Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.     

Secretion of collagen by insects: Autoradiographic study of l-proline 3H-5 incorporation by the firebrat Thermobia domestica

The biosynthesis of collagenous endoskeletal endosterna by fibroblasts was studied by electron microscope autoradiography after tritium-labelled proline administration in the firebrat Thermobia domestica at various time intervals. Autoradiographs were quantitatively analyzed and the relative concentration of label was determined for various cellular compartments. The labelling in the rough endoplasmic reticulum, ground cytoplasm, Golgi apparatus and endosterna was compared during the secretion of radioactive products. The label, initially located in the rough endoplasmic reticulum was found in the Golgi apparatus before it accumulated in the endosternum. The involvement of the rough endoplasmic reticulum and Golgi apparatus in the transport of secreted collagen is discussed, comparing our results with current knowledge of collagenous secretion.     

Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport

The hemagglutinin of influenza virus is synthesized as a monomeric subunit that is cotranslationally translocated across the membrane of the rough endoplasmic reticulum. We show that folding and assembly of hemagglutinin monomers into trimeric structures takes approximately 7-10 min and is completed before the protein leaves the endoplasmic reticulum. Mutants of hemagglutinin that fail to be transported from the endoplasmic reticulum are blocked at different stages of the folding pathway. Unfolded molecules of hemagglutinin are associated with a cellular protein of 77 kd that has been shown previously to bind to IgG heavy chain in the endoplasmic reticulum of certain myelomas. We discuss why assembly of native structures is required for transport of proteins through the exocytotic pathway.     

Subcellular fractionation studies on the post-translational processing of pro-adrenocorticotropic hormone/endorphin in rat intermediate pituitary

The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.     

Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

The relationship between the morphology of cell organelles and kinetics of the secretory process in male sex accessory glands of mice

Summary Two male sex accessory glands of the mouse, seminal vesicle and coagulating gland, were compared with the aim of relating differences in the morphology of organelles to the kinetics of the secretory process. The epithelial cells of the two glands were assessed by morphometric analysis, cytochemical staining, and electron-microscopic autoradiography after administration of a labeled amino acid. The rough endoplasmic reticulum of the seminal vesicle comprised narrow parallel cisternae, while that of the coagulating gland was greatly distended and occupied a much larger percentage of the cytoplasmic volume. Radioactively labeled products were secreted much more rapidly in the seminal vesicle than in the coagulating gland. The primary point of difference in kinetics of intracellular transport between the two glands was in exit of material from the rough endoplasmic reticulum. The more rapid drainage of the rough endoplasmic reticulum may be related to its relatively greater membrane surface density and lesser internal volume. In contrast, similarities in size and cytochemical staining in the Golgi apparatus of the two glands were accompanied by similar kinetics of intracellular transport of secretory protein through this organelle.     

Ultrastructural changes in the liver of the sand lamprey,Lampetra reissneri,during sexual maturation

Ultrastructural changes of hepatocytes were examined in the sand lamprey,Lampetra reissneri, during various phases of the life cycle. In hepatocytes of ammocoetes, the rough endoplasmic reticulum was composed of short cisternae and the Golgi apparatus were scarcely developed, showing no sexual differences at this stage of life cycle. In hepatocytes of female lampreys at the metamorphic stages 4 to 5, the rough endoplasmic reticulum was developed to form long parallel cisternae and the Golgi apparatus were well-developed. The rough endoplasmic reticulum developed further to form stacks of long parallel cisternae extending over the cytoplasm in hepatocytes of females at the young adult stage, and became composed of both long parallel and vesicular cisternae in the cells of females at the adult stage. The Golgi apparatus were invariably welldeveloped in hepatocytes of young adult and adult females. No consipcuous development was observed in profiles of the rough endoplasmic reticulum and the Golgi apparatus in hepatocytes of males during and after metamorphosis. The ultrastructural changes of the rough endoplasmic reticulum and the Golgi apparatus observed in hepatocytes of female sand lampreys are considered to have an intimate relation to the activity of vitellogenin synthesis in the liver, and it is suggested that the hepatocytes begin to rapidly synthesize vitellogenin in the sand lamprey at the metamorphic stages 4 to 5.     

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.     

The overexpression of GMAP-210 blocks anterograde and retrograde transport between the ER and the Golgi apparatus

Golgi Microtubule-Associated Protein (GMAP)-210 is a peripheral coiled-coil protein associated with the cis -Golgi network that interacts with microtubule minus ends. GMAP-210 overexpression has previously been shown to perturb the microtubule network and to induce a dramatic enlargement and fragmentation of the Golgi apparatus (Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM. J Cell Biol 1999; 145: 83–98). We now report that overexpressing GMAP-210 blocks the anterograde transport of both a soluble form of alkaline phosphatase and the hemagglutinin protein of influenza virus, an integral membrane protein, between the endoplasmic reticulum and the cis /medial (mannosidase II-positive) Golgi compartment. Retrograde transport of the Shiga toxin B-subunit is also blocked between the Golgi apparatus and the endoplasmic reticulum. As a consequence, the B-subunit accumulates in compartments positive for GMAP-210. Ultrastructural analysis revealed that, under these conditions, the Golgi complex is totally disassembled and Golgi proteins as well as proteins of the intermediate compartment are found in vesicle clusters distributed throughout the cell. The role of GMAP-210 on membrane processes at the interface between the endoplasmic reticulum and the Golgi apparatus is discussed in the light of the property of this protein to bind CGN membranes and microtubules.     

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.     

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.     

A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.

ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB which causes hindlimb paralysis in mice. Previously, it had been shown that the temperature-sensitive defect resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly into two cleavage products, gp70 and Prp15E. This inefficient processing of gPr80env is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25----Ile in gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env at the restrictive temperature, and neurovirulence of ts1. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr80env remained in the endoplasmic reticulum of cells infected by ts1, but no endoglycosidase H-resistant gPr80env and only trace amounts of gp70 were detected in the infected cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus, inefficient processing of gPr80env at the restrictive temperature is most likely due to inefficient transport of gPr80env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80env being a poor substrate for the processing protease at the restrictive temperature.     

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.     

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.     

Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.     

Immunocytochemical localization of ras p21 product in thyroid follicular cells of normal rats using monoclonal antibody RAP-5

Summary The ultrastructural localization ofras p21 product was studied immunocytochemically in thyroid follicular cells of normal rats using pre-embedded peroxidase-labelled antibody techniques and a monoclonal antibody, RAP-5, which had been raised against a synthetic peptide corresponding to amino acid positions 10–17 of theras p21 protein. Theras p21 product was detected in cisternae of rough endoplasmic reticulum, Golgi apparatus and the subapical portion of apical plasma membrane, in which it was most concentrated. This study indicated that the p21 product may be synthesized in the rough endoplasmic reticulum and finally localized at the subapical portion of the thyroid follicular cells, and also that the apical plasma membrane may be a major site for the reception of environmental stimuli.     

Analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope

We isolated mutants whose vaccinia hemagglutinin (HA) accumulates on nuclear envelopes and the rough endoplasmic reticulum. Mutant HA must be blocked at a pre-Golgi step because it has high-mannose-type carbohydrates but no fucose. Neither N- nor O-glycosidically linked carbohydrates are involved in the transport defect of the mutant HA, because tunicamycin, an inhibitor of N-type glycosylation, has no effect, and O-type glycosylation takes place in the Golgi organelle. The unglycosylated form of the mutant HA synthesized in the presence of tunicamycin is 3000 daltons larger than the wild type. This higher molecular weight is related to the transport defect. HAs translated in vitro also show this difference, evidence that it reflects mutation in the HA structural gene. Portions of HAs that project into the cytoplasm seem to account for this weight difference. Thus the cytoplasmic tail of glycoprotein has an important function in transport out of the rough endoplasmic reticulum.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid