Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.     

Fine Structural Analysis of Brefeldin A-Induced Compartment Formation After High-Pressure Freeze Fixation of Maize Root Epidermis: Compound Exocytosis Resembling Cell Plate Formation during Cytokinesis

Formation of large perinuclear brefeldin A (BFA)-induced compartments is a characteristic feature of root apex cells, but it does not occur in shoot apex cells. BFA-induced compartments have been studied mostly using low resolution fluorescence microscopy techniques. Here, we have employed a high-resolution ultrastructural method based on ultra rapid freeze fixation of samples in order to study the formation of BFA-induced compartments in intact maize root epidermis cells in detail. This approach reveals five novel findings. Firstly, plant TGN/PGN elements are not tubular networks, as generally assumed, but rather vesicular compartments. Secondly, TGN/PGN vesicles interact with one another extensively via stalk-like connections and even fuse together via bridge-like structures. Thirdly, BFA-induced compartments are formed via extensive homotypic fusions of the TGN/PGN vesicles. Fourthly, multivesicular bodies (MVBs) are present within the BFA-induced compartments. Fifthly, mitochondria and small vacuoles accummulate abundantly around the large perinuclear BFA-induced compartments.Key Words: brefeldin A, BFA-induced compartments, golgi, endosomes, root apex     

Contractile vacuoles in cells of a fresh water sponge,Spongilla lacustris

Summary Forty or more independently functioning contractile vacuoles (CVs) occupy the central region of fresh water sponge pinacocytes. Each CV undergoes a cycle of enlargement by fusion, movement, shape change, rounding up, and emptying over the course of 5–30 min. Diameter at discharge varies between 1 and 13 m. CVs in all cell types are associated with submicroscopic coated vesicles. Filled CVs are bounded by an unmodified trilaminar membrane, but vacuoles with excess membrane frequently show coated evaginations. These evaginations are thought to pinch off as coated vesicles, providing an avenue for membrane recycling in the CV system.Supported by NIH grants AS-T01-GM-0723 and GM-23708-CBY     

Brefeldin A-dependent membrane tubule formation reconstituted in vitro is driven by a cell cycle-regulated microtubule motor

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus, trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5'-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of approximately 100 and approximately 130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.     

Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus

The release of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A (BFA) action, preceding the movement of Golgi membrane into the ER. ATP depletion also causes the reversible redistribution of the 110-kD protein from Golgi membrane into the cytosol, although no Golgi disassembly occurs. To further define the effects of BFA on the association of the 110-kD protein with the Golgi apparatus we have used filter perforation techniques to produce semipermeable cells. All previously observed effects of BFA, including the rapid redistribution of the 110-kD protein and the movement of Golgi membrane into the ER, could be reproduced in the semipermeable cells. The role of guanine nucleotides in this process was investigated using the nonhydrolyzable analogue of GTP, GTP gamma S. Pretreatment of semipermeable cells with GTP gamma S prevented the BFA-induced redistribution of the 110-kD protein from the Golgi apparatus and movement of Golgi membrane into the ER. GTP gamma S could also abrogate the observed release of the 110-kD protein from Golgi membranes which occurred in response to ATP depletion. Additionally, when the 110-kD protein had first been dissociated from Golgi membranes by ATP depletion, GTP gamma S could restore Golgi membrane association of the 110-kD protein, but not if BFA was present. All of these effects observed with GTP gamma S in semipermeable cells could be reproduced in intact cells treated with AlF4-. These results suggest that guanine nucleotides regulate the dynamic association/dissociation of the 110-kD protein with the Golgi apparatus and that BFA perturbs this process by interfering with the association of the 110-kD protein with the Golgi apparatus.     

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes.

We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.     

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.     

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.     

Brefeldin A promotes hydrolysis of sphingomyelin.

The hydrolysis of sphingomyelin (SM) is a key reaction in the "sphingomyelin cycle," which plays a role in the regulation of cell proliferation and differentiation (Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080). SM is produced from endoplasmic reticulum-derived ceramide and is delivered to organelle membranes in a regulated manner, presumably through the same endomembrane trafficking system used for sorting and delivery of proteins. Since brefeldin A (BFA) interferes with this endomembrane trafficking system and thus alters normal membrane and organelle distribution, we investigated the effect of BFA on SM levels in HL-60 leukemia cells. BFA caused a dose-dependent decrease of 20-25% in cellular SM levels, with effects observed at concentrations of BFA as low as 0.10 microgram/ml. BFA effects on SM levels were noted as early as 5 min and were maximal by 20 min, with no further SM hydrolysis observed up to 60 min following treatment with BFA, suggesting the presence of a fixed SM-sensitive pool. BFA did not cause SM hydrolysis at 16 degrees C, a temperature that inhibits the effects of BFA on endomembrane mixing. The very early effects and temperature dependence of BFA-induced SM hydrolysis suggest that the mechanism of hydrolysis may be closely related to endomembrane mixing. These studies are beginning to define important interrelationships between membrane trafficking and topology, SM metabolism, and cell regulation.     

Brefeldin A induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum

The fungal drug brefeldin A (BFA) has recently been found to induce a redistribution of medial- and cis-Golgi components to the endoplasmic reticulum (ER), raising the possibility of the existence of a retrograde pathway from the Golgi complex to the ER. Here, we demonstrate a BFA-induced reversible rearrangement of the trans-Golgi membrane protein galactosyltransferase (Gal-T) to the ER in HeLa cells. With immunofluorescence microscopy we have shown that BFA first caused a rapid change of Gal-T immunolabelling from a normal Golgi complex pattern to long and slender structures emanating from the cell centre and co-localizing with tubulin. Then immunofluorescence became ER-like. This effect was not dependent on ongoing protein synthesis and was reversed to normal within 120 min after removal of the drug. Restoration of the Golgi complex after removal of brefeldin A was energy-dependent but not mediated by microtubules nor dependent on protein synthesis. BFA-induced backflow of Gal-T was inhibited by nocodazole, a microtubule-disrupting agent. Immunoelectron microscopy showed that BFA treatment resulted in the fusion of Gal-T-containing vesicles with the ER. Furthermore, sucrose gradient centrifugation showed a significant shift in density of mature Gal-T polypeptides upon BFA treatment: about 40% of the enzyme migrated from its original density (1.13 g/ml) to the density of rough ER (1.19 g/ml). Thus, BFA caused microtubule-dependent vesicular backflow from a trans-Golgi component to the ER followed by fusion of the Golgi-derived vesicles with the ER.     

Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin

The effects of the fungal metabolite brefeldin A (BFA) on the endocytic routes of internalised wheat germ agglutinin (WGA) were studied in human HepG2 hepatoma cells, drawing particular attention to the application times in relation to the membrane dynamics occurring at the trans Golgi face during endocytosis. As shown in previous studies, transport of internalised WGA into the Golgi apparatus can be classified in three stages being characterised by predominance of vesicular endosomes (stage I), formation of an extended endocytic trans Golgi network (stage II) and uptake of WGA into the stacked Golgi cisternae (stage III). BFA treatment of the cells led to rapid tubular-reticular transformations of the Golgi stacks. Retrograde transport and further destinations of internalised WGA depended on the time of BFA application. When BFA was administered during stages I or II, WGA was localised within the BFA-induced tubules and networks, but never was found within the endoplasmic reticulum. By contrast, BFA treatment during stage III led to a redistribution of internalised WGA into cisternae of the endoplasmic reticulum. These results show that BFA administered according to a precise time schedule can be used as a regulatory agent that allows to control retrograde traffic of internalised molecules into the endoplasmic reticulum.     

Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.     

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.     

Inhibition of endosomal trafficking by brefeldin A interferes with long-distance interaction between chloroplasts and plasma membrane transporters

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic streaming. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indicated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive electrodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and distribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the aggregation of organelles into BFA compartments within 30–120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting formation of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illuminated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.     

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

Summary An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding ofS. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4±3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bindS. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74±9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.     

Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress.

In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting double-membraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.