A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Staphylococcal ADP-ribosyltransferase-sensitive small G protein is involved in brefeldin A action.

An early event in the action of brefeldin A (BFA) is the dissociation of beta-coat protein (beta-COP) from the Golgi membrane. We have recently shown that staphylococcal ADP-ribosyltransferase (epidermal cell differentiation inhibitor (EDIN)), which specifically modifies a small G protein, rho, mimics the action of BFA and disassembles the Golgi apparatus in Vero cells (Sugai, M., Chen, C-h., and Wu, H. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8903-8907). Three independent BFA-resistant cell lines (BER-40 from Vero cells, PtK1, and MDCK) showed cross-resistance to EDIN regarding the release of the beta-COP from the Golgi membrane by EDIN or BFA. BFA as well as EDIN induced disassembly of the actin microfilaments in Vero cells, and they both failed to induce the disassembly of actin microfilaments in BER-40, PtK1, and MDCK cells. BFA inhibited protein secretion in Vero cells but not in BFA-resistant cell lines, whereas EDIN did not inhibit protein secretion in either Vero or other cell lines. AlF-4 inhibited the effect of EDIN as well as that of BFA on the distribution of the beta-COP. These results suggest that an EDIN-sensitive rho protein together with trimeric and other small G protein(s) is involved in the regulation of the assembly of coated vesicles and vesicular transport in the Golgi apparatus.     

Isolation and characterization of a brefeldin A-resistant mutant of monkey kidney Vero cells.

Brefeldin A (BFA) is a fungal antibiotic which disrupts protein transport between the endoplasmic reticulum and the Golgi. A BFA-resistant mutant of monkey kidney Vero cells, BER-40, which exhibited about a 90-fold increase in the LD50 of BFA (5.2 ng/ml for Vero cells versus 460 ng/ml for BER-40 cells), has been isolated. The increased resistance of BER-40 cells toward BFA was also manifested in a greatly reduced inhibition of protein secretion by BFA in the mutant and a lack of protection by BFA of the mutant cells from ricin cytotoxicity. Somatic cell hybridization between the Vero and BER-40 cells showed that the BFA-resistance in BER-40 behaved as a codominant trait. The structure of the Golgi region, as examined by immunofluorescence microscopy with antibodies against Golgi markers (the 110-kDa protein and mannosidase II) or with fluorescent lipid NBD-ceramide, was unchanged in the mutant cells as compared to that in the wild-type cells. Treatment of Vero cells with BFA (1 micrograms/ml) or with 2-deoxyglucose plus sodium azide resulted in a rapid release of the 110-kDa protein, mannosidase II, and NBD-ceramide from the Golgi membrane to a more diffuse distribution in the cytosol. In contrast, these three Golgi markers remained to be Golgi-associated following treatment of BER-40 cells with BFA or with 2-deoxyglucose plus sodium azide. Immunoblotting of cell extracts from Vero and BER-40 cells with monoclonal antibody against the 110-kDa protein did not reveal any significant difference in the level of this Golgi marker in the mutant cells. These data suggest that the BFA-resistance mutation in BER-40 has rendered the cyclic pathway of the 110-kDa protein assembly to the Golgi membrane resistant to both BFA and 2-deoxyglucose plus sodium azide.     

Brefeldin A inhibits protein synthesis in cultured cells.

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml. Inhibition was both time-dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.     

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.     

7-Dehydrobrefeldin A, a naturally occurring brefeldin A derivative, inhibits secretion and causes a cis-to-trans breakdown of Golgi stacks in plant cells.

7-Dehydrobrefeldin A (7-oxo-BFA) is a brefeldin A (BFA) analog that, like BFA, is a potent phytotoxin of Alternaria carthami, a fungal pathogen of safflower (Carthamus tinctorius L.) plants. Both BFA and 7-oxo-BFA have been shown to be causal agents of the leaf spot disease of these plants. We have investigated the effects of 7-oxo-BFA on the secretion and the structure of the Golgi stacks of sycamore maple (Acer pseudoplatanus) suspension-cultured cells to determine whether 7-oxo-BFA affects these cells in the same manner as BFA. When applied at 10 micrograms/mL for 1 h, 7-oxo-BFA inhibits secretion of proteins by approximately 80%, the same value obtained for BFA. However, electron micrographs of high-pressure frozen/freeze-substituted cells demonstrated that 7-oxo-BFA is a more potent disrupter of the Golgi stacks of sycamore maple cells than BFA. In cells treated for 1 h with 10 micrograms/mL 7-oxo-BFA, very few Golgi stacks can be discerned. Most of those that are left consist of fewer than three cisternae, all of which stain like trans-Golgi cisternae. They are surrounded by clusters of large (150-300 nm in diameter), darkly staining vesicles that are embedded in a fine-filamentous, ribosome-excluding matrix. Similarly sized and stained vesicles are seen budding from the rims of the residual trans-Golgi cisternae. Both the large vesicles and the residual Golgi stack buds stain with anti-xyloglucan polysaccharide antibodies. Recovery of Golgi stacks after removal of 7-oxo-BFA from 1-h-treated cells takes 2 to 6 h, compared with 1 to 2 h for cells treated with BFA. In contrast to 7-oxo-BFA, the BFA breakdown product BFA acid had no effect either on secretion or on the secretory apparatus. This is the first report, to our knowledge of a BFA analog inhibiting secretion in a eukaryotic cell system.     

Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells.

Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [3H]xylose and [3H]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mu g/mL BFA causes the following major changes in the secretory pathway of sycamore maple cells: (a) it inhibits the transport of secretory proteins to the cell surface by about 80% and of hemicelluloses by about 50%; (b) it changes the patterns of glycosylation of N-linked glycoproteins and hemicelluloses; (c) it reduces traffic between trans Golgi cisternae and secretory vesicles; (d) it produces a major block in the transport of XG-containing, dense secretory vesicles to the cell surface; and (e) it induces the formation of large aggregates of Golgi apparatus of plant and animal cels share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.     

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.     

Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect

Whereas brefeldin A (BFA) protected a number of cell lines against the protein toxin ricin, two of the cell lines tested were not protected but rather sensitized to ricin by BFA. EM studies revealed that upon addition of BFA the Golgi stacks in cells which were protected against the toxin rapidly transformed into a characteristic tubulo-vesicular reticulum connected to the endoplasmic reticulum, and subcellular fractionation experiments showed that galactosyl transferase disappeared from the Golgi fractions where it was normally located. EM and subcellular fractionation also indicated that in contrast to the Golgi stacks, the trans-Golgi network (TGN) remained intact and that internalized ricin was still localized in the TGN both when BFA was added before and after the toxin. Thus, BFA does not prevent fusion of ricin-containing vesicles with the TGN, and unlike resident proteins in Golgi stacks, ricin is not transported back to ER upon treatment of cells with BFA. Two kidney epithelial cell lines, MDCK and PtK2, were not protected against ricin by BFA, and EM studies of MDCK cells revealed that BFA did not alter the morphology of the Golgi complex in these cells. Also, subcellular fractionation revealed that, in contrast to the other cell types tested, the localization of galactosyl transferase in the gradients was not affected by BFA treatment. The data show that there is a correlation between BFA-induced disassembly of the Golgi stacks and protection against ricin, and they demonstrate that the structural organization of the Golgi apparatus is affected by BFA to different extents in various cell lines.     

Differential Effects of the Putative GBF1 Inhibitors Golgicide A and AG1478 on Enterovirus Replication

The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication.The Enterovirus genus, belonging to the Picornaviridae family, includes many important human pathogens, such as poliovirus, human rhinovirus, echovirus, and coxsackievirus. These are small, nonenveloped viruses that contain a single-stranded RNA genome of positive polarity. The genome is approximately 7.5 kb in length and encodes a single large polyprotein, which is processed into capsid proteins, encoded in the P1 region, and the nonstructural P2 and P3 region proteins that mediate viral RNA replication.Brefeldin A (BFA), a fungal metabolite, is a well-known inhibitor of enteroviruses. BFA blocks transport of cargo from the endoplasmic reticulum (ER) to the Golgi vesicles by disrupting the Golgi vesicles and ER-Golgi intermediate compartment (ERGIC) integrity through inhibition of several guanine nucleotide exchange factors (GEFs), including Golgi-specific BFA resistance factor 1 (GBF1), BFA-inhibited GEF 1 (BIG1), and BIG2 (3, 18). These GEFs regulate the activity of GTPase ADP-ribosylation factor 1 (Arf1) by stimulating GTP exchange. Upon activation, Arf1-GTP binds to Golgi membranes where it induces formation of secretory vesicles via recruitment of coatomer protein complex I (COP-I), a coatomer protein involved in the transport between the Golgi vesicles and the ER. The inhibitory effect of BFA on enterovirus replication is attributed to the inhibition of GBF1 and does not seem to involve BIG1 or BIG2 (2, 11). Besides enteroviruses, other plus-strand RNA viruses, such as mouse hepatitis virus and hepatitis C virus, also seem to rely on GBF1 for efficient replication (2, 8, 11, 21). The viral protein 3A of the enteroviruses poliovirus and coxsackievirus B3 (CVB3) has been shown to interact directly with GBF1 (22, 22a, 23), but the exact function of this interaction remains to be established.Recently, two compounds, AG1478 and Golgicide A (GCA), have been proposed to specifically inhibit GBF1. AG1478 was identified by screening a library of compounds for their ability to induce Golgi complex disassembly (13). AG1478, known as an inhibitor of the epidermal growth factor receptor (EGFR), had effects on the Golgi membranes highly similar to those of BFA through a mechanism not involving the inhibition of EGFR. Arf1-GTP pulldown assays showed that AG1478 inhibited Arf1 activation. Furthermore, overexpression of GBF1 was shown to counter the effect of AG1478 on COP-I localization. Based on these results, AG1478 was proposed to be a GBF1 inhibitor.GCA was identified in a high-throughput screen for small molecules that protected Vero cells from the effects of Shiga toxin (15). Similar to AG1478 and BFA, GCA was reported to fragment the Golgi vesicles and to inhibit Arf1 activation. Furthermore, overexpression of either wild-type GBF1 or the BFA-resistant mutant GBF1-M832L relieved the effects of GCA. In addition, the authors constructed a structural model of the catalytic Sec7 domain of GBF1 in complex with GCA, showing that GCA binds GBF1 at the same site as BFA. Collectively, their results provided convincing lines of evidence that GCA specifically inhibits GBF1 in a manner similar to BFA and does not act on BIG1 and BIG2.BFA has been instrumental in elucidating the membrane requirements for enterovirus replication. Therefore, we investigated the effects of AG1478 and GCA on enterovirus replication after first characterizing the effects of these drugs on BGM cells, the cell line that we routinely use in our studies on coxsackievirus B3 replication. Treatment with other AG1478 or GCA fragmented the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet these drugs had different effects on GBF1 localization. Interestingly, the effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1. Next, GCA was found to abrogate enterovirus replication, whereas surprisingly AG1478 did not affect replication at all. Together these results indicate that AG1478 on one hand and GCA and BFA on the other hand have different mechanisms of action, leading to a disparate effect on enterovirus replication.     

Brefeldin A effects on the trans-Golgi network and Golgi bodies inBotryococcus braunii are not uniform during the cell cycle

Summary Using cryo-fixation and freeze-substitution electron microscopy, the effects of brefeldin A (BFA) on the structure of the trans-Golgi network (TGN), the endoplasmic reticulum (ER), and Golgi bodies in the unicellular green algaBotryococcus braunii were examined at various stages of the cell cycle. In the presence of BFA, all the TGNs of interphase and dividing cells aggregated to form a single tubular mass. In contrast, the TGNs decomposed just after cell division and disappeared during cell wall formation. Throughout the cell cycle, the TGN produced at least six kinds of vesicles, of which two were not formed in the presence of BFA: vesicles with a diameter of 200 nm and fibrillar substances, which formed in interphase cells; and vesicles with a diameter of 180–240 nm, which may participate in septum formation. In addition, the number of clathrin-coated vesicles attaching to the TGN decreased. In interphase cells, BFA induced the disassembly of Golgi bodies and an increase in the smooth-ER cisternae at the cis-side of Golgi bodies. This result may suggest the existence of retrograde transport from the Golgi bodies to the ER in the presence of BFA. These drastic structural changes in the Golgi bodies and the ER of interphase cells were not observed in BFA-treated dividing cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TGN trans-Golgi network     

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.     

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.     

Bfr1, a Multicopy Suppressor of Brefeldin a-Induced Lethality, Is Implicated in Secretion and Nuclear Segregation in Saccharomyces Cerevisiae

Brefeldin A (BFA) blocks protein transport out of the Golgi apparatus and causes disassembly of this organelle in mammalian cells. The primary effect of BFA is the release of the non-clathrin coat from Golgi membranes and vesicles. We sought to elucidate the mechanism of BFA action using a genetic approach in Saccharomyces cerevisiae. When an erg6 S. cerevisiae strain is treated with BFA, cell growth is arrested, cells lose viability and secretory proteins are accumulated in the endoplasmic reticulum (ER) and early Golgi compartments. We demonstrate that the mutant sec21 (defective in the S. cerevisiae homolog of γ-COP, a non-clathrin coat protein) is supersensitive to BFA. Hence BFA probably affects the same processes in S. cerevisiae as in mammalian cells. We used a multicopy genomic DNA library to search for multicopy suppressors of BFA-induced lethality. We identified one such gene, BFR1, that, in addition, partially suppresses the growth and secretion defects of the ER-to-Golgi secretion mutant sec17. A bfr1-Δ1::URA3 deletion strain is viable, but has defects in cell morphology and nuclear segregation, and the mutation accentuates the growth and secretion defects of a sec21 mutant.     

Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators.

Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., gamma-adaptin) associated with the clathrin-coated vesicles that bud from the trans-Golgi network (TGN), while the clathrin-coated vesicles that bud from the plasma membrane are unaffected. gamma-Adaptin redistributes with the same kinetics as beta-COP, a coat protein associated with the non-clathrin-coated vesicles that bud from the Golgi complex. Upon removal of BFA, however, gamma-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AlF4-. Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilized cell system and is increased by addition of GTP gamma S and blocked by addition of BFA. These results suggest a role for G proteins in the control of the clathrin-coated vesicle cycle at the TGN and further extend the similarities between clathrin-coated vesicles and non-clathrin-coated vesicles.     

Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf . GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf . GTP, rather than the formation of an Arf . GDP . BFA . GBF1 complex.     

CSFV proliferation is associated with GBF1 and Rab2

The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. In this study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi was disturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA), and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particle generation after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected with GBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using a lentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate in CSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlying CSFV replication.