supplemented with 16% FCS and 1 mM L-glutamine. CHO cells were cultured in
DME supplemented with 10% FCS.
Antibodies and Other Reagents
NAD+, NADP+, NADH, BFA, and GAPDH from skeletal rabbit muscles were obtained from Sigma Chemical Co. (St. Louis, MO). Tissue culture materials were from GIBCO BRL (Grand Island, NY) and Seromed (Berlin, Germany). GTP and ATP were from Boehringer Mannheim
(Mannheim, Germany). Rabbit anti- Cell Permeabilization
RBL (grown in glass chamber slides) were placed on ice and immediately
washed with the permeabilization buffer (PB: 25 mM Hepes-Koh, pH 6.95, 125 mM KOAc, 2.5 mM Mg[OAc]2, 10 mM glucose, 1 mM DTT, 1 mM
EGTA, and 0.5 µM taxol). Cells were then incubated with 3 U/ml of
streptolycin O (SLO) (Biomerieux, Marcy l'Etoile, France), previously activated for 5 min at room temperature in PB for 8 min on ice. Unbound
SLO was removed and cell monolayer was washed with cold PB, and then
treated with permeabilization buffer supplemented with 1 mg/ml rat brain
cytosol, 1 mM ATP, 250 µM UTP, 2 mM creatine phosphate, 7.3 U/ml creatine phosphokinase at 37°C for between 20-30 min (in the presence of the
indicated treatments). To check the extent of permeabilization, cells were stained with Trypan blue (and propidium iodide) and the leakage of the
cytosolic enzyme lactic dehydrogenase was measured. With the adopted
schedule of SLO treatment, 95% of cells were stained with Trypan blue or
propidium iodide and >80% of the lactic dehydrogenase activity was recovered in the supernatant of the permeabilized cell monolayer. Rat brain
cytosol was prepared according to Malhotra et al. (1989) BFA-dependent ADP-Ribosylation
ADP-Ribosylation in Permeabilized Cells.
RBL cells were plated in 24-well
plates and used after 24 h at 90% confluency (300,000 cells/well per 250 µl). They were permeabilized as described above and then exposed for 20 or
60 min to PB containing 500 µM tymidine, 30 µM 32P-NAD+ (3 µCi/sample) and, where specified, BFA. At the end of the incubations the supernatant and the cell proteins were precipitated with 10% TCA, dissolved in
sample buffer, and separated on SDS-PAGE. The radioactivity bound to
BARS-50 and GAPDH was evaluated by fluorography.
ADP-Ribosylation of Cytosol.
Cytosol and membranes were prepared
from rat brain as described (De Matteis et al., 1994
Immunofluorescence and Lectin Staining
Intact or permeabilized RBL cells were fixed in 4% paraformaldehyde in
PBS at room temperature for 10 min, quenched in 10 mM NH4Cl for 10 min, washed in PBS, and permeabilized with 0.05% saponin, 0.2% BSA in
PBS for 30 min at room temperature. The cells were stained with FITC-conjugated helix pomatia lectin (100 µg/ml in PBS containing 0.2% BSA)
for 45 min or incubated with primary antibody for 1 h at room temperature, washed thoroughly with PBS, and incubated with specific FITC-,
TRITC-, or Cy3-conjugated secondary antibody for 30 min at room temperature. After thorough washing, slides were mounted in Mowiol 4-88 (Calbiochem-Novabiochem, La Jolla, CA) and examined using a microscope equipped with a Plan-Neofluar 40× objective (Axiophot; Carl Zeiss,
Thornwood, NY). RBL or CHO cells grown in glass chamber slides
(Nunc, Roskilde, Denmark; intact or permeabilized as described above),
were fixed in 4% paraformaldehyde in PBS (pH 7.4) at room temperature
for 10 min, quenced in 10 mM NH4Cl for 10 min and then washed in PBS
and permeabilized with 0.05% saponin, 0.2% BSA in PBS for 30 min at
room temperature. Cells were stained with FITC-conjugated helix pomatia
lectin (100 µg/ml in PBS containing 0.2% BSA) for 45 min or incubated
with primary antibody for 1 h at room temperature, washed thoroughly with PBS and incubated with specific FITC-, TRITC-, or Cy3-conjuagted secondary antibody for 30 min at room temperative as described earlier
(Buccione et al., 1996 Electron Microscopy
Cells were fixed with 2% glutaraldehyde in PBS (pH 7.4), postfixed with
reduced osmium (1% of OsO4 and 1.5% of potassium ferrocianide in 0.1 M
cacodilate buffer, pH 7.4), and embedded in Epon 812 as described earlier
(Buccione et al., 1996 Preparation of BARS-50-enriched Cytosolic Fractions
Rat brain cytosol (Malhotra et al., 1989 NAD+ Is Required for the BFA-induced Tubular
Reticular Transformation of the Golgi Complex and the
Redistribution of Golgi Enzymes into the ER
To examine the effects of NAD+ on the Golgi structure,
cells were depleted of the nucleotide by a permeabilization protocol designed to selectively porate the plasma
membrane. The resulting membrane damage and cell
rounding caused a less than perfect resolution of the Golgi
complex at the immunofluorescence level; however, the
redistribution of Golgi markers into the ER by BFA remained easily detectable. Moreover, the fine structure of
the organelle was very well preserved (see Figs. 1 and 2). It
is known that the permeabilization with SLO results in the
rapid loss of most low molecular weight soluble molecules,
including NAD+, from the cell interior (Bhakdi et al.,
1993
The effect of adding NAD+ to the permeabilization medium was then examined. In the presence of the nucleotide
(15-450 µM) and dialyzed cytosol (1 mg/ml), BFA strikingly regained its ability to induce the redistribution of the
Golgi complex (Fig. 1 f), albeit with a lower potency than
in intact cells (EC50: ~5 µg/ml). Both NAD+ and cytosol
were necessary for BFA to express its activity. NAD+ had
no visible effect in the absence of BFA (Fig. 1 e). Very high concentrations of BFA (>50 µg/ml) or long incubations with the toxin were able to induce slow Golgi disassembly even in the absence of NAD+ in the incubation
medium (not shown). Possibly, at high concentrations of
BFA, the cellular NAD+ presumably remaining after permeabilization might be sufficient to sustain Golgi disassembly. To investigate whether this effect of NAD+ might
be due to the participation of the nucleotide in redox reactions, NADH (which is inactive as a substrate of ADP-
ribosylation) was added together with NAD+ at concentrations up to twice those of the oxidized nucleotide. NADH
had no effect on Golgi morphology both in the presence
and the absence of BFA (not shown).
The effects of BFA and NAD+ were also investigated at
the ultrastructural level. In thin sections of CHO and RBL
cells the Golgi complex appears as one or more stacks of
cisternae and a number of associated tubular and vesicular
profiles (Fig. 2 a). In intact cells, BFA rapidly produced its
typical effect consisting of the conversion of the Golgi stacks
into a tubular network (Fig. 2 b) and, later, of the disappearance of the Golgi complex. Upon SLO permeabilization, the cells visibly lost their cytoplasmic content but
subcellular organelles maintained a nearly normal appearance. The stacked cisternae were fairly well preserved, albeit rather thin, with rare loci of detachment from each
other, and with a small population of short tubular and vesicular profiles in their vicinity (Fig. 2 c). In permeabilized
cells incubated in the absence of NAD+, in line with the
results seen at the immunofluorescence level, BFA had no
effect on Golgi morphology even when used at concentrations up to 100-fold higher than those active in intact cells
(Fig. 2 d). However, the readdition of NAD+ in the permeabilization buffer strikingly restored the ability of BFA
to induce its typical phenotype albeit with a potency lower than that found in intact cells (the effects were visible at 5 µg/ml) (Fig. 2 f). NADP+ and NADH did not modify the
effects of NAD+ (not shown). Most of the above experiments in RBL cells were repeated in CHO cells; the findings were similar in the two cell lines (not shown), suggesting a widespread role of NAD+ in the regulation of the
Golgi structure.
BFA-dependent ADP-Ribosylation in
Permeabilized Cells
Since ADP-ribosylation had been previously studied only
in cell homogenates, it was important to verify that the reaction also occurs in permeabilized cells. Cells were porated by SLO under the same conditions used for morphological experiments, exposed to radioactive NAD+ and
BFA at concentrations twice the EC50 in permeabilized
cells, and the labeling of GAPDH and BARS-50 was evaluated. Fig. 3 A shows that ADP-ribosylation (at ~10% of
the maximal level) was clearly detectable after 20 min. The
lack of a stronger signal might be because of slow exchange of BARS-50 (native Mol Wt: ~200 kD; Di Girolamo et al., 1995 Involvement of the ADP-Ribosylation Substrates in the
Effects of BFA on Golgi Morphology
If the effect of BFA on the Golgi structure requires ADP-ribosylation, pre-ADP-ribosylated cytosol should mimic
the effect of NAD+. ADP-ribosylated cytosol was prepared by incubating brain cytosol with ADP-ribosylating
enzyme-containing membranes in the presence of BFA and
NAD+. Control cytosols were prepared with only BFA or
NAD+ or their vehicles. As previously described, BARS-50
and GAPDH were selectively ADP-ribosylated when
both BFA and NAD+ were present during the incubation
but not under control conditions (De Matteis et al., 1994
Role of Coatomer
It is known that BFA induces the cytosolic redistribution of
coatomer from Golgi membranes and that this effect precedes the disassembly of the Golgi complex. Based on this
temporal association, it has been proposed that coatomer
redistribution is a major cause of the effects of BFA on
Golgi morphology (Donaldson et al., 1991
Inhibitors of BFA-dependent ADP-Ribosylation
Prevent the BFA-induced Golgi Disassembly. Role of
the ADP-Ribosylation Substrates
As recently reported, several compounds belonging to two
different chemical groups, one containing a coumarin, and
the other a quinone ring, act as inhibitors of the BFA-
dependent ADP-ribosylation in vitro (Weigert et al., 1997
NAD+ Is Required for Golgi Disassembly by BFA
In permeabilized cells exposed to BFA at concentrations
active in intact cells and in the presence of ATP and dialyzed cytosol, the addition of physiological concentrations
of NAD+ is required for disassembly of the Golgi complex. The effect of NAD+ was not mediated by changes in
ATP levels or the state of microtubules (both are factors
already known to affect Golgi dynamics; for review see
Klausner et al., 1992 ADP-Ribosylated Cytosol Replaces NAD+ in
Supporting BFA-induced Golgi Disassembly
The main such piece of evidence is that ADP-ribosylated
cytosol can substitute for NAD+ in enabling BFA to alter
the Golgi structure. Several observations indicate that the
ADP-ribosylation of the cytosolic substrates, rather than
some unknown concomitant BFA-induced modification, is
responsible for the activity of ADP-ribosylated cytosol.
First, only the coincubation of cytosol with both BFA and
NAD+, but not with BFA or NAD+ alone, is effective in
causing the cytosol to support Golgi disassembly. While
BFA and NAD+ may have different effects, the only known
consequence of combining the two agents is the ADP-ribosylation of BARS-50 and GAPDH. Second, the effects of
BFA and NAD+ in permeabilized cells are prevented by
ADP-ribosylation inhibitors. Third, these inhibitors become unable to antagonize BFA in the presence of ADP-ribosylated cytosol. A question raised by these results is
whether the effects of ADP-ribosylation depend on the loss or gain of function of the target proteins. Complementing ADP-ribosylated cytosol with extracts enriched
in native BARS-50 reversed the effects of the pre-ADP-ribosylated cytosol, despite the presence of the cytosolic
ADP-ribosylated BARS-50. It appears, therefore, that this
protein normally acts to prevent the action of BFA and
that it loses its activity in the ADP-ribosylated state. Interestingly, GAPDH was inactive in its native and ADP-ribosylated forms. Thus, although a role for GAPDH cannot
yet be excluded, these results also suggest that BARS-50 is
likely to be the ADP-ribosylation substrate involved in
modulating the action of BFA on the Golgi structure.
ADP-Ribosylation Inhibitors Prevent the Effects
of BFA on Golgi Structure and Act Via the Cytosolic
ADP-Ribosylation Substrates
We have previously described a series of synthetic molecules endowed with the properties to prevent the BFA-
dependent ADP-ribosylation in vitro, and reported that
they inhibit the effects of BFA on the Golgi in vivo (Weigert
et al., 1997 ADP-Ribosylation of the Substrate Proteins Occurs in
Permeabilized Cells
Another evidence for a role of ADP-ribosylation in Golgi
disassembly is that this reaction occurs under the same
conditions used for morphological experiments in permeabilized cells. Thus, the ADP-ribosylated substrates are
generated concomitantly with the developement of the Golgi
disorganization by BFA. A difficulty concerning these experiments is the limited extent of ADP-ribosylation: BFA,
at a concentration twice the EC50 for inducing Golgi disruption, caused only a fraction of of BARS-50 to become
ADP-ribosylated (Fig. 3). This, in view of the fact that
ADP-ribosylation seems to act via loss of function of the
protein, as discussed above, would appear inconsistent with
a major role for ADP-ribosylation in the action of BFA.
This discrepancy may be resolved by the fact that the exchange of BARS-50 (with a native mol wt of ~200 kD; Di
Girolamo et al., 1995 Altogether, the above findings on the role of NAD+ and
ADP-ribosylated cytosol strongly support a role for NAD+
and ADP-ribosylation in the BFA-sensitive machinery controlling the Golgi architecture. ADP-ribosylation, however, is not sufficient to explain the morphological effects
of the toxin, since the ADP-ribosylated cytosol was unable
to induce the BFA phenotype alone. Additional mechanisms, most likely including the BFA-induced dissociation of the coatomer from Golgi membranes, must be required
for Golgi disruption.
Role of the Coatomer and of NAD+-dependent
Mechanisms in Golgi Disassembly
In the absence of NAD+, BFA, while inactive on the morphology of the Golgi complex, preserved its ability to
cause the detachment of coatomer-based coats from Golgi
stacks (Figs. 1-3). The dissociation revealed that in these
experiments between coatomer detachment and Golgi disassembly may seem at odds with recent literature suggesting a requirement for the coatomer to maintain the Golgi structure. It has been reported that (a) isolated Golgi
stacks incubated in vitro with coatomer-depleted mitotic
cytosol lose their structure and change into tubular networks (Misteli and Warren, 1994 What might be the process affected by the NAD+ and
ADP-ribosylation-dependent mechanism? The disassembly and redistribution of the Golgi apparatus induced by
BFA is a complex event, the key steps of which have not
been clearly identified. One possibility is that the primary
effect of BFA is the induction or stabilization of tubules
emanating from the Golgi stacks. Alternately, the disorganization of the organelle might result from the disruption
of a protein scaffold involved in maintaining the stack, and
tubulation might be secondary to the loss of structure. The
nature of this putative scaffold is unknown. Morphological
studies reveal that proteinaceous bridges resembling triad
junctions between transverse tubules and the sarcoplasmic
reticulum, as well as less structured matrix, seem to connect adjacent cisternae (Cluett and Brown, 1992 The Golgi apparatus, despite its complexity, is a very
dynamic organelle, as observed most dramatically by the
rapid and reversible effects of BFA. This study proposes
that NAD+ and ADP-ribosylation are novel factors in the
machinery controlling the structure of the Golgi complex
and, in particular, of its tubular-reticular transformation in
response to BFA. It also opens new questions concerning
the significance of the NAD+-dependent regulation in the
physiology of this organelle, and the precise role(s) of the
ADP-ribosylation protein substrates.
; Rambourg and Clermont, 1990
; Clermont et al., 1994
). A notable feature of these structures is
that despite their complexity they are highly dynamic:
stacks can rapidly change shape and tubules can be seen to
emanate from, or retract to, the cisternae under a variety
of conditions (Lippincott-Schwartz et al., 1989
; Cole et al.,
1996
). Given the central role of the Golgi complex in the
secretory process, there is much interest in understanding
the molecular mechanisms responsible for generating and
maintaining the organelle's structure as well as the relationships existing between such structure and the organelle's functions. However, although recent significant
progress mainly based on studies of Golgi reassembly after
fragmentation induced by the toxin ilimaquinone or during mitosis (Lucocq and Warren, 1987
; Lucocq et al., 1987
,
1989
; Moskalewski and Thyberg, 1990
; Souter et al., 1993
;
Acharya et al., 1995a
,b; Rabouille et al., 1995a
,b; Warren
et al., 1995
; Kondo et al., 1997
), these mechanisms are still
largely obscure.
; Misumi et al., 1986
;
Fujiwara et al., 1988
; Magner and Papagiannes, 1988
; Doms
et al., 1989
). BFA causes an impressively rapid and extensive disruption of the Golgi morphology, consisting of the
transformation of Golgi stacks into a tubular-reticular network (Lippincott-Schwartz et al., 1989
, 1990
; Orci et al.,
1991
) as early as 30 s after its application (Pavelka and Ellinger, 1993
). This is followed by the formation of long microtubule-dependent tubules connecting the Golgi region with the cell periphery, and by the redistribution of most
of the Golgi resident proteins into the ER (Lippincott-Schwartz et al., 1989
, 1990
; Alcalde et al., 1992
; Pavelka
and Ellinger, 1993
). It seems likely that understanding
how BFA disorganizes the Golgi complex would provide
important clues as to how the organelle's structure is normally maintained. It has been hypothesized that the structural effects of BFA are due to the release of coat proteins (COP) from Golgi membranes (Donaldson et al., 1991
;
Klausner et al., 1992
). Indeed, a well-characterized molecular action of BFA is to release a set of polypeptides from
the Golgi complex including the coatomer (a major protein complex involved in COPI-coated vesicle formation)
and the small GTP binding protein ARF (ADP-ribosylation factor) (Donaldson et al., 1992
; Helms and Rothman,
1992
). Other proteins released by BFA from Golgi membranes are
-adaptin, p200, the low-density lipoprotein C
(LDLC)-encoded protein, spectrin, and cyclin B (Narula
et al., 1992
; Robinson and Kreis, 1992
; Beck et al., 1994
;
Podos et al., 1994
; Jackman et al., 1995
; Erickson et al.,
1996
). Although the coatomer is indeed likely to play a
role in Golgi structure (Guo et al., 1994
; Misteli and Warren, 1994
, 1995a
,b), the evidence that its detachment from
the organelle is the sole cause of BFA-induced Golgi disruption is far from direct or clear. It is not easy to understand, for instance, how coatomer dissociation could mediate the very rapid BFA-induced tubular transformation of
Golgi stacks, since the coatomer is localized almost exclusively on cisternal rims, vesicles, buds, and tubule tips,
whereas it is virtually absent from the core of Golgi cisternae (Oprins et al., 1993
). It seems more likely that multiple
factors are involved in the dynamic control of the Golgi
shape.
). The 50-kD ADP-ribosylation substrate (BARS-50; Di Girolamo et al., 1995
) binds
GTP and is regulated by
subunits of trimeric G proteins; it has been proposed, therefore, to be a novel G protein involved in membrane transport (Di Girolamo et al.,
1995
). BFA activates ADP-ribosylation both in intact and Triton-solubilized Golgi membranes through a site with a
ligand selectivity identical to that involved in the BFA-
dependent inhibition of ARF binding (Di Girolamo et al.,
1995
). Moreover, a series of chemical inhibitors of ADP-ribosylation antagonizes the BFA-induced redistribution
of the Golgi complex in intact cells (Weigert et al., 1997
).
Thus, there is correlative evidence suggesting that ADP-
ribosylation plays a role in the cellular actions of BFA. In
this study, we use a direct approach to investigate whether ADP-ribosylation is involved in the BFA-induced disassembly of the Golgi complex by using manipulations aimed
at controlling the ADP-ribosylated state of BARS-50 and
GAPDH in the intracellular space to then examine if the
structural effects of BFA are modified. We find that treatments designed to inhibit ADP-ribosylation by depleting
permeabilized cells of NAD+ (the ADP-ribose donor in
ADP-ribosylation reactions) strongly inhibit the ability of
BFA to rapidly transform Golgi stacks into a tubular-reticular network, and that NAD+ restores the ability of BFA
to disassemble Golgi stacks. Moreover, the use of ADP-ribosylated cytosol in permeabilized cells mimicked this effect of NAD+ and prevented the effects of ADP-ribosylation inhibitors on Golgi morphology. These results implicate
NAD+, ADP-ribosylation, and the proteins involved in this
reaction in the mechanisms controlling the structure of the
Golgi complex.
Materials and Methods
-mannosidase II (Man II) antibody
was provided by K. Moremen (University of Georgia, Athens, GA), and a
rabbit anti-
-COP antibody by J. Donaldson and J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD). All other chemicals were
obtained from commercial sources at the highest available purity. BFA
was stored at
20°C in stock solutions in DMSO. Dicumarol was prepared before use as an aqueous solution.
.
). Cytosol (10 mg/ml)
and salt-washed membranes (2 mg/ml) were incubated in the presence or
absence of 200 µM NAD+ or 100 µM BFA or both for 60 min at 37°C.
Under these experimental conditions the ADP-ribosylation of BARS-50
(evaluated in parallel experiments run in the presence of 32P-NAD+) was
maximal (>90%), whereas that of GAPDH was only partial (3-4%). No
other proteins were detectably ADP-ribosylated by BFA (see Fig. 3). At
the end of the incubation the samples were centrifuged at 100,000 g for 60 min and then the supernatants (cytosol) were dialyzed for 16 h at 4°C and
used in immunofluorescence experiments in permeabilized cells as described below.
Fig. 3.
BFA induces the ADP-ribosylation of BARS-50 and
GAPDH in permeabilized cells.
(A) RBL cells were permeabilized
with 3 U/ml SLO and exposed to
10 µg/ml BFA in the presence of
32P-NAD+ for 20 or 60 min at
37°C. At the end of the incubation, proteins were separated on
SDS-PAGE, and the radioactivity bound to BARS-50 and
GAPDH in the presence of BFA
was evaluated by fluorography. Similar results were obtained in
four experiments. (B) Cytosol was
ADP-ribosylated exactly as described in Materials and Methods. Proteins were separated on SDS-PAGE and the radioactive bands revealed by fluorography. Only
BARS-50 and GAPDH were
detectably ADP-ribosylated by
BFA. GAPDH was also weakly
modified in the absence of the
toxin, due to a nonenzymatic
ADP-ribosylation different from
that induced by BFA (De Matteis
et al., 1994).
[View Larger Versions of these Images (56 + 25K GIF file)]
).
).
) was precipitated with 35% saturated (NH4)2SO4. The precipitate was dissolved in 25 mM Hepes, pH 8.0, containing 5% glycerol, 0.5 M (NH4)2SO4 and 1 mM DTT (buffer A) and
applied to a phenyl sepharose HP column (Pharmacia Biotech, Piscataway, NJ) equilibrated with buffer A. Proteins were eluted with a linear
gradient of buffer A minus (NH4)2SO4. The fractions containing BARS-50
were identified by the BFA-dependent ADP-ribosylation assay (De Matteis et al., 1994
). These fractions (containing a 45-fold enriched BARS-50 and
no GAPDH) were concentrated and dialyzed against buffer B (25 mM Hepes, pH 7.2, 50 mM K, and 1 mM Mg acetate) overnight. The final protein concentration was 2-3 mg/ml.
Results
). In addition, the brain cytosol normally used in experiments with permeabilized cells was extensively dialyzed. Fig. 1 shows that whereas in intact cells BFA caused
the expected rapid and complete diffuse redistribution of
Golgi markers (with ~50% effective concentration [EC50]
of 0.3 µg/ml), in NAD+-depleted cells (permeabilized and
exposed to dialyzed cytosol), the toxin dramatically lost
activity (Fig. 1 d) even when used at concentrations up to
100-fold higher than those active in intact cells (not shown).
The loss of BFA activity was not due to ATP depletion or
microtubule depolymerization (both conditions are known
to impede Golgi redistribution), because these experiments were routinely conducted in the presence of an
ATP-regenerating system and the microtubule stabilizer
taxol.
Fig. 1.
NAD+ is required for the BFA-induced redistribution
of Golgi markers in permeabilized cells. Intact RBL cells (a and
b) were treated with 3 µg/ml BFA for 15 min (b), or were permeabilized with 3 U/ml SLO (c-f), and incubated for 20 min at 37°C
as described in Materials and Methods in the absence (c) or the
presence of 150 µM NAD+ (e), or of 10 µg/ml BFA alone (d), or
of BFA plus 150 µM NAD+ (f). Cells were then stained with an
anti-Man II antibody. Similar results were obtained using the helix pomatia lectin, a marker of the cis Golgi compartment (not
shown). Experiments were repeated four times in duplicate with
similar results. Bar, 5 µm
[View Larger Version of this Image (68K GIF file)]
Fig. 2.
Ultrastructure of the Golgi complex in permeabilized
cells: NAD+ is required for the effect of BFA. Intact RBL cells (a
and b) were treated with 3 µg/ml BFA for 15 min (b), or were
permeabilized with 3 U/ml SLO (c-f), and incubated for 20 min
at 37°C in the absence (c) or in the presence of BFA alone (d), or
of 150 µM NAD+ (e), or of BFA in combination with 150 µM
NAD+ (f). The cells were then processed for electron microscopy. Experiments were repeated at least three times in duplicate
with similar results. Bar, 0.5 µm.
[View Larger Version of this Image (130K GIF file)]
) through the SLO-induced pores. This would result in long permanence of BARS-50 within the
cell (in close proximity of the ADP-ribosylating enzyme)
and, therefore, in high intracellular concentrations of ADP-ribosylated protein, whereas the extracellular cytosol might
be ADP-ribosylated to a much lower extent.
;
Di Girolamo et al., 1995
) (Fig. 3 B). It might be noticed
that GAPDH was weakly modified also in the absence of
the toxin, but this is known to be due to a nonenzymatic ADP-ribosylation reaction different from the specific modification induced by BFA (De Matteis et al., 1994
). Notably, BARS-50 was ADP-ribosylated exhaustively whereas
GAPDH was modified to a minor extent (2-4%). No other
protein was detectably ADP-ribosylated by BFA (Fig. 3 B).
ADP-ribosylated or control cytosols were then separated
from membranes by centrifugation and dialyzed extensively. They were indistinguishable from each other in terms
of protein composition (as determined by SDS-PAGE), and
behaved identically in a functional test measuring their ability to support the vesiculation of the Golgi complex by the
nonhydrolyzable analogue of GTP, GTP
S in permeabilized cells (not shown). Both control and ADP-ribosylated cytosols were then tested in experiments of BFA-dependent Golgi disassembly in the absence of NAD+.
Whereas control cytosols were inactive (Fig. 4 b), pre-
ADP-ribosylated cytosol supported the BFA-induced
Golgi tubular transformation (Fig. 4 e). The addition of
NAD+ did not noticeably change the activity of pre-ADP-ribosylated cytosol (Fig. 4 f) whereas, in agreement with
previous experiments (Fig. 1 f), the nucleotide restored
the ability of control cytosols to support the activity of
BFA (Fig. 4 c). It was next tested whether the addition of
extracts enriched in native (nonADP-ribosylated) BARS-50
and/or GAPDH would reverse the effects of the ADP-
ribosylated cytosol: ADP-ribosylated cytosol was mixed
with either purified GAPDH or enriched native BARS-50
(Silletta, M.G., manuscript in preparation) and assayed for
its ability to support the activity of BFA (Fig. 4, g-i).
GAPDH had no effect (not shown); instead, native
BARS-50 (in the absence of NAD+), reversed the effects
of ADP-ribosylated cytosol (Fig. 4 g). Pre-ADP-ribosylated BARS-50 or native BARS-50 with NAD+ (Fig. 4, h
and i) were, as expected, without effect. Altogether, the
data indicate that BARS-50, in the native form, acts to
prevent the Golgi-disassembling action of BFA, and that
ADP-ribosylation inactivates the protein. The role of
GAPDH, if any, remains unclear.
Fig. 4.
Pre-ADP-ribosylated cytosol replaces NAD+ in sustaining the Golgi-disassembling activity of BFA. Effects of
BARS-50-containing extracts. RBL cells were permeabilized
with 3 U/ml SLO and incubated with control (a-c) or ADP-ribosylated (d-i) cytosol (1 mg/ml) for 20 min at 37°C in the absence
(a and d) or in the presence (b, c, and e-i) of 10 µg/ml BFA. Native BARS-50 (an extract prepared as described in Materials and
Methods and diluted 10-fold in ADP-ribosylated cytosol) was
added in i (with NAD+) and in g without NAD+. ADP-ribosylated BARS-50 (an extract identical to that containing native
BARS-50 but prepared from ADP-ribosylated cytosol; see Materials and Methods) was added to h. Cells were fixed and labeled with anti-Man II antibody. Similar results were obtained in four different experiments. Bar, 5 µm.
[View Larger Version of this Image (128K GIF file)]
). Therefore, we
wanted to test whether the effect of BFA on coatomer dissociation requires NAD+. Fig. 5 shows, however, that BFA
in permeabilized cells induces the cytosolic redistribution
of the coatomer (as revealed by antibodies against
-COP),
with a potency similar to that reported in intact cells (Fig.
5 b), both in the presence and in the absence of NAD+
(Fig. 5, d and f). Also, the use of pre-ADP-ribosylated cytosol did not influence the effect of BFA on coatomer
(Fig. 5, g and h). The effect of BFA was clearly detectable
although permeabilization itself induced partial dissociation of
-COP from the Golgi apparatus in some cells
(Donaldson et al., 1991
). As noted above, by contrast, BFA
requires NAD+ or pre-ADP-ribosylated cytosol to cause
Golgi disassembly (Figs. 1 and 4). Thus, in the absence of
NAD+, BFA can dissociate coatomer from the Golgi complex without affecting the structure of the organelle; only the
presence of the nucleotide or of ADP-ribosylated substrates
allows the toxin to express its effects on Golgi morphology.
Fig. 5.
NAD+ is not required for BFA-induced coatomer dissociation from the Golgi complex in permeabilized cells. Intact
RBL cells (a and b) were treated with 3 µg/ml BFA (b), or were
permeabilized with 3 U/ml SLO and then exposed to control
buffer (c), or to a buffer containing 150 µM NAD+ (e), or 10 µg/ml
BFA alone (d), or BFA in combination with 150 µM NAD+ (f).
The cells were fixed, permeabilized with saponin, and stained with anti--COP antibody. SLO permeabilization induces a partial detachment of
-COP from Golgi complex (c) compared to
intact cells (a), but BFA is completely effective in inducing the
total cytosolic redistribution of
-COP independently of the presence of NAD+ in the permeabilization buffer (d and f). Pre-ADP-ribosylated cytosol (g and h) behaves indistinguishably from control cytosol. Similar results were obtained in permeabilized CHO
cells (not shown). The experiments were repeated four times in
duplicate with similar results. Bar, 5 µm.
[View Larger Version of this Image (65K GIF file)]
).
Dicumarol and ilimaquinone, a marine sponge metabolite
that causes the gradual and reversible breakdown of the
Golgi complex (Takizawa et al., 1993
), are relatively potent and nontoxic representatives of the two classes of
compounds. Remarkably, ADP-ribosylation inhibitors are
able to antagonize the BFA-induced redistribution of Golgi
enzymes into the ER in intact cells (Weigert et al., 1997
),
with potencies similar to those observed in assays of BFA-dependent ADP-ribosylation in vitro. This suggests that
they inhibit the BFA-induced Golgi disassembly by inhibiting ADP-ribosylation. We wanted to directly test this possibility by assessing whether ADP-ribosylation inhibitors would lose their effect in permeabilized cells exposed
to pre-ADP-ribosylated cytosol. The effect of these agents
on the fine structure of the Golgi complex, both in intact
and permeabilized cells, was first characterized. In intact
cells, as expected, a prominent early effect of BFA was the
disorganization and tubular-vesicular transformation of
the Golgi complex (Fig. 2 b); dicumarol (Fig. 6 a) or ilimaquinone (not shown) strongly inhibited these alterations and, in fact, afforded a remarkable preservation of
the stack structure. The effects of the inhibitors, in line with
previous data (Weigert et al., 1997
), were dose-dependent
and dependent on the dose of BFA, since higher concentrations of the toxin overcame the inhibition (Fig. 6 b). In
permeabilized cells, dicumarol had very similar effects to
those seen in vivo in that it inhibited the BFA and NAD+-induced Golgi disassembly in the presence of control cytosol (Fig. 7 b). It was then tested whether dicumarol would
maintain its ability to antagonize BFA in the presence of
pre-ADP-ribosylated cytosol. Remarkably, under these
conditions, the effect of dicumarol was largely prevented
(Fig. 7 d). Furthermore, when the pre-ADP-ribosylated cytosol was complemented with enriched native (nonADP-ribosylated) BARS-50 and NAD+ (Fig. 7 f), dicumarol regained its property to prevent the BFA-induced redistribution of the Golgi apparatus. GAPDH had no effect (not shown). The addition of pre-ADP-ribosylated BARS-50
under the same conditions was unable to restore dicumarol
activity (Fig. 7 h). These experiments indicate that dicumarol
either acts by preventing the ADP-ribosylation of BARS-50 or that it requires unmodified BARS-50 to exert its effects.
Fig. 6.
Dicumarol prevents the tubular-reticular transformation of the Golgi apparatus induced by BFA. RBL cells were
treated with the indicated BFA concentrations for 15 min after a
30-min pretreatment with 200 µM dicumarol. They were then processed for electron microscopy. Dicumarol (and ilimaquinone,
not shown) prevents the tubular-reticular transformation and disappearance of the Golgi stacks induced by moderate (a), but not
by high concentrations of BFA (b). Similar results were obtained
in three independent experiments run in duplicate. Bar, 0.5 µm.
[View Larger Version of this Image (147K GIF file)]
Fig. 7.
Pre-ADP-ribosylated cytosol prevents and native
BARS-50 rescues the anti-BFA effects of dicumarol on the Golgi
complex in permeabilized cells. RBL cells were permeabilized with
3 U/ml SLO, and incubated for 20 min at 37°C in a medium containing BFA (10 µg/ml) and NAD+ (150 µM) without (a, c, e, and
g) or with 200 µM dicumarol (b, d, f, and h) in the presence of
control (a and b) or pre-ADP-ribosylated (c-h) cytosol (1 mg/ml).
A native BARS-50-enriched extract (see Fig. 4 legend) was added
to (e) and (f), whereas ADP-ribosylated BARS-50 was added to
(g) and (h). The cells were fixed and labeled with anti-Man II antibody. Similar results were obtained in three independent experiments. Bar, 5 µm.
[View Larger Version of this Image (61K GIF file)]
Discussion
), since it was observed under conditions where both microtubules and ATP levels are kept
stable. It remains unclear whether the requirement for
NAD+ is absolute under all conditions, because very high
concentrations of BFA (50 or more µg/ml) can cause a
partial disorganization of the Golgi apparatus even without addition of the pyridine nucleotide in the permeabilization medium. This can be explained in two ways. One is
that very high levels of BFA can somehow bypass the
NAD+ requirement. Alternately, and, in our view, more
likely, NAD+-depleted cells most probably still contain
low amounts of the nucleotide. Our procedure for NAD+
depletion (cell permeabilization by SLO and dialysis of
the cytosol) is unlikely to be completely effective, because
of tight binding of NAD+ to cytosolic proteins and because
a large fraction of the pyridine nucleotide is trapped inside
organelles, mainly mitochondria, that are not porated by
SLO (Bhakdi et al., 1993
). Thus, conceivably, high BFA
concentrations can activate ADP-ribosylation to an extent sufficient to sustain partial Golgi disorganization even in
the presence of low levels of NAD+. More work is needed
to establish whether NAD+ is a necessary, or a potent facilitatory component, of the machinery involved in Golgi
disassembly. For brevity, however, in the rest of the discussion, the role of NAD+ will be referred to as required
for Golgi disassembly. The requirement for NAD+ has
been used in the past as one of the criteria to define the role of ADP-ribosylation in the mechanism of action of
bacterial toxins such as cholera and pertussis toxins (Moss
and Vaughan, 1988
; Okazaki and Moss, 1994
). Clearly, it is
not sufficient alone; it must be combined with converging
lines of evidence to establish the role of ADP-ribosylation in
the action of BFA.
). Certain indirect lines of evidence including
the facts that (a) the profile of activity of these drugs as inhibitors of ADP-ribosylation in vitro is similar to their profile as antagonists of BFA in vivo, and (b) they inhibit Golgi redistribution by antagonizing BFA in an apparently selective fashion, rather than through toxic or nonspecific effects, suggesting that the block of ADP-ribosylation and the inhibition of BFA in vivo are causally linked
(Weigert et al., 1997
). In this report, we have built on these
findings to provide more direct evidence that these inhibitors indeed act through the ADP-ribosylation substrates.
First, in permeabilized as well as intact cells, the inhibitors
antagonized the effect of BFA and NAD+. Second and
more important, when the roles of ADP-ribosylation in
their action were directly tested by replacing NAD+ with
pre-ADP-ribosylated cytosol as a means to support the
effect of BFA, the inhibitors dramatically lost activity.
Moreover, they recovered activity when native BARS-
50-containing extracts were added to the ADP-ribosylated
cytosol in the presence of NAD+. Altogether, these findings indicate that the ADP-ribosylation inhibitors act as
BFA antagonists by preventing the ADP-ribosylation reaction. Whether they do so by binding the enzyme or the
substrates remains to be defined.
) through the SLO-induced pores is most probably slow. Therefore, once it has entered the
cell, the protein is likely to be rapidly ADP-ribosylated and
then reside for a relatively long time in intracellular compartments, in the proximity of the ADP-ribosylation enzyme, before leaking out back into the extracellular cytosol.
This would result in a much higher ratio of ADP-ribosylated
over nonADP-ribosylated protein molecules in the cytoplasmic space than that detected int he external or the total cytosol. The intracellular levels of ADP-ribosylation, therefore, might be sufficient to support Golgi disassembly.
); and, (b) that CHO cells
with a defective
-COP (a coatomer component) exhibit a
tubular-vesicular dissociation of the Golgi complex (Guo
et al., 1994
). We believe that the seeming discrepancy between these results and ours can be explained by differences between the experimental systems used in the three laboratories: for instance, in permeabilized cells (our conditions), the overall cellular structure and the cytoskeleton
were preserved whereas Misteli and Warren (1994)
used
isolated Golgi stacks in vitro; in cells with mutated
-COP
(Guo et al., 1994
), the disorganization of the Golgi structure develops over long periods of time and may involve
mechanisms that are quite different from those rapidly triggered by BFA, with which we are concerned. It seems
reasonable to conclude that both the coatomer and a
NAD+-dependent factor(s) participate in the regulation of
the Golgi structure, and that their relative importance
might depend on experimental conditions. Moreover, our
data do not exclude; in fact, they suggest that coatomer detachment by BFA might be necessary for NAD+ to express its disassembling action on the Golgi complex.
). Large polymers of Golgi enzymes have been proposed to form in
the flat portions of the cisternae, to bind to the intercisternal matrix, and play a role in maintaining the cisternal
structure (Nilsson and Warren, 1994
; Nilsson et al., 1994
;
Slusarewicz et al., 1994
); putative components of the intercisternal matrix proteins have been identified by various
means (Kooy et al., 1992
; Fritzler et al., 1993
; Rios et al.,
1994
; Slusarewicz et al., 1994
; Nakamura et al., 1995
).
Moreover, cytoskeletal proteins (comitin, spectrin, and
ankyrin) have been found to be associated to the Golgi
complex (Weiner et al., 1993
; Beck et al., 1994
, 1997
; Devarajan et al., 1996
; Viel and Branton, 1996
). It is possible
that one or some of these putative Golgi scaffold proteins
may be regulated by NAD+ and ADP-ribosylation. This
hypothesis can now be tested by examining the effects of
NAD+ and of the two ADP-ribosylation protein substrates
(BARS-50 and GAPDH; De Matteis et al., 1994
; Di Girolamo et al., 1995
) on the state of the above Golgi protein
complexes. The properties of BARS-50 are compatible
with a regulatory role in the dynamics of the Golgi structure. BARS-50 binds GTP and is regulated by the
subunit of trimeric GTPases (Di Girolamo et al., 1995
); it has
been suggested, therefore, to be a novel G protein involved in controlling the secretory pathway. GAPDH also has interesting features in that it is a multifunctional protein
that, in addition to functioning in glycolysis, displays a
number of other unrelated activities: for instance, it associates with the anion exchanger (band 3) on the plasma
membrane as well as with microtubules and microfilaments,
and it promotes the formation of triad junctions between
transverse tubules and terminal cisternae of the sarcoplasmic reticulum (Caswell and Corbett, 1985
). The apparent
lack of effect of pure GAPDH in our assays might be because of the inability of the commercial protein to mimic
the activity of the endogenous one, or, more interestingly,
the possibility that GAPDH might be involved in the effects of BFA on the endocytic pathway, which we do not
follow with our assays. Intriguingly, it has been reported that CHO cells expressing a mutated form of GAPDH exhibit tubular extensions emanating from late endocytic compartments that are reminiscent of the BFA-induced tubules in the endocytic compartments (Peters et al., 1995
).
Received for publication 14 August 1997 and in revised form 12 September 1997.
This research was supported in part by a grant from the Italian National Research Council (Convenzione Consiglio Nazionale delle Ricerche-Consorzio Mario Negri Sud and Progetto Finalizzato contract #96.00755.PF39) and the Italian Association for Cancer Research. R. Weigert is the recipient of a fellowship from the Centro di Formazione e Studi per il Mezzogiorno.We thank G. Lenaz and M. Cavazzoni (University of Bologna, Bologna,
Italy) for helpful comments and discussion, K.W. Moremen (University of
Georgia) for the anti-Man II polyclonal antibody, J. Donaldson and J. Lippincott-Schwartz (National Institutes of Health) for the polyclonal antibody to -COP, and R. Bertazzi (Consorzio Mario Negri Sud) for preparation of the figures.
BARS-50, BFA-dependent ADP-ribosylation substrate of 50 kD; BFA, brefeldin A; COP, coat proteins; EC, effective concentration; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Man, mannosidase; PB, permeabilization buffer; RBL, rat basophilic leukemia cells; SLO, streptolycin O.
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