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.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday.     

Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth,cell-wall extensibility and secretion of cell-wall proteins

Brefeldin A (BFA), a fungal metabolite causing dysfunction of the Golgi apparatus in plant and animal cells, was used to investigate the role of secretory processes at the plasma membrane in auxin-mediated elongation growth of maize (Zea mays L.) coleoptiles. In abraded coleoptile segments BFA produced, within less than 30 min, a decrease in the incorporation of [³H]leucine into tightly bound cell-wall proteins, accompanied by an increased incorporation into the intracellular pool of putative cell-wall glycoproteins. Total protein synthesis was not affected. Electron micrographs revealed striking morphological changes in dictyosomes (especially vesiculation of trans-cisternae), accumulation of Golgi vesicles and dilation of the endoplasmic reticulum. These effects are taken as indication that BFA interferes with the secretion of cell-wall components. Elongation growth of coleoptile segments in the presence and absence of auxin was inhibited by 80% in 20 mg·l^–1 BFA. If BFA was applied to segments growing in the presence of auxin, maximum inhibition was reached after about 30 min, indicating that the growth response depends on an uninterrupted supply of a cell-wall or plasma-membrane component (wall-loosening factor) delivered by the secretory pathway. After its secretion, this factor has a rather short growth-effective life time. The inhibition of auxin-mediated growth by BFA was accompanied by an elimination of auxin-induced cell-wall extensibility and by an inhibition of auxin-induced proton excretion. Fusicoccin-induced proton excretion was similarly affected by BFA. It is concluded that both the wall-loosening process underlying elongation growth as well as proton excretion depend on an intact secretory pathway from the Golgi apparatus to the cell wall; however, a causal relationship between these processes is not warranted by the data.Abbreviations BFA brefeldin A - FC fusicoccin - TCA trichloroacetic acid - WLF wall-loosening factor Supported by Deutsche Forschungsgemeinschaft (SFB 206). We thank Ms. B. Huvermann and Mrs. C. Plachy for conducting growth and proton excretion measurements.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Golgi regeneration after brefeldin A treatment in BY-2 cells entails stack enlargement and cisternal growth followed by division

Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. This effect is reversible upon washing out the drug, providing a situation for studying Golgi biogenesis. In this investigation Golgi regeneration in synchronized tobacco BY-2 cells was followed by electron microscopy and by the immunofluorescence detection of ARF1, which localizes to the rims of Golgi cisternae and serves as an indicator of COPI vesiculation. Beginning as clusters of vesicles that are COPI positive, mini-Golgi stacks first become recognizable 60 min after BFA washout. They continue to increase in terms of numbers and length of cisternae for a further 90 min before overshooting the size of control Golgi stacks. As a result, increasing numbers of dividing Golgi stacks were observed 120 min after BFA washout. BFA-regeneration experiments performed on cells treated with BFA (10 microg mL(-1)) for only short periods (30-45 min) showed that the formation of ER-Golgi hybrid structures, once initiated by BFA treatment, is an irreversible process, the further incorporation of Golgi membranes into the ER continuing during a subsequent drug washout. Application of the protein kinase A inhibitor H-89, which effectively blocks the reassembly of the Golgi apparatus in mammalian cells, also prevented stack regeneration in BY-2 cells, but only at very high, almost toxic concentrations (>200 microm). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling

Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between trans-Golgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20 degrees C. Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K+ depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y. Misumi, K. Miki, A Takatsuki, G. Tamura, and Y. Ikehara. 1986. J. Biol. Chem. 261:11398-11403). These and earlier results suggest that the exo/endocytic pathway of mammalian cells consist of two similar but distinct endomembrane systems: an ER/Golgi system and a post-Golgi system. BFA prevents forward transport without affecting return traffic in both systems.     

Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein-Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atgamma-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 microg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atgamma-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)-Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

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.     

Brefeldin A-resistant mutants of human epidermoid carcinoma cell line with structural changes of the Golgi apparatus.

We have isolated brefeldin A (BFA)-resistant cell lines, KB/BF-1 and KB/BF-2, from the human epidermoid carcinoma KB cell line. The BFA-resistant phenotypes have been stably maintained for more than 3 months in the absence of BFA. KB/BF-1 and KB/BF-2 showed 10-30-fold higher resistance to cytotoxicity of BFA but were 2-3-fold more sensitive to monensin and nigericin, than KB cells. KB/BF-1 showed aberrant structures of the Golgi complex with poorly developed cisternae surrounded by many small vesicles. Immunocytochemical studies were done with antibodies against a Golgi-specific antigen (chronic rheumatoid arthritis antigen) and a coatomer subunit (beta-subunit for coat proteins of non-clathrin-coated vesicles). Golgi-specific markers were distributed into the small vesicles which were localized diffusedly in cytoplasm of KB/BF-1 cells. Such Golgi markers were observed in a strictly confined perinuclear region of the parental KB cells, whereas in the mutant cells the markers were distributed more diffusedly in dot-like structures at perinuclear regions. In addition, when exposed to BFA, the mutant and parental cells showed a different distribution of these markers. Synthesis and maturation of low density lipoprotein receptor showed apparently slower rates in processing of low density lipoprotein receptor in KB/BF-1 and KB/BF-2 cells than those observed in their parental KB cells. Protein secretion in KB/BF-1 and KB/BF-2 cells was about 30% less than that in KB cells. Much less inhibition by BFA on the secretion was observed in KB/BF-1 and KB/BF-2 cells. A BFA-resistant mutation in BFA-resistant KB cell lines appears to affect assembly of the Golgi apparatus as well as some Golgi-specific functions.     

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.     

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.     

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.     

Disruption of the Golgi apparatus and secretory mechanism in the desmid, Closterium acerosum by brefeldin A

The mucilage-secreting desmid, Closterium acerosum, is sensitive to the secretory inhibiting drug, brefeldin A (BFA). After 5 min of treatment with 5 g ml^-1 of BFA, the Golgi body displays the following alterations: the number of cisternae decreases from 12-15 to 6-7; peripheries of cisternae from the same Golgi body often fuse to yield unique profiles; secretory vesicles still merge from the Golgi body; the cisternal stack dissociates to form irregular masses in the alleys of cytoplasm created by the lobes of the chloroplast. Fluoresbrite bead labelling shows that mucilage production ceases in cells treated with BFA even after only 5 min of treatment. Immunogold labelling using anti-mucilage antibody reveals that mucilage production still occurs in the Golgi body and associated vesicles. Helix pomatia lectin-gold labelling shows that wall synthesis still occurs in BFA-treated Golgi bodies and wall precursors accumulate in the perforate cisternal/vesicular masses seen in the TGN region of the Golgi stack.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

Localized accumulation of cell wall mannoproteins and endomembranes induced by brefeldin A in hyphal cells of the dimorphic yeastCandida albicans

Summary Candida albicans, a dimorphic yeast, has the abililty to switch its growth form between budding growth and hyphal growth. Since fungal growth involves secretory processes, spatial control of secretion should play a crucial role in such a morphogenetic transition. Brefeldin A (BFA), an inhibitor of the membrane trafficking system of eukaryotes, increases the occurrence of Golgi-like cisternae in the yeast. In the present study, BFA was used to obtain further insights into the spatial organization of secretory processes in hyphal growth ofC. albicans. BFA completely inhibited the formation and growth of germ tubes at a concentration of 35 M or higher. Electron microscopy of BFA-untreated germinated cells revealed many vesicles in the apical region and Golgi-like cisternae in the cytoplasm. In cells treated with 35 M BFA, the vesicles disappeared from the apical region, and, instead, stacked membrane cisternae and membrane-enclosed spherical dense bodies accumulated in the subapical region. These accumulated structures were positive for both polysaccharide staining and immunocytochemical staining with antibodies raised against cell surface antigens ofC. albicans, as were Golgi cisternae in BFA-untreated cells. In cells treated with a higher concentration of BFA (140 M), the structures that appeared in cells treated with 35 M BFA were no longer observed and the endoplasmic reticulum was extended and positive for polysaccharide staining. These results suggested that BFA affects different steps of membrane trafficking in a concentration-dependent manner. The accumulated structures induced by 35 M BFA seemed to be the altered forms of Golgi cisternae. Their accumulation in the subapical region of the germ tube might indicate that the step(s) in membrane trafficking that are associated with the Golgi pathway are vectorially organized in hyphal growth ofC. albicans.Abbrevations BFA brefeldin A - BSA bovine serum albumin - CBB Coomassie brilliant blue - Con A concanavalin A - HRP horseradish peroxidase     

Differential effects of monensin on the plant secretory pathway

The sodium ionophore monensin was used as an inhibitor of Golgifunction to study the secretory event in suspension-culturedsycamore cells. The morphological changes induced by monensinwere recorded. Concurrent studies of the disturbance causedby the drug were carried out by biochemical, cytochemical andimmunocytochemical techniques. Monensin induced accumulationof smooth and coated vesicles in the vicinity of the Golgi apparatus,swelling of Golgi cisternae and also provoked the formationof extracytoplasmic pockets between the plasma membrane andthe cell wall. Cytochemical tests for polysaccharides and immunolocalizationof secreted pectins, recognized by the monoclonal antibody JIM7, were performed, and the results indicated that the biosynthesisand/or processing machinery of polysaccharides was affectedby the drug. In contrast, immunolocalization of glycoproteins,recognized by the monoclonal antibodies JIM 84 and JIM 13 demonstratedthat the accumulation of vesicles in the cytoplasm were Golgiderived, and that the secretion of glycoproteins was not drasticallyaffected by monensin. Fluorography of radiolabelled proteinsdemonstrated that in this system the secretion of proteins wasnot qualitatively changed by monensin. The implications of theseresults are discussed in the light of the current hypotheseson the effects of monensin on the secretory pathway. Key words: Golgi apparatus, immunocytochemistry, monensin, secretion