(Received for publication, June 29, 1995; and in revised form, November 1, 1995 )
From the
Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 µM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.
Proteins which enter the central vacuolar system are synthesized
in the endoplasmic reticulum (ER) ()where they acquire N-linked glycans which are then processed and matured as they
are sequentially transported through the cis-, medial-, and
trans-cisternae of the Golgi complex. From here they move to an array
of tubulo-vesicular structures, the trans-Golgi network (TGN) from
where sorting occurs to lysosomes, regulated secretory vesicles, and to
the plasma membrane.
The use of specific inhibitors has been highly valuable for cellular biologists, allowing the study of intracellular transport processes in intact cells in a convenient way. Nocodazole and other agents that depolymerize microtubules have been widely used to study the role of the cytoskeleton in intracellular transport (Kelly, 1990). In the last years, brefeldin A (BFA) has attracted the attention of many researchers for its effects on intracellular transport (Pelham, 1991). It was first documented that BFA inhibited the secretion of proteins at an early step. In the presence of BFA, secretory proteins were retained in the endoplasmic reticulum (Lippincott-Schwartz et al., 1989, 1990, 1991). It was found that even Golgi enzyme markers were located in the ER after a few minutes of treatment with BFA (Misumi et al., 1986; Fujiwara et al., 1988; Doms et al., 1989). In addition, no recognizable Golgi stacks were seen in BFA-treated cells. These data suggested that BFA promoted the redistribution of Golgi stacks into the ER. Markers of the TGN did not redistribute into the ER and were, however, found to fuse with endosomes. BFA did not affect the cycling between the plasma membrane and endosomes, although the traffic between endosomes and lysosomes was impaired (Lippincott-Schwartz et al., 1991). BFA produced the tubulation of Golgi stacks, endosomes, and lysosomes, and it has been suggested that these tubules may target membrane fusions (Pelham, 1991). The redistribution of Golgi markers to the ER produced by BFA is microtubule-mediated, requires ATP, and is suppressed by the addition of nonhydrolizable GTP analogs.
Other compounds that have been
lately used as probes of intracellular processes are the macrolide
antibiotics bafilomycin (Bowman et al., 1988; Yoshimori et
al., 1991; Johnson et al., 1993; Pakolangas et
al., 1994) and concanamycin (Yilla et al., 1993). These
antibiotics are highly selective and specific inhibitors of vacuolar
proton ATPases (V-H-ATPases) (Nelson and Taiz, 1989)
identified in organelles belonging to the central vacuolar system such
as lysosomes (Moriyama and Nelson, 1989a) and the Golgi complex
(Moriyama and Nelson, 1989b), as well as in coated vesicles (Xie and
Stone, 1986; Arai et al., 1987). Those ATPases are likely
responsible for the generation and maintenance of the acidity in those
organelles. The acidic lumenal environment has been suggested to be
important to assure the fidelity of vesicular transport. In this sense,
Yilla et al.(1993), showed that concanamycin B significantly
impaired intra-Golgi trafficking and plasma membrane delivery in HepG2
cells without affecting the endoplasmic reticulum to Golgi transport.
Bafilomycin A
(Yoshimori et al., 1991) and
concanamycin B (Woo et al., 1992) have been found to inhibit in vivo lysosomal protein degradation through inhibition of
the ATP-dependent acidification of endosomes and lysosomes, without an
apparent inhibition of the intracellular protein transport, including
the endocytic pathway. However other authors have shown an inhibition
of protein transport in the endocytic pathway (Oda et al.,
1991; Pakolangas et al., 1994; Johnson et al., 1993).
In addition, Xu and Shields(1994) provided evidence that
prosomatostatin processing in the TGN is inhibited by bafilomycin
A
.
Two other types of compound have been added to the
list of inhibitors of vesicular transport. The sponge metabolite
ilimaquinone (IQ) caused the breakdown of the Golgi into small
vesicles, allowing the transport of secretory vesicle proteins to the
cis-Golgi, but not further (Takizawa et al., 1993).
Interestingly, IQ inhibited the association of -COP to transport
vesicles but, unlike BFA, did not produce the fusion of the Golgi into
the ER. In addition, a product isolated from the culture broth of Streptomyces sp. originally described as an inhibitor of
inflammation has been recently reported as an inhibitor of the
intracellular transport of VSV G glycoprotein (Seog et al.,
1994), although there is no indication on the mechanism or site of
action.
Here we describe that megalomicin (MGM), a macrolide antibiotic with wide antibacterial spectrum (Weinstein et al., 1972) and antiviral activity (Alarcón et al., 1988), produces profound morphological and functional effects on the Golgi complex of cultured cells, causing the inhibition of the last steps of glycoprotein processing in the Golgi without an apparent effect on the processing enzimes (glycosyltransferases) themselves, but more probably affecting the intra-Golgi transport.
Bafilomycin A was a kind gift of Dr. L. Carrasco (Centro
de Biologia Molecular, Madrid, Spain) and BFA was from Epicentre
Technologies (Madison, WI). Erythromycins A, B, and C were a generous
gift of Dr. J. Corbalan, J. (Lilly).
Figure 1:
Distribution of
Golgi markers in COS and NRK cells upon MGM treatment. Cells were
treated with 50 µM MGM or 1 µM bafilomycin
for 2 h and stained with the cis-Golgi marker MG-160 (MG-160),
with an anti-mannosidase II antibody (Man II) as a
medial-Golgi marker and with antibodies to the trans-Golgi markers
galactosyl-transferase (GalT) and sialyltransferase (SSST). ERD2 is a marker distributed along the ER,
intermediate compartment, and Golgi stacks. NRK cells were used for
MG-160 and Man II stainings and COS cells for GalT staining. COS cells
transfected with the corresponding plasmids were used for the SSST and
ERD2 markers. All photographs were taken at
630.
Confocal laser scanning and electron microscopy were performed to evaluate the effect of MGM on Golgi morphology. Confocal microscopy of COS cell samples stained for the trans-Golgi marker sialyltransferase showed that upon incubation with MGM for periods as short as 1 h, the distribution of the marker was well spread and a dilation and engorging of the compartment became evident as noticed by the larger size of the stained vesicles extending through a reticular-like structure (Fig. 2, compare a with b and c). Fig. 2also shows that the effect of MGM on the trans-Golgi is reversible, returning, although slowly, to normal morphology after a 1-3 h washout (d and e).
Figure 2: The effect of MGM on the trans-Golgi is reversible. COS cells transfected with SSST were treated with 50 µM MGM for 1 h (b), for 3 h (c), or left untreated (a). Alternatively, COS cells treated with 50 µM MGM for 3 h were washed and incubated in the absence of MGM for 1 h (d) or 3 h (e). Photographs were taken in a confocal microscope.
The immunofluorescence data presented above showed that MGM caused a more intense alteration on the trans-Golgi. This was confirmed by electron microscopy of NRK cells treated with MGM and stained with a Golgi mannosidase II-specific antibody by the immunoperoxidase method. As shown in Fig. 3, the stacks of flattened cisternae seen in the control sample (left panel) were dramatically altered in MGM-treated cells (right panel). In these cells, the Golgi apparatus appeared to be formed by stacks of normal size cisternae, mostly located on the cis-side of the Golgi, which were infiltrated by dilated cisternae that were specially abundant on the trans-side.
Figure 3:
Electron microscopy structure of the Golgi
apparatus in MGM-treated NRK cells. NRK cells were stained with the
antibody to mannosidase II by the immunoperoxidase method and
contrasted with uranyl and lead salts. In control cells (left
panel), the Golgi is seen as a stack of flattened cisternae (arrowhead) whereas in MGM-treated cells (right
panel), the Golgi is seen as a series of swollen cisternae (arrow) intercalated among flattened cisternae (arrowheads). Both pictures were taken at 30,000. N indicates the position of the
nucleus.
Figure 4:
Acquisition of endo-H resistance of VSV G
protein in MGM-treated cells. NRK cells were infected with VSV at a
multiplicity of infection of 20 and labeled 4 h later with
[S]methionine for 10 min. After pulse labeling,
cells were chased for the times indicated. MGM (50 µM) was
added from time 0 of infection. An acetone precipitate of each sample
was obtained and treated (+) with endo-H or left
untreated (-). Samples were run by SDS-polyacrylamide gel
electrophoresis on a 10% polyacrylamide gel. The position of VSV G
glycoprotein is indicated with an arrow.
Figure 5:
Analysis of glycans synthesized in the
presence of MGM. A and B, oligosaccharides were
isolated from COS cells that had been labeled overnight with
[H]mannose in the presence of 50 µM MGM or mock-treated. The oligosaccharides from the cell lysates (A) or from the culture supernatants (B) were
fractionated by concanavalin A-Sepharose chromatography. Complex type
corresponded to the unbound material. Hybrid type make the major
fraction eluted with 10 mM
-methylglucoside, and the high
mannose fraction was eluted with 500 mM
-methylmannoside. C, VSV-infected NRK cells were labeled for 4 h with
[
H]mannose in the presence (closed bars)
or absence (open bars) of 50 µM MGM. Cell lysates
were immunoprecipitated with VSV G protein antibody P5D4, and
oligosaccharides on the VSV G protein were fractionated on a
Q-Sepharose column. Fraction 1 was the unbound material and
corresponded to neutral oligosaccharides. Fractions 2, 3, and 4 were
obtained by elution with increasing concentrations of NaCl (20, 75, and
120 mM) and corresponded to monosialylated, disialylated, and
trisialylated oligosaccharides,
respectively.
Concomitantly to the inhibition in the formation of complex type oligosaccharides, MGM caused a lower secretion of glycoproteins containing this type of sugars (Fig. 5B). In this figure, it can be clearly seen that the composition of the secreted glycoproteins from MGM-treated cells was richer in the forms with a higher content in mannose (hybrid and mannose-rich). Interestingly, the addition of MGM resulted in the secretion of proteins containing high mannose oligosaccharides, which were not detected in culture supernatants from untreated cells.
The apparent discrepancy between the endo-H sensitivity data of VSV G protein and the concanavalin A chromatography data of total NRK glycoprotein glycans could be due to different times of exposure to the drug or to the different sensitivity of the labeling techniques. Furthermore, VSV G protein could be converted to an endo-H-resistant form in the medial-Golgi, but still be defectively matured in the presence of MGM as their transport to the trans-cisternae of the Golgi is impaired.
The addition of sialic acid is the last step in the maturation of glycoproteins taking place in the trans-Golgi. To determine how MGM affected this process, oligosaccharide preparations of MGM-treated VSV-infected NRK cells were subjected to chromatography on Q-Sepharose. This system allows to distinguish between oligosaccharides that have incorporated different number of sialic acid residues. As shown in Fig. 5C, the fractions corresponding to oligosaccharides containing a low number of sialic acids (0-1) were higher in MGM-treated cells than in control samples, and conversely, MGM inhibited the formation of oligosaccharides with a higher level of sialylation. These data suggest that MGM is acting preferentially on the trans-side of the Golgi or just before it.
The MGM effect on the sialylation of surface proteins was also evaluated by staining NRK cells with sialic acid-specific lectins, such as M. amurensis, L. poliphemus, and S. nigra agglutinins. After an overnight treatment with MGM, the lectin staining of the cell surface was reduced to a 13-24% of the staining in control cells (Fig. 6), further supporting the idea that MGM reduces the sialylation of glycoproteins.
Figure 6: Inhibition of glycoprotein sialylation in MGM-treated cells. Flow cytometry analysis of NRK cells stained with fluoresceinated lectins M. amurensis agglutinin (MAA), S. nigra agglutinin (SNA) and L. polyphemus agglutinin (LPA). The bar represents the point of maximum fluorescence for 98% of the cell population in unstained samples used as controls. The numbers in each quadrant represent the percentage of fluorescence positive cells (numbers on top) and the mean fluorescence intensity (numbers at bottom).
Figure 7: Intra-Golgi transport inhibition by MGM. Standard transport reactions were carried out as described under ``Materials and Methods.'' Where indicated the reactions were performed in the absence of one of the components of the reaction. In the other samples the assays were performed with all the components in the presence of the indicated concentrations of MGM or of 1 mM NEM. A shows the effect of MGM on cis- to medial-Golgi transport where the donor membranes were from CHO 15B cells, and the acceptor membranes were from CHO wild type cells. B shows the effect on medial- to trans-Golgi transport, where the donor membranes were obtained from BHK ricin-resistant mutant 17 and the acceptor membranes from BHK wild type cells. 1 µg/ml of MGM roughly equals 1 µM.
Figure 8:
Effect of MGM on Golgi acidification.
H pumping activity was determined by fluorescence
quenching of quinacrine with Golgi-enriched membrane vesicles as
described under ``Materials and Methods.'' MGM (A)
or bafilomycin A
(B) were added at the
concentrations indicated, and the reaction was started by addition of
ATP. Finally, 5 mM nigericin was added to reverse the
acidification of the Golgi vesicles.
The results presented in this paper show that MGM causes
profound effects on the Golgi apparatus morphology and function.
Standard fluorescent and confocal laser scanning microscopy showed that
MGM produced a general dilation of the Golgi cisternae, although the
effect was most dramatic on the trans-Golgi. Electron micrographs
showed the presence of swollen cisternae of the Golgi mostly located on
the trans-side, coexisting with cis-located cisternae of normal
appearance. The morphological alteration of MGM on the Golgi was
reflected in an impaired function, producing an anomalous glycosylation
of VSV G protein and cellular glycoproteins. Interestingly, the MGM
effects on glycosylation correlated well with its morphological
effects, causing a higher inhibition of the later processes of glycan
maturation, which take place on the trans-side of the Golgi. Thus, MGM
caused an inhibition in the formation of complex-type oligosaccharides
paralleled to an increase of the high mannose type. In addition, after
shorter incubations, MGM had no detectable effect on the maturation of
VSV G protein to an endo-H-resistant form, a process that is dependent
on the activity of the medial-Golgi located enzyme mannosidase II,
indicating that MGM does not inhibit, to a substantial degree, the
access to the medial-Golgi compartment. Furthermore, in the presence of
MGM, VSV G protein was undersialylated. A possible explanation for
these results could be that MGM inhibits the activities of either
sialyltransferase or the previous acting galactosyltransferase that
would generate the substrate for the sialyltransferase. However, MGM
did not inhibit the activities of galactosyl- and sialyltransferases in vitro (Table 1). An alternative non-excluding
explanation would be that the swelling induced by the drug could result
in a decreased active lumenal concentration of the sugar nucleosides
and hence reduced modification rates. MGM could directly inhibit sugar
nucleotide transporters which would account for some of the MGM effects
described. Such idea would imply that MGM preferentially inhibits the
UDP-Gal and/or CMP-NeuAc transporters but not the GDP-Man or the
UDP-GlcNac transporters. However, as far as the literature surveyed
(for review, see Hirschberg and Snider(1987) and Milla et
al.(1989)), all the sugar nucleoside transporters described so far
share the same biochemical properties, and there is no structural
homology between MGM to any sugar nucleotide to act as a selective
inhibitor. It is clear that direct testing of MGM in a sugar nucleotide
transport assay will answer that point. However, we have tested CHO
mutant cell lines lacking UDP-Gal (CHO Lec 2, Deutscher et
al., 1984) or CMP-NeuAc (CHO Lec 8, Deutscher and Hirschberg,
1986) transporters for their MGM sensitivity. Both cell lines behave
like the parental cell line in the sense that MGM induces the cisternal
swelling to the same extent. ()Finally, MGM could be
inhibiting the access of the proteins to the compartments where the
glycosyltransferases are located. This possibility is strongly
supported by the effect of MGM in an established in vitro intra-Golgi transport assay. Although in the in vitro assay, MGM inhibited cis- to medial- and medial- to trans-Golgi
transport of VSV G protein, only this last effect was correlated with
the effect of MGM in vivo. This is easily explained,
considering that the maturation of glycans is a sequential process
relying on the sequential action of compartmentalized enzymes, and
therefore, the effects of transport inhibition accumulate through
successive compartments.
The inhibition of glycoprotein sialylation was in agreement with previous data showing that MGM inhibited the addition of galactose to HSV glycoproteins (Alarcón et al., 1988). The intra-Golgi transport inhibition shown in this paper could explain the activity of MGM as an antiviral agent. MGM inhibited normal glycosylation of HSV, but not the synthesis of viral proteins nor the formation of viral particles, which were, nevertheless, non-infectious (Alarcón et al., 1988). The effect of MGM on glycoprotein maturation could also explain why was MGM active against enveloped viruses such as HSV, VSV, and Semliki forest virus, but not against naked ones, such as polio or encefalomiocarditis virus (Alarcón et al., 1984). The antiviral effect of MGM is nevertheless difficult to explain, because, as shown in this paper, MGM was not selective for viral glycoproteins, causing the secretion of incompletely processed glycoproteins and an inhibition in the sialylation of cell membrane glycoproteins. Although dramatic, the effects of MGM on the Golgi of noninfected cells were not correlated with an apparent toxicity to cells grown with MGM added once a week (data not shown). The observed effect could be due to an adaptation of the cells to the continuous presence of the inhibitor and also to a possible loss of activity of MGM in culture conditions. A possible mechanism of adaptation has been suggested for concanamycin B (Yilla et al., 1993), where an acidification inhibition of the TGN could cause a default pathway to be utilized. The authors propose that proteins would be shunted into a different pathway causing proteins to reach the cell surface and to be secreted although at a lower rate.
The effects of MGM on the Golgi complex organization
were different to those seen with BFA and IQ, which inhibit vesicular
transport by acting on early steps of vesicle formation. BFA causes the
cis-, medial-, and trans-Golgi to redistribute into the ER by blocking
the anterograde vesicular transport, whereas the trans-Golgi network is
fused with endosomes (for a review, see Klausner et
al.(1992)). IQ, on the other hand, causes the fragmentation and
vesiculation of the Golgi and inhibits the transport beyond the
cis-Golgi (Takizawa et al., 1993). The molecular target of MGM
is at present unknown. It seems clear that it is different to the
target recognized by BFA, because -COP is not dissociated from the
Golgi upon MGM treatment (data not shown). In addition, transport took
place at a normal extension when cytosol from MGM-treated cells was
used in the in vitro transport assay (data not shown). This
suggested that MGM does not inhibit intra-Golgi transport by binding to
a soluble component.
On the other hand, it is difficult to understand how the swelling of Golgi cisternae may impair the access of docking vesicles from earlier Golgi compartments. In this regard, the effect of MGM would be similar to the effects of mastoparan and ARFp13, which have been recently described to inhibit intra-Golgi transport by damaging Golgi membranes (Weidman and Winter, 1994). However, unlike these peptides, the effect of MGM was reversible and affected specifically the last steps of glycoprotein maturation.
In accordance with the effect on intra-Golgi transport, MGM at 50 µM produced a partial inhibition (25-35%) of total protein secretion (data not shown), pointing to an impairment in the transport of secretory proteins beyond the trans-Golgi. The presence of undersialylated glycoproteins in the secreted proteins may be due to sialylation by sialyltransferases in earlier Golgi compartments, perhaps due to mislocalization of the enzymes caused by MGM. This would not be surprising since other drugs, like BFA, produce anomalous processing of glycoproteins, due to the accumulation of Golgi enzymes in the ER (Chawla and Hughes, 1991).
The effect of concanamycin B, a
macrolide antibiotic, on the Golgi apparatus has been described (Yilla et al., 1993). Concanamycin B, as well as bafilomycins, is a
V-H-ATPase inhibitor (Bowman et al., 1988;
Woo et al., 1992). The almost complete blockade of
glycoprotein secretion by concanamycin B (Yilla et al., 1993)
and the impairment in the transport of viral particles to the membrane
by bafilomycin (Pakolangas et al., 1994) suggested that
V-H
-ATPases maintain a low pH in the trans-Golgi,
which is fundamental for protein trafficking through the trans-Golgi
and for the activity of sialyltransferases (Yilla et al.,
1993). The undersialylation of proteins induced by MGM also could be
explained by an indirect inhibition of the sialyltransferase(s), due to
an alteration of the intra-Golgi milieu essential to the enzymatic
activity caused by the drug. In this regard, as concanamycin B inhibits
sialylation (Yilla et al., 1993), it could be argued that MGM
could also act by raising the intraluminal pH of the Golgi through an
inhibition of the V-H
-ATPases. An efficient
recognition of the transported proteins (soluble and membrane) by the
components of the sorting machinery may rely on the compartment pH.
However, in contrast to concanamycin and bafilomycin, MGM inhibited
poorly the V-type H
-ATPase of the Golgi in an in
vitro test at the concentrations that were inhibitory in the
intra-Golgi transport assays. In addition, bafilomycin A
,
which did inhibit Golgi acidification, had no detectable effects on
Golgi morphology as concanamycin and MGM do. The swelling of the Golgi
induced by MGM could be due to the alteration of other ion gradients,
different from proton, across the Golgi membrane. In this regard,
monensin has been shown to cause a swelling of the trans-Golgi stacks
that result from the dissipation of Na
gradients (for
review, see Mollenhauer et al.(1990)). The swelling of the
Golgi cisternae is produced because there is a net flow of water into
the Golgi stacks to compensate for the H
gradient that
is still maintained in the presence of monensin. Thus, the addition of
a proton ionophore prior to monensin results in the abrogation of
monensin-caused swelling (Mollenhauer et al., 1993). By
contrast, the addition of the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not prevent the swelling
effects of MGM on the trans-Golgi (data not shown), suggesting that the
mechanism of MGM-induced swelling is different from that of monensin.
Monensin, on the other hand, totally inhibits the acquisition of endo-H
resistance, while MGM did not.
So, at this point, the subtle
differences between other macrolide antibiotics and MGM are more
interesting than their similarities, and one relevant issue raised is
that they are possibly revealing some hitherto unappreciated complexity
in the family of vesicular ATPases not equally susceptible to
inhibition by the drugs, with a defined substrate specificity or acting
on unrelated processes, located in all of the Golgi compartments. It is
important to mention here that the vacuolar H-ATPase
is a minor component of the total ATPase activity on Golgi fractions
(Moriyama and Nelson, 1989b). Furthermore, in a recent report it has
been shown that even after bafilomycin A
inhibition of the
V-H
-ATPase, the pH of the endosome/late endosome was
not completely neutral (van Weert et al., 1995), and Yoshimori et al.(1991) have reported a lysosomal pH of 6.3 in the
presence of Bafilomycin indicating that V-H
-ATPase may
not be the only factor responsible for pH homeostasis in late
endosomes/lysosomes and probably also in the Golgi.
Finally, the use of drugs like MGM can be useful tools to dissect the complexity of the mechanisms responsible of maintaining the homeostasis of the Golgi.