Cloning and characterization of beta-COP from Dictyostelium discoideum

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.     

Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.

Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton.     

Homology modelling of the core domain of the endogenous lectin comitin: structural basis for its mannose-binding specificity

The N-terminal core domain of comitin from the slime mold Dictyostelium discoideum has been modelled from the X-ray coordinates of the monocot mannose-binding lectin from snowdrop (Galanthus nivalis). Docking experiments performed on the three-dimensional model showed that two of the three mannose-binding sites of the comitin monomer are functional. They are located at both ends of the comitin dimer whereas the actin-interacting region occurs in the central hinge region where both monomers are non covalently associated. This distribution is fully consistent with the bifunctional character of comitin which is believed to link the Golgi vesicles exhibiting mannosylated membrane glycans to the actin cytoskeleton in the cell.     

Indirect immunofluorescence localization of ponticulin in motile cells

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Phospholipase D2 is localized to the rims of the Golgi apparatus in mammalian cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid, a molecule known to have multiple physiological roles, including release of nascent secretory vesicles from the trans-Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. We recently demonstrated that PLD1 is localized to the Golgi apparatus, nuclei, and to a lesser extent, plasma membrane. Due to its low abundance, the intracellular localization of PLD2 has been characterized only indirectly through overexpression of chimeric proteins. Using antibodies specific to PLD2, together with immunofluorescence microscopy, herein we demonstrate that a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and was also distributed throughout cells in dense cytoplasmic puncta; a fraction of which colocalized with caveolin-1 and the plasma membrane. On treatment with brefeldin A, PLD2 translocated into the nucleus in a manner similar to PLD1, suggesting a potential role in nuclear signaling. Most significantly, cryoimmunogold electron microscopy demonstrated that in pituitary GH(3) cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively. The data are consistent with a model whereby PLD2 plays a role in Golgi vesicular transport.     

Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport

Cortactin is an actin-binding protein that has recently been implicated in endocytosis. It binds directly to dynamin-2 (Dyn2), a large GTPase that mediates the formation of vesicles from the plasma membrane and the Golgi. Here we show that cortactin associates with the Golgi to regulate the actin- and Dyn2-dependent transport of cargo. Cortactin antibodies stain the Golgi apparatus, labelling peripheral buds and vesicles that are associated with the cisternae. Notably, in vitro or intact-cell experiments show that activation of Arf1 mediates the recruitment of actin, cortactin and Dyn2 to Golgi membranes. Furthermore, selective disruption of the cortactin-Dyn2 interaction significantly reduces the levels of Dyn2 at the Golgi and blocks the transit of nascent proteins from the trans-Golgi network, resulting in swollen and distended cisternae. These findings support the idea of an Arf1-activated recruitment of an actin, cortactin and Dyn2 complex that is essential for Golgi function.     

Localization of actin in Dictyostelium amebas by immunofluorescence

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant.     

Golgi-associated filament networks in duct epithelial cells of rabbit submandibular glands: immunohistochemical light and electron microscopic studies

The duct epithelial cells of rabbit submandibular glands expressed keratin 8, keratin 14, and actin in the supranuclear region, and these cytoskeletal proteins formed ring structures, approximately 1-2.5 microm in diameter. Ultrastructurally, these ring structures were observed as a 'Golgi-associated filament network' surrounding Golgi apparatuses. Double immunofluorescent studies showed that keratin 8 and keratin 14 formed keratin 8/14 filaments, and that these filaments and actin filaments colocalized as components of the Golgi-associated filament network. Our studies suggested that the Golgi-associated filament network maintains the complex structure and location of the Golgi apparatus of the duct epithelium of rabbit submandibular glands. Tubulin was distributed diffusely throughout the cytoplasm of columnar cells, but no special relationship was found between tubulin and the Golgi apparatus.     

Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.     

Cholesterol loading induces a block in the exit of VSVG from the TGN

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.     

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 μg/ml) for 1 h at 37 °C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a marked perinuclear fluorescence. In addition, MAM-6 fluorescence was observed in swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-μm secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement

The human hepatoma cell line HepG2 was used to study the effect of cetaben, a non-fibrate hypolipidemic drug, on cell morphology and vesicle distribution. Cetaben treatment correlated with a fragmentation and/or condensation of Golgi cisternae and the appearance of large electron-lucent vesicles. The Golgi apparatus, demonstrated, for example, by fluorescence-lectin histochemistry, was fragmented after cetaben treatment. The lectin-positive remnants were dispersed throughout the cytoplasm, but with a preference for being transported to tips of cells. However, microtubules and the intermediate filaments as well as the actin microfilaments were unchanged after cetaben treatment indicating that changes in Golgi morphology are not caused by alterations in the cytoskeleton. Cetaben decreases the cholesterol content due to inhibition of cholesterol biosynthesis. Changes in the intracellular cholesterol content are known to influence the intracellular vesicle distribution and are most likely responsible for cetaben-induced Golgi alterations, as depletion of cellular cholesterol by starvation or lovastatin and/or cyclodextrin treatment resulted in a similar redistribution of Golgi-derived wheat germ agglutinin vesicles. These lectin-stained vesicles colocalized with lysosomal marker proteins such as Limp-1 and Lamp-2, but not with the early endosomal markers Rab5 and EEA1. Upon removal of cetaben the lectin- and Limp-1/Lamp-2-costained vesicles dissociated and were transported back to the perinuclear region. Thus, cetaben-induced changes such as fragmentation of the Golgi apparatus and the dispersion of lysosomes away from their juxtanuclear location were reversible.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0689-6     

Involvement of a triton-insoluble floating fraction in Dictyostelium cell-cell adhesion

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals that Dictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cell-cell adhesion during development.     

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.     

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.     

The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms

Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.     

Activated ADP-ribosylation factor assembles distinct pools of actin on golgi membranes

The small GTP-binding protein ADP-ribosylation factor (ARF) has been shown to regulate the interaction of actin and actin-binding proteins with the Golgi apparatus. Here we report that ARF activation stimulates the assembly of distinct pools of actin on Golgi membranes. One pool of actin cofractionates with coatomer (COPI)- coated vesicles and is sensitive to salt extraction and the plus end actin-binding toxin cytochalasin D. A second ARF-dependent actin pool remains on the Golgi membranes following vesicle extraction and is insensitive to cytochalasin D. Isolation of the salt-extractable ARF-dependent actin from the Golgi reveals that it is bound to a distinct repertoire of actin-binding proteins. The two abundant actin-binding proteins of the ARF-dependent actin complex are identified as spectrin and drebrin. We show that drebrin is a specific component of the cytochalasin D-sensitive, ARF-dependent actin pool on the Golgi. Finally, we show that depolymerization of this actin pool with cytochalasin D increases the extent of the salt-dependent release of COPI-coated vesicles from the Golgi following cell-free budding reactions. Together these data suggest that regulation of the actin-based cytoskeleton may play an important role during ARF-mediated transport vesicle assembly or release on the Golgi.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.