Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.     

Glucose deprivation induces the selective accumulation of hexose transporter protein GLUT-1 in the plasma membrane of normal rat kidney cells

Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.     

Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea cotyledons in different transport vesicles

In the parenchyma cells of developing legume cotyledons, storage proteins are deposited in a special type of vacuole, known as the protein storage vacuole (PSV). Storage proteins are synthesized at the endoplasmic reticulum and pass through the Golgi apparatus. In contrast to lysosomal acid hydrolases, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-dense vesicles rather than in clathrin-coated vesicles. By combining isopycnic and rate zonal sucrose density gradient centrifugation with phase partitioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage proteins. In dense vesicles, prolegumin occurred in a more aggregated form than it did in the endoplasmic reticulum. The putative vacuolar sorting receptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly reduced in the dense vesicle fraction. This result was confirmed by quantitative immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of the dense vesicles could not be detected. In contrast, 90% of the dense vesicles were labeled with antibodies raised against alpha-TIP (for tonoplast intrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and alpha-TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.     

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.     

Presence of an immunologically-defined class of peri-Golgi vesicles in cultured mammalian cells

Immunsera of mice injected with clathrin-depleted coated vesicle membranes, purified from rat liver, revealed a preferential labeling of some perinuclear structures by immunofluorescence microscopy on NRK cells. Subsequent production of 4 monoclonal antibodies was achieved. The antigen was strictly located in the Golgi area of the cell but was not an intrinsic element of the Golgi complex. The restricted location of the structures excluded these were lysosomes which appeared more dispersed in these cells. After nocodazole treatment the material was found dispersed in the cytoplasm. This provided a means of distinguishing the antigen from clathrin-coated structures and Golgi intrinsic elements. Immunolocalization at the electron microscope level confirmed the data obtained at the light level. Some peroxidase reaction product was rarely associated with Golgi elements, but predominantly stained small neighboring Golgi vesicles (50 nm diameter), as well as tubulo-elongated structures and some large (500 nm) irregular-shaped vesicles. A 32 kDa molecular weight antigen was characterized by immunopurification from NRK cells metabolically labeled with 35S-Met. This 32 kDa antigen appeared as part of a higher multimolecular membrane component of 300 kDa. A 170 kDa and a 70 kDa components were immunodetected in a semi-purified membrane fraction from rat liver, demonstrating that the antigen was a minor but very antigenic contaminant of the coated vesicle preparation used as immunogen. In conclusion, the labeled peri-Golgi structures may be part of the newly characterized trans-Golgi network and/or of the reticular/vesicular endosomal, prelysosomal structure recently described.     

Role of Microtubules in the Organization of the Golgi Complex

The Golgi complex of mammalian cells is composed of cisternal stacks that function in processing and sorting of membrane and luminal proteins during transport from the site of synthesis in the endoplasmic reticulum to lysosomes, secretory vacuoles, and the cell surface. Even though exceptions are found, the Golgi stacks are usually arranged as an interconnected network in the region around the centrosome, the major organizing center for cytoplasmic microtubules. A close relation thus exists between Golgi elements and microtubules (especially the stable subpopulation enriched in detyrosinated and acetylated tubulin). After drug-induced disruption of microtubules, the Golgi stacks are disconnected from each other, partly broken up, dispersed in the cytoplasm, and redistributed to endoplasmic reticulum exit sites. Despite this, intracellular protein traffic is only moderately disturbed. Following removal of the drugs, scattered Golgi elements move along reassembling microtubules back to the centrosomal region and reunite into a continuous system. The microtubule-dependent motor proteins cytoplasmic dynein and kinesin bind to Golgi membranes and have been implicated in vesicular transport to and from the Golgi complex. Microinjection of dynein heavy chain antibodies causes dispersal of the Golgi complex, and the Golgi complex of cells lacking cytoplasmic dynein is likewise spread throughout the cytoplasm. In a similar manner, kinesin antibodies have been found to inhibit Golgi-to-endoplasmic reticulum transport in brefeldin A-treated cells and scattering of Golgi elements along remaining microtubules in cells exposed to a low concentration of nocodazole. The molecular mechanisms in the interaction between microtubules and membranes are, however, incompletely understood. During mitosis, the Golgi complex is extensively reorganized in order to ensure an equal partitioning of this single-copy organelle between the daughter cells. Mitosis-promoting factor, a complex of cdc2 kinase and cyclin B, is a key regulator of this and other events in the induction of cell division. Cytoplasmic microtubules depolymerize in prophase and as a result thereof, the Golgi stacks become smaller, disengage from each other, and take up a perinuclear distribution. The mitotic spindle is thereafter put together, aligns the chromosomes in the metaphase plate, and eventually pulls the sister chromatids apart in anaphase. In parallel, the Golgi stacks are broken down into clusters of vesicles and tubules and movement of protein along the exocytic and endocytic pathways is inhibited. Using a cell-free system, it has been established that the fragmentation of the Golgi stacks is due to a continued budding of transport vesicles and a concomitant inhibition of the fusion of the vesicles with their target membranes. In telophase and after cytokinesis, a Golgi complex made up of interconnected cisternal stacks is recreated in each daughter cell and intracellular protein traffic is resumed. This restoration of a normal interphase morphology and function is dependent on reassembly of a radiating array of cytoplasmic microtubules along which vesicles can be carried and on reactivation of the machinery for membrane fusion.     

IMMUNOGLOBULIN SYNTHESIS AND SECRETION : II. Radioautographic Studies of Sites of Addition of Carbohydrate Moieties and Intracellular Transport

The subcellular sites of synthesis and route of intracellular transfer of immunoglobulin G (IgG) have been investigated by electron microscope radioautography with precursors used for the polypeptide chain (leucine-³H) and for the carbohydrate moieties (galactose-³H and glucosamine-³H). For this purpose, plasma cells from a mouse myeloma tumor were labeled with appropriate precursors and the distribution of radioautographic grains was determined at the end of the labeling period and after varying times of incubation in unlabeled medium. The results indicated that the polypeptide backbone is synthesized in a region of the cell occupied by the rough endoplasmic reticulum (RER) and is transported from there to the region of the Golgi complex. Galactose is incorporated in IgG primarily at the level of the Golgi complex, whereas the incorporation of glucosamine appears to take place both in the RER and in the Golgi complex. From the Golgi complex, the completed IgG molecules reach the plasma membrane and are discharged extracellularly. The latter route of transport and the mechanism of discharge are not understood but may be mediated via smooth-surfaced vesicles.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.     

Microtubules and the organization of the Golgi complex

Electron microscopic and cytochemical studies indicate that microtubules play an important role in the organization of the Golgi complex in mammalian cells. During interphase microtubules form a radiating pattern in the cytoplasm, originating from the pericentriolar region (microtubule-organizing centre). The stacks of Golgi cisternae and the associated secretory vesicles and lysosomes are arranged in a circumscribed juxtanuclear area, usually centered around the centrioles, and show a defined orientation in relation to the rough endoplasmic reticulum. Exposure of cells to drugs such as colchicine, vinblastine and nocodazole leads to disassembly of microtubules and disorganization of the Golgi complex, most typically a dispersion of its stacks of cisternae throughout the cytoplasm. These alterations are accompanied by disturbances in the intracellular transport, processing and release of secretory products as well as inhibition of endocytosis. The observations suggest that microtubules are partly responsible for the maintenance and functioning of the Golgi complex, possibly by arranging its stacks of cisternae three-dimensionally within the cell and in relation to other organelles and ensuring a normal flow of material into and away from them. During mitosis, microtubules disassemble (prophase) and a mitotic spindle is built up (metaphase) to take care of the subsequent separation of the chromosomes (anaphase). The breaking up of the microtubular cytoskeleton is followed by vesiculation of the rough endoplasmic reticulum and partial atrophy, as well as dispersion of the stacks of Golgi cisternae. After completion of the nuclear division (telophase), the radiating microtubule pattern is re-established and the rough endoplasmic reticulum and the Golgi complex resume their normal interphase structure. This sequence of events is believed to fulfil the double function to provide tubulin units and space for construction of the mitotic spindle and to guarantee an approximately equal distribution of the rough endoplasmic reticulum and the Golgi complex on the two daughter cells.     

The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment

There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.     

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.     

Passage of viral membrane proteins through the Golgi complex

BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes.     

Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.     

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.     

The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum.

The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2. alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX). Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum. 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi. The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase. The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

Synthesis,secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac

Summary Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.Abbreviations apo apolipoprotein - ER endoplasmic reticulum - IDL intermediate density-lipoprotein - LDL low density-lipoprotein - VLDL very low density-lipoprotein - PBS phosphate-buffered salt solution - RER rough endoplasmic reticulum - TEM transmission electron microscopy     

Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface.

The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane.