(Received for publication, July 24, 1995; and in revised form, August 25, 1995)
From the
Ribophorin I is a type I transmembrane glycoprotein specific to
the rough endoplasmic reticulum. We have previously shown that, when
expressed in transfected HeLa cells, a carboxyl-terminally truncated
form of ribophorin I that contains most of the luminal domain
(RI) is, like the native protein, retained in the
endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa
cells leads to O-glycosylation of RI
by
glycosyltransferases that are redistributed from the Golgi apparatus to
the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S.,
Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of
glycosylation of RI
as a measure for the BFA-induced
backflow of enzymes of the Golgi apparatus to the ER, we now
demonstrate that the retrograde transport is inhibited when cells are
treated with various agents that affect intracellular Ca
concentrations, such as the dipeptide benzyloxycarbonyl
(Cbz)-Gly-Phe-amide, the Ca
ionophore A23187, and
thapsigargin, an inhibitor of the Ca
-transporting
ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI
. Immunofluorescence
localization of the Golgi markers, MG-160 and galactosyltransferase,
shows that when BFA is applied in the presence of Ca
modulating agents, the markers remain confined to the Golgi
apparatus and are not redistributed to the ER, as is the case when BFA
alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the
BFA-induced release of
-COP from the Golgi apparatus. We conclude
that the maintenance of a Ca
gradient between the
cytoplasm and the lumen of the ER and the Golgi apparatus is required
for the BFA-induced retrograde transport from the Golgi apparatus to
the ER to occur.
In eukaryotic cells a series of cytoplasmic organelles, which
extend from the endoplasmic reticulum (ER) ()to the plasma
membrane and includes the Golgi apparatus, forms an endomembrane system
through which flow of material can take place in both the anterograde
and retrograde directions. Anterograde flow is necessary for the
distribution of newly synthesized proteins throughout the cell and
takes place by means of vesicles that bud from a donor organelle and
fuse with an acceptor membrane (for review see Sabatini and Adesnik
(1994) and Rothman(1994)). Retrograde flow, on the other hand, must be
invoked as a mechanism to maintain the steady state composition of the
organelles and, in particular, to return to their site of origin
constitutive components (i.e. nonpassengers) of the vesicles
that effect anterograde flow (for review see Lippincott-Schwartz(1993)
and Pelham(1989)).
Forward transport between the ER and the Golgi apparatus is blocked by the drug brefeldin A (BFA) (for review see Klausner et al. (1992)). This drug acts by preventing the activation of the small GTP-binding protein ARF, which is required for the assembly of a protein coat on the donor membrane and the ensuing vesicle formation (Donaldson et al., 1992; Helms and Rothman, 1992). Under these circumstances, when anterograde transport is suppressed, retrograde transport from the Golgi to the ER becomes apparent or is, possibly, greatly induced. This leads to the redistribution of many resident Golgi constituents to the ER and to the nearly complete resorption of the Golgi apparatus into the ER (Klausner et al., 1992; De Lemos-Chiarandini et al., 1992). A striking manifestation of the redistribution of Golgi components to the ER is the modification by Golgi enzymes of ER resident proteins that are not normally accessible to them. These include the conversion of N-linked oligosaccharides to endo-H resistant forms and the addition of O-linked sugars to proteins susceptible to this modification (Lippincott-Schwartz et al., 1989; Doms et al., 1989; Ulmer and Palade, 1989; Ivessa et al., 1992).
The retrograde transport that takes place in the presence of BFA is thought to be mediated by tubular processes that in drug-treated cells are seen to emerge from Golgi cisternae, rather than by discrete vesicular carriers analogous to those that mediate anterograde transport (Lippincott-Schwartz, 1993). The biochemical requirements for anterograde transport have been studied in considerable detail using semi-intact cells (Pind et al., 1994) in which it is possible to monitor the modifications of oligosaccharides that accompany the forward flow of glycoproteins. On the other hand, very little is known of the biochemical requirements for Golgi to ER retrograde transport.
In previous work (Ivessa et al., 1992) we have observed
that the backflow of Golgi enzymes into the ER induced by BFA can be
easily measured by following the addition of O-linked sugars
to a truncated non-membrane-anchored form (RI) of the
resident ER transmembrane protein ribophorin I. As is the case with the
native protein, the truncated polypeptide is retained in the ER and
does not contain O-linked oligosaccharides. In contrast to
ribophorin I, which appears to be only a poor substrate for the Golgi
glycosyltransferases, RI
is efficiently modified by O-glycosylation when cells that express it are treated with
BFA. This may be due to the fact that the native protein is
incorporated into a macromolecular complex that renders it inaccessible
to the relocated Golgi glycosyltransferases. In this paper we have
monitored the O-glycosylation of RI
to assess
the effect of Ca
perturbing agents on the backflow of
Golgi enzymes induced by BFA. Applying this biochemical criterion as
well as results from immunofluorescence studies using Golgi markers, it
was concluded that benzyloxycarbonyl (Cbz)-Gly-Phe-amide, A23187, and
thapsigargin, three agents that perturb Ca
homeostasis in cells, suppress the BFA-induced fusion of the Golgi
elements with the ER. The importance of the regulation of cytosolic
Ca
concentrations is supported by recent findings
showing that the Ca
-binding protein calmodulin plays
a role in this process (de Figueiredo and Brown, 1995). Our results
indicate that sequestration of Ca
to intracellular
stores is required for retrograde transport between the ER and the
Golgi apparatus.
To examine the effect of Cbz-Gly-Phe-amide on the BFA-induced
retrograde flow of Golgi enzymes to the ER, permanent HeLa cell
transformants expressing RI were pulse-labeled with
[
S]methionine and then incubated for 30 min in
medium containing various concentrations of the dipeptide. BFA was then
added (5 µg/ml), and the incubation continued for up to 90 min (Fig. 1). As we previously observed (Ivessa et al.,
1992), in the absence of the dipeptide, addition of BFA led to the
conversion of RI
to a more slowly migrating form with a
half-time of approximately 30 min (Fig. 1, lanes a-c and m). We have shown (Ivessa et al., 1992) that
this modification results from the O-glycosylation of
RI
by the relocated Golgi enzymes. However, the addition
of Cbz-Gly-Phe-amide led to a dose-dependent inhibition of the
BFA-induced modification (Fig. 1, lanes d-l), and O-glycoslation was completely suppressed when the dipeptide
was present at a concentration of 3 mM (lanes
j-l). As may be expected from its metalloprotease inhibitor
activity, in parallel with its suppression of the O-glycosylation, the dipeptide also halted the degradation of
RI
as seen in the fuzzy bands in lane c or f in Fig. 1. In the presence of BFA, this degradation takes
place with a half-life of approximately 40 min (Ivessa et al.,
1992). The specificity of the inhibitory effects of Cbz-Gly-Phe-amide
was demonstrated by the complete lack of effect of the dipeptide
Cbz-Gly-Gly-amide, an inactive analogue of Cbz-Gly-Phe-amide (Strous et al., 1988; Gravotta et al., 1990; Brostrom et
al., 1991; Pitt and Schwartz, 1991) on either the BFA-induced O-glycosylation or the degradation of RI
(Fig. 2A, compare lanes d, h, and l with e, i and m, respectively). Fig. 2A also shows that the active dipeptide completely
suppresses the effects of BFA even when added simultaneously with the
drug. Since BFA is known to act very rapidly (Lippincott-Schwartz et al., 1989; De Lemos-Chiarandini et al., 1992), the
dipeptide must exert its effects almost instantaneously.
Figure 1:
The dipeptide metalloendoprotease
inhibitor Cbz-Gly-Phe-amide inhibits the BFA-induced redistribution of
Golgi glycosyltransferases to the ER as well as the degradation of a
truncated form (RI) of ribophorin I. HeLa-RI
cells, plated 1 day before the experiment in 35-mm wells of
6-well dishes at a density of 5
10
cells/well, were
incubated in serum- and methionine-free medium for 30 min and
pulse-labeled with the same medium containing
[
S]methionine (125 µCi/ml) for 10 min (see schematic at the bottom). The subsequent chase period
was carried out in medium containing 7% fetal calf serum and 5
mM unlabeled methionine in the absence (lanes a-c and m) or presence (lanes d-l) of
Cbz-Gly-Phe-amide (GP) at various concentrations (0.3, 1, and
3 mM). After 30 min BFA (5 µg/ml) was added, and the chase
period in the presence of BFA was continued for up to 90 min. Cells
were lysed and processed for immunoprecipitation using a polyclonal
rabbit anti-ribophorin I antibody that was used for all
immunoprecipitation experiments. The samples were analyzed by SDS-PAGE
(6-11% gradient gel) and fluorography (24 h exposure of Kodak
X-Omat AR film).
Figure 2:
Cbz-Gly-Phe-amide, but not the inactive
analog Cbz-Gly-Gly-amide, prevents the BFA-induced redistribution of
Golgi glycosyltransferases to the ER. HeLa-RI cell
cultures were incubated in methionine-free medium for 30 min and
subsequently labeled with
[
S]methionine-containing medium for 5 min. These
two incubations were carried out in the absence (panel A, lanes
a-n, and panel B, lane j) or presence (panel
B, lanes a-i) of BFA (5 µg/ml). The cells were
then chased for up to 90 min (A) or 60 min (B) in the
absence of drugs (panel A, lanes a, b, f, j, and n; panel B, lane
j), in the presence of BFA alone (panel A, lanes c, g, and k), in the presence of both Cbz-Gly-Gly-amide (GG; 3 mM) and BFA (panel A, lanes
d, h, and l; panel B, lanes b, d, f, and h), or in the presence of both
Cbz-Gly-Phe-amide (GP; 3 mM) and BFA (panel
A, lanes e, i, and m; panel B, lanes c, e, g, and i).
Immunoprecipitates obtained from cell lysates were analyzed by SDS-PAGE
followed by fluorography.
To establish whether Cbz-Gly-Phe-amide suppresses O-glycosylation by inhibiting the glycosyltransferases or by preventing the BFA-induced retrograde transport of Golgi enzymes, we determined whether the dipeptide was effective in suppressing O-glycosylation when added after the relocation of the Golgi enzymes had taken place. To this effect, cells that were pretreated with BFA for 30 min and then pulse-labeled and chased in the presence of BFA received either the active Cbz-Gly-Phe-amide dipeptide (Fig. 2B, lanes c, e, g, and i) or its inactive analogue (lanes b, d, f, and h) at the beginning of the chase period. In this case, the active dipeptide did not prevent O-glycosylation from proceeding, although it did cause a slight delay. These results established that the active dipeptide does not inhibit the glycosylation reaction. A direct demonstration that Cbz-Gly-Phe-amide blocks the relocation of Golgi enzymes to the ER was obtained by immunofluorescence microscopy (Fig. 3). In HeLa cells the Golgi apparatus is not a compact structure confined to a crescent-shaped perinuclear region of the cytoplasm, but rather it appears to be composed of many distinct, although smaller, Golgi complexes that are dispersed throughout the cytoplasm (De Lemos-Chiarandini et al., 1992). This is demonstrated by immunofluorescence (Fig. 3A) using an antibody to MG-160, a sialoglycoprotein of the medial cisternae of the Golgi apparatus (Croul et al., 1990; Gonatas et al., 1989). As in the case with other cell types (Lippincott-Schwartz et al., 1990), treatment of HeLa cells with BFA for 30 (D) or 60 (G) min leads to the redistribution of the Golgi marker to the ER, as indicated by the reticular fluorescence pattern of the cytoplasm obtained with the antibody to MG-160. This effect was completely abolished when the active dipeptide (F, I), but not its inactive analogue (C), was added simultaneously with BFA. Neither dipeptide when added alone, however, altered significantly the distribution of the marker observed in control cells (compare B, E, and H with control in A). Similar results (not shown) were obtained using an antibody directed against galactosyltransferase, a trans Golgi marker (Roth and Berger, 1982). Electron micrographs of cells treated with Cbz-Gly-Phe-amide in the presence of BFA show stacks of Golgi cisternae but no accumulation of vesicles (not shown), indicating that the budding stage, and not the fusion of the vesicles with ER membranes, is inhibited.
Figure 3:
The BFA-induced redistribution of Golgi
constituents to the ER is inhibited by Cbz-Gly-Phe-amide, but not by
Cbz-Gly-Gly-amide. HeLa-RI cells were incubated in the
absence (A) or presence of 5 µg/ml BFA for 30 (D)
or 60 min (G) at 37 °C. Other cultures were treated with
Cbz-Gly-Gly-amide (3 mM) for 60 min (B) or with
Cbz-Gly-Phe-amide for 30 (E) or 60 min (H).
HeLa-RI
cells were also incubated simultaneously with BFA
and Cbz-Gly-Gly-amide for 60 min (C) or with Cbz-Gly-Phe-amide
(3 mM) for 30 (F) or 60 min (I). The cells
were fixed, permeabilized, and labeled for immunofluorescence
microscopy by incubating the samples sequentially with an antibody
directed against MG-160, a marker of the medial cisternae of the Golgi
apparatus, followed by FITC-conjugated goat anti-rabbit F(ab`)
-IgG. Bar, 10.3
µm.
Present evidence suggests that BFA prevents
anterograde vesicular transport by inhibiting the assembly of coatomers
on Golgi membranes (for review see Lippincott-Schwartz, 1993). On the
other hand, treatment of cells with BFA leads to the formation of
tubular extensions that have been implicated in the backflow of Golgi
components to the ER (Lippincott-Schwartz et al., 1989, 1990).
It was conceivable that Cbz-Gly-Phe-amide inhibits the BFA-mediated
retrograde transport from the Golgi apparatus to the ER by inhibiting
the release of coatomer protein (COP) components such as -COP from
the Golgi membranes (Donaldson et al., 1990). Therefore, an
immunofluorescence study was carried out using an anti-
-COP
antibody (Allan and Kreis, 1986; Duden et al., 1991) on cells
treated with Cbz-Gly-Phe-amide and BFA. In order to facilitate the
interpretation of these experiments normal rat kidney cells were used,
which, in contrast to HeLa cells (De Lemos-Chiarandini et al.,
1992), have a discrete Golgi apparatus when immunolabeled with the
anti-MG-160 antibody (Fig. 4A). As expected, in untreated
control cells
-COP was concentrated mainly in the perinuclear
region (B) corresponding to the Golgi apparatus (A).
After treatment with BFA for 30 min the
-COP staining had a
diffuse cytoplasmic granular appearance (D), and the Golgi
marker MG-160 assumed the reticular pattern typical for ER staining (C). Treatment of the cells for 30 min with Cbz-Gly-Phe-amide
alone did not release
-COP from the Golgi apparatus (F),
and the Golgi apparatus stayed largely intact (E), although
tubular extensions were seen (see arrowheads in panel
E) similar to those observed immediately after BFA treatment
(Lippincott-Schwartz et al., 1990). Immunostaining of cells
treated with both the active dipeptide and BFA shows the typical Golgi
pattern (G) with MG-160 but diffuse labeling for the cytoplasm
when the antibody directed against
-COP was used (H).
Similar staining patterns were seen when these treatments were extended
to 60 min (not shown). Therefore, Cbz-Gly-Phe-amide exerts its
inhibitory effect on the BFA-mediated retrograde transport even though
-COP has been released from Golgi membranes. Since intact
microtubules are a prerequisite for the BFA-mediated retrograde
transport from the Golgi apparatus to the ER (Lippincott-Schwartz et al., 1990; Klausner et al., 1992), we considered
the possibility that Cbz-Gly-Phe-amide causes a breakdown of this
cytoskeletal system. However, this was not seen when cells were
analyzed by immunofluorescence using anti-
-tubulin antibodies or
by thin section electron microscopy (not shown).
Figure 4:
Cbz-Gly-Phe-amide does not inhibit the
BFA-induced redistribution of Golgi elements to the ER by preventing
the release of -COP from the Golgi apparatus. Normal rat kidney
cells were kept untreated (A and B) or were incubated
with 5 µg/ml of BFA for 30 min (C and D) or with
3 mM of Cbz-Gly-Phe-amide for 30 min (E and F). In addition cultures were treated with a combination of
both drugs for 30 min (G and H). Cells were fixed and
then labeled for immunofluorescence microscopy using the antibody
directed against the medial Golgi marker MG-160 (A, C, E, and G) or against
-COP (B, D, F, and H) followed by
FITC-conjugated goat anti-rabbit F(ab`)
-IgG or
FITC-conjugated sheep anti-mouse F(ab`)
-IgG,
respectively. Arrowheads in panels E and G indicate tubular extensions of the Golgi apparatus. Bar,
21 µm.
Although it was
initially thought (Strous et al., 1988) that the capacity of
Cbz-Gly-Phe-amide to inhibit various processes, such as protein
synthesis and the transport of secretory proteins through the cell, was
a direct consequence of its inhibition of an as yet unidentified
metalloprotease, it has been subsequently shown that the active
dipeptide, but not its inactive analogue, affects Ca homeostasis, causing the release of 70% of the total
intracellular stores, and that it lowers the cytosolic Ca
by 30% (Brostrom et al., 1991; Kuznetsov et
al., 1993). Moreover, several of the effects of the
metalloprotease inhibitors can be reproduced by treatment with the
Ca
ionophores A23187 (Brostrom and Brostrom, 1990)
and ionomycin (Kuznetsov et al., 1993). We therefore
considered the possibility that the effect of the active dipeptide on
retrograde transport was a consequence of its effect on Ca
homeostasis. Cytosolic Ca
levels decrease when
Ca
is removed from the extracellular medium, and this
effect is enhanced by the addition of the chelating agent EGTA. We
found, however, that this treatment had no effect on the BFA-induced
redistribution of Golgi enzymes as assessed by the O-glycosylation of RI
(data not shown). On the
other hand, treatment of the cells with the Ca
ionophore A23187 in a Ca
-containing medium (1.8
mM), which leads to a rapid equilibration of Ca
concentrations across cellular membranes (including ER and plasma
membrane) (Brostrom and Brostrom, 1990), completely prevented the
BFA-induced O-glycosylation of RI
(Fig. 5, lanes e-h). Again, this was due to
an inhibition of the redistribution of the Golgi glycosyltransferases
and not of their activity, since the ionophores had little inhibitory
effect on O-glycosylation when added 30 min after BFA, a time
at which the relocation of the enzymes had already taken place (Fig. 5, lanes i-l). It has been shown before
that the active dipeptide lowers the cytosolic Ca
concentration, while the ionophore, under the conditions used
(1.8 mM extracellular Ca
) raises it
(Brostrom and Brostrom, 1990; Brostrom et al., 1991). Since
both drugs caused an inhibition of the BFA-induced retrograde
transport, it appears that the effects observed may be due to the
depletion of intracellular Ca
stores. The notion that
both the active dipeptide and the ionophore act by a common mechanism,
possibly by causing a decrease in the Ca
concentration within the ER, was supported by an experiment in
which both agents were added together at suboptimal concentrations, at
which each of them appeared to only slow down the BFA-induced
retrograde transport (Fig. 6, a-h). In this case
the combined treatment almost completely prevented the relocation of
the Golgi enzymes responsible for the O-glycosylation of
RI
(Fig. 6, lanes j-l). The fuzzier
appearance of bands in lanes k and l may be due to
residual glycosylation that was not completely suppressed by the two
drugs after 70 and 90 min of treatment.
Figure 5:
In HeLa cells the BFA-induced retrograde
Golgi to ER transport is inhibited by the Ca ionophore A23187. HeLa-RI
cells were incubated in
methionine-free medium for 30 min and then pulse-labeled with
[
S]methionine for 10 min in the absence of BFA (lanes a-h). After a 30-min incubation in the absence (lanes a-d) or presence (lanes e-h) of
the Ca
ionophore A23187 (2 µM), BFA (5
µg/ml) was added, and the chase period continued for up to 90 min.
Four cultures (lanes i-l) were methionine-starved and
pulse-labeled for 5 min, both in the presence of BFA. These samples
were chased for up to 60 min in medium containing both BFA and A23187.
All samples were processed for immunoprecipitation and analyzed by
SDS-PAGE followed by fluorography.
Figure 6:
Low concentrations of Cbz-Gly-Phe-amide
and A23187 inhibit the BFA-induced O-glycosylation of
RI in a synergistic fashion. HeLa-RI
cells
were pulse-labeled with [
S]methionine for 10 min
followed by incubation for 30 min with chase medium containing 1 mM Cbz-Gly-Phe-amide (GP; lanes a-d), 0.2 µM Ca
ionophore A23187 (lanes e-h)
or both agents simultaneously (lanes i-l). BFA (5
µg/ml) was then added, and the chase period was continued for up to
90 min. Immunoprecipitates obtained from cell lysates were analyzed by
SDS-PAGE and fluorography.
The observations just
described led us to examine the effect of thapsigargin on the
BFA-induced retrograde flow of Golgi enzymes. This sesquiterpene
lactone is a selective inhibitor of the ER Ca-ATPase,
a Ca
pump that normally maintains the high
concentration of Ca
in the lumen of the organelle
(Thastrup, 1990; Thastrup et al., 1990). This drug, when added
30 min before BFA, almost completely suppressed the O-glycosylation of RI
(Fig. 7, compare lanes e-h with lanes a-d), but had no
effect when added after the BFA-induced relocation of the enzymes had
already occurred (not shown). The inhibitory effect of thapsigargin on
the relocation of Golgi enzymes was also demonstrated by
immunofluorescence using an antibody directed against
galactosyltransferase. Treatment with thapsigargin for 50 (Fig. 8D), 70 (E), and 90 (F) minutes had
little effect on the organization of the Golgi apparatus compared with
that of control HeLa cells. Pretreatment with thapsigargin for 30 min,
however, retarded considerably the effect of BFA added subsequently.
Thus, whereas 20 min of incubation with BFA alone was sufficient to
totally redistribute the Golgi marker to the ER (C), in the
thapsigargin-treated cells at this time (G) the Golgi pattern
was only somewhat altered, showing frequent tubular processes (see arrowheads in G) emanating from Golgi cisternae
similar to those that can be observed in HeLa cells after 5 min of
treatment with BFA alone (Fig. 8B; see also
Lippincott-Schwartz et al.(1990)). Even after treatment with
BFA for 40 and 60 min (H and I, respectively) in the
presence of thapsigargin the distribution of the Golgi markers had not
been fully converted to that typical for ER localization. Thus,
although thapsigargin suppressed the backflow of Golgi enzymes to the
ER, it was not as effective as the active dipeptide or the
Ca
ionophore. This may reflect the fact that
inhibition of the Ca
-ATPase by thapsigargin causes
only a slow release of Ca
from the ER lumen (Bian et al., 1991).
Figure 7:
Treatment of cells with thapsigargin
inhibits the BFA-induced Golgi to ER transport. HeLa-RI cells were pulse-labeled with
[
S]methionine for 10 min and then incubated with
chase medium in the absence (lanes a-d) or presence (lanes e-h) of thapsigargin (0.5 µM). After
30 min, BFA (5 µg/ml) was added to the cell cultures, and the
incubations were continued for up to 60 min chase time. The samples
were analyzed by fluorography of SDS-polyacrylamide gels that had been
loaded with the immunoprecipitates obtained from cell
lysates.
Figure 8:
Treatment of cells with thapsigargin
inhibits the BFA-induced redistribution of Golgi components to the ER.
HeLa-RI cells were incubated in the absence (A)
or presence of 5 µg/ml BFA for 5 (B) or 20 min (C) at 37 °C. Other cultures were treated with
thapsigargin (0.5 µM) for 50 (D), 70 min (E), or 90 min (F). HeLa-RI
cells were
also preincubated with thapsigargin for 30 min and then incubated
simultaneously with BFA and thapsigargin for 20 (G), 40 (H), or 60 min (I). The cells were fixed,
permeabilized, and labeled for immunofluorescence microscopy by
incubating the samples sequentially with an antibody directed against
galactosyltransferase, a marker of the trans cisternae of the Golgi
apparatus, followed by FITC-conjugated goat anti-rabbit F(ab`)
-IgG. Arrowheads in panels B and G indicate tubular extensions of the Golgi apparatus. Bar, 19.8 µm.
The existence of a recycling pathway that functions in the
retrieval of membranes and luminal content from the Golgi apparatus to
the ER has been extensively characterized (for review, see Pelham(1989,
1991). This retrograde pathway is greatly amplified when cells are
treated with BFA (Klausner et al., 1992). Although a strict
cytoplasmic Ca requirement for the anterograde
transport from the ER to the Golgi apparatus has been established
(Beckers and Balch(1989), Pind et al.(1994), and for review,
see Balch(1989, 1990), it was not known whether Ca
was also required for the retrograde pathway. The results
presented here demonstrate that Ca
sequestered in
intracellular stores is indeed necessary for retrograde transport to
occur. The assay used to determine the BFA-induced backflow of
membranes from the Golgi apparatus to the ER consists of HeLa cells
expressing a truncated form of ribophorin I (RI
) that is
retained in the ER and upon treatment of the transfected cells with BFA
becomes accessible to O-glycosylating enzymes from the Golgi
apparatus (Tsao et al., 1992; Ivessa et al., 1992).
We have tested the effect on the BFA-induced retrograde transport of
three compounds, Cbz-Gly-Phe-amide, A23187, and thapsigargin, all of
which are known to affect intracellular Ca
homeostasis (Brostrom and Brostrom, 1990; Thastrup, 1990).
Recently, the effects of calmodulin-specific antagonists on the
BFA-induced retrograde Golgi apparatus to ER transport have been
investigated (de Figueiredo and Brown, 1995). These results support the
notion that the regulation of intracellular Ca
levels
plays a major role in this mechanism.
In eukaryotic cells
Ca has been shown to be an important second messenger
(for review, see Brostrom and Brostrom (1990); Berridge(1993)). For
example, increases in cytosolic Ca
levels are
involved in triggering the exocytosis of storage granules or of
synaptic vesicles (for review see Rindler(1992)) where synaptotagmin
may function as a Ca
sensor (Kelly, 1995).
Mobilization of Ca
from intracellular stores affects
protein synthesis (Brostrom et al., 1991), and Ca
sequestered in the lumen of the ER is thought to be involved in
protein folding (for review see Sambrook (1990) and processing of the N-linked oligosaccharide chains of glycoproteins (Kuznetsov et al., 1992). Cytoplasmic Ca
levels are
regulated by different Ca
-ATPases located in the
plasma membrane and the membranes of the ER, the mitochondria (for
review see Carafoli and Chiesi(1992)), and the Golgi apparatus (Virk et al.(1985); see also Antebi and Fink(1992) for a homologous
yeast protein). Since mitochondria transport Ca
with
low affinity, they are thought to play no major role in the
intracellular Ca
homeostasis (Carafoli and Chiesi,
1992). Cells maintained at physiological concentrations have low levels
of cytosolic Ca
, and it is thought that most of the
cellular Ca
is sequestered in the ER (for review see
Brostrom and Brostrom, 1990). Treatment of cells with thapsigargin, a
specific inhibitor of the Ca
-ATPase of the ER
(Thastrup et al., 1990), results in the loss of Ca
from the lumen of this organelle. Since under these conditions,
the Ca
transporting systems, especially the
Ca
pumps in the plasma membrane, are functional,
Ca
levels in the ER and the cytosol are expected to
equilibrate at a low level.
We have shown that Cbz-Gly-Phe-amide
inhibits the BFA-mediated retrograde transport. This agent has the
capacity to deplete cells of intracellular Ca (Lelkes
and Pollard, 1987; Brostrom et al., 1991), and it has been
suggested that this change of cellular Ca
levels is
related to the activity of metalloendoproteases (Couch and
Strittmatter, 1983), which may be inhibited by active dipeptides, such
as Cbz-Gly-Phe-amide (Lelkes and Pollard, 1987). Metalloendoproteases
have also been directly implicated in the fusion of different cellular
membranes (for review see Lennarz and Strittmatter(1991)). Histamine
and catecholamines are released from mast cells or from adrenal
chromaffin cells, respectively, by regulated exocytosis. These events,
which are triggered by an increase of intracellular
Ca
concentrations, are blocked by metalloendoprotease
inhibitors (Mundy and Strittmatter, 1985; Lelkes and Pollard, 1987).
Lelkes and Pollard (1987) concluded, however, that metalloendoproteases
are not directly involved in membrane fusion events but at best
function in the regulation of intracellular Ca
homeostasis.
We have also shown that in HeLa-RI cells that had been exposed to thapsigargin followed by
incubation of this drug together with BFA, O-glycosylation of
RI
, which is seen in the presence of BFA alone, was
suppressed. Furthermore, we have demonstrated that this effect was not
due to an inhibition of Golgi glycosyltransferases by thapsigargin.
That this inhibitor of the ER Ca
-ATPase interfered
with the backflow of Golgi elements to the ER was supported by
immunofluorescence studies showing that staining patterns
characteristic of the Golgi apparatus were preserved to a large extent,
even after 60 min of treatment. These results suggest that the
depletion of intracellular Ca
stores that are
sensitive to thapsigargin suppresses the BFA-induced redistribution of
Golgi elements to the ER.
An entirely different class of agents that
perturbs the Ca homeostasis in cells are the
Ca
-specific ionophores, such as A23187 or ionomycin.
They increase the permeability of all cellular membranes, and at the
higher extracellular levels of Ca
, elevated cytosolic
Ca
concentrations are observed. On the other hand,
when EGTA, a chelator of divalent cations, is added to the medium in
addition to the ionophore, cytosolic Ca
concentrations will be low, and intracellular Ca
stores will be depleted (for review see Brostrom and Brostrom,
1990). While A23187 in the presence of high extracellular
Ca
levels is expected to result in high levels of
Ca
in the cytoplasm and in the lumen of the
endomembrane system, treatment with thapsigargin would lead to low
Ca
concentrations in these compartments. We have
shown here that both A23187 and thapsigargin inhibit the BFA-mediated
retrograde Golgi to ER transport. It appears, therefore, that a
Ca
gradient between the cytosol and the lumen of the
endomembrane compartments is required for retrograde transport to
occur. It has been shown that in cells pretreated with the
Ca
-specific ionophore ionomycin, addition of BFA did
not result in the conversion of the high mannose oligosaccharides of
-antitrypsin into endoglycosidase H-resistant forms
(Kuznetsov et al., 1993). The interpretation given by these
authors was that ionomycin had interfered with the proper processing of
the high mannose oligosaccharides, thus preventing further
glycosylation steps to be carried out by the Golgi glycosyltransferases
that had presumably been redistributed to the ER. However, these data
are also compatible with an inhibition, by
Ca
-specific ionophores, of the BFA-induced retrograde
transport of Golgi glycosyltransferases, an interpretation favored by
our results. In fact, a Golgi-specific Ca
-ATPase has
been identified in yeast, and deletion of the corresponding gene causes
pleotropic secretory defects (Antebi and Fink, 1992), suggesting that
elevated levels of Ca
in the lumen of the Golgi
apparatus may affect vesicular transport. Furthermore, in Golgi
fractions obtained from rat mammary glands a
Ca
-ATPase activity has been characterized (Virk et al., 1985), and microprobe analysis demonstrated elevated
Ca
levels in the Golgi apparatus of rat exocrine
pancreas cells (Roos, 1988). Since our investigations are concerned
with the retrograde transport from the Golgi apparatus to the ER, it
may be speculated that the drugs used to perturb Ca
homeostasis were also affecting the Ca
levels
in the lumen of the Golgi apparatus.
It has been observed that
Cbz-Gly-Phe-amide, but not an inactive analog, inhibits protein
transport in intact cells (Strous et al., 1988; Brostrom, et al., 1991), and endosomal transport in vitro (Pitt
and Schwartz, 1991). Interference with intracellular transport may be
achieved not only by inhibiting fusion events at the level of the
acceptor organelle but also by interfering with budding from the donor
membrane. The latter scenario is supported by experiments where
semipermeabilized Madin-Darby canine kidney cells infected with
influenza virus were incubated with Cbz-Gly-Phe-amide (Gravotta et
al., 1990). It was found that the trans Golgi network to plasma
membrane transport was inhibited mainly at early times, suggesting that
the dipeptide affects the budding stage and not the fusion event
(Gravotta et al., 1990). In analogy, our results demonstrating
an inhibition of the BFA-induced retrograde flow from the Golgi
apparatus to the ER may be due to an inhibition of the formation of
vesicles and tubules, a process that depends on the tight regulation of
intracellular Ca levels.