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J E Bergmann

Publications and source records attributed to J E Bergmann.

29 records · Page 2Linked to original sources
PubMed1983

Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein.

We have altered the structure of the COOH-terminus of the vesicular stomatitis virus (VSV) glycoprotein (G) by introducing deletions into a cDNA clone encoding G protein. We examined the effects of these deletions on intracellular transport of G protein after expression of the deleted genes in eucaryotic cells under control of the SV40 late promoter. To prevent readthrough of translation into vector sequences, we introduced synthetic DNA linkers containing translation stop codons at the site of the deletion. G proteins that lacked the cytoplasmic domain and most of the transmembrane domain were secreted slowly from the cells. Deletion mutants affecting the structure of the cytoplasmic domain fell into two classes. The first class completely arrested transport of the protein to the cell surface at a stage prior to acquisition of complex oligosaccharides. The second class showed severely reduced rates of complex sugar addition although the proteins were eventually transported to the cell surface. Indirect immunofluorescence microscopy suggested that mutant proteins in both classes may accumulate in the rough endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1983

Membrane insertion at the leading edge of motile fibroblasts.

We are concerned with the mechanisms involved in the directed migration of eukaryotic cells. Previously we found that, inside cells at the edge of an experimental wound, the Golgi apparatus and the microtubule-organizing center were rapidly repositioned forward of the nucleus in the direction of subsequent cell migration into the wound. This repositioning was proposed to serve the purpose of introducing new membrane mass at the leading edge of the cell, by directing Golgi apparatus-derived vesicles bound for the plasma membrane to that edge. We now provide evidence to support this proposal. Cultured fibroblastic cells at the edge of a wound were infected with a temperature-sensitive mutant (0-45) of vesicular stomatitis virus. It is known that the G-protein, an integral membrane protein of the virus, is synthesized and remains in the rough endoplasmic reticulum at the nonpermissive temperature, but when the infected cells are shifted to the permissive temperature, the G-protein moves through the Golgi apparatus to the plasma membrane. By immunofluorescence microscopy, we here show that the first appearance of the G-protein at the cell surface corresponds to the leading edge of the motile cell. These observations are incorporated into a coherent scheme for the mechanisms involved in cell migration.

Animals↗
PubMed1983

Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein.

We have characterized two stable transformed mouse cell lines (CG1 and CTG1) that express either the normal vesicular stomatitis virus glycoprotein (G) or a truncated form of the G protein (TG) that lacks the COOH-terminal anchor sequences and is secreted from the cells. These cell lines were obtained using a hybrid vector consisting of the transforming DNA fragment of bovine papilloma virus linked to a segment of the SV40 expression vector pSV2 containing cloned cDNA encoding either the normal or truncated form of the vesicular stomatitis virus G protein. Using indirect immunofluorescence we have found that greater than 95% of the cells in each line express the G protein(s), although the level of expression within the population is variable. The normal G protein expressed in these cells obtains its complex oligosaccharides in less than 30 min and is transported to the cell surface. In contrast, the TG protein obtains its complex oligosaccharides with a half-time of about 2.5 h. Immunofluorescence data show an apparent concentration of the TG protein in the rough endoplasmic reticulum. These data together suggest that transfer of this anchorless protein from the rough endoplasmic reticulum to the Golgi apparatus is the rate-limiting step in its secretion. We observed, in addition to normal G protein, two smaller G-related proteins produced in the CG1 cell line. We suggest that these proteins could result from aberrant splicing from sites within the G mRNA sequence to the downstream acceptor in the pSV2 vector.

Amino Acid Sequence↗
PubMed1983

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.

Animals↗
PubMed1982

Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells.

A cDNA clone of the mRNA encoding the glycoprotein (G) of vesicular stomatitis virus was inserted into plasmid vectors under the control of either the SV40 early promoter (pSV2G) or the SV40 late promoter (pSVGL). Synthesis of G protein was observed in mouse L cells injected with pSV2G DNA or in COS1 cells transfected with pSVGL DNA. Immunofluorescent staining of G protein produced in both cell types showed a pattern of internal and cell-surface staining indistinguishable from that seen in cells infected with vesicular stomatitis virus. The G protein produced in transfected COS1 cells was the size of normal G protein and was glycosylated. Expression of a G protein lacking 79 amino acids from the COOH terminus was also examined. This G protein lacks the transmembrane domain and the hydrophilic COOH terminus, which, we postulated, anchor G protein in the lipid bilayer. This "anchorless" protein is glycosylated and is secreted, albeit slowly.

Amino Acid Sequence↗
PubMed1981

Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane.

The intracellular pathway of biogenesis of the vesicular stomatitis virus transmembrane glycoprotein was investigated in situ by using indirect immunofluorescence of whole infected Chinese hamster ovary cells and immunoelectron microscopy of ultrathin frozen sections of infected cells. Transport of the glycoprotein was synchronized by using the temperature-sensitive virus mutant Orsay-45 and a temperature shift-down protocol. Sequential appearance of the glycoprotein in the rough endoplasmic reticulum, Golgi apparatus, and plasmalemma was demonstrated. The potential of this system for further studies is discussed.

Animals↗
PubMed1980

Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells.

It was previously shown that the poliovirus-induced inhibition of translation of capped mRNAs can be reversed by a protein found in preparations of the eukaryotic initiation factor eIF-4B [Rose, J. K., Trachsel, H., Leong, K. & Baltimore, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2732--2736]. This "restoring factor" has now been purified from a high-salt wash of rabbit reticulocyte ribosomes by taking advantage of its tight association with factor eIF-3 at low salt concentrations. It did not copurify with the major Mr 80,000 polypeptide of eIF-4B preparations but did copurify with a Mr 24,000 polypeptide previously shown to bind to the cap structures of mRNAs [Sonenberg, N., Rupprecht, K. M., Hecht, S. M. & Shatkin, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 4345--4349]. Both the electrophoretic mobility and the tryptic peptide pattern of the restoring factor were indistinguishable from those of the cap-binding protein, and the restoring factor could be crosslinked to the 5'-terminal cap on mRNA. Thus, is appears that poliovirus inhibits cellular protein synthesis by inactivation of some crucial property of the cap-binding protein.

Carrier Proteins↗
PubMed1979

A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control.

We present a kinetic model of protein synthesis which encompasses initiation, elongation, and termination parameters. We have investigated the dependence of the total rate of protein synthesis and the size of the translating polysomes on each of these parameters and in particular on the level of active 40 S ribosomes and initiation factors. This model qualitatively fits experimental data for the ratio of alpha- to beta-globin synthesis in reticulocytes, both under normal conditions and in the presence of inhibitors of chain initiation. This model has also been used to examine the effect that limiting amounts of certain tRNAs might have on the total rate of protein synthesis. In addition, the role of initiation factor discrimination and mRNA length are examined with respect to the differential translation of mRNAs.

Animals↗
PubMed1979

Characterization of rabbit reticulocyte factor(s) that stimulates the translation of mRNAs lacking 5'-terminal 7-methylguanosine.

Purified reticulocyte initiation factors were assayed for their ability to stimulate the translation of uncapped vesicular stomatitis virus (VSV) mRNA relative to capped VSV mRNA in reticulocyte lysates. Both eIF-3 and eIF-4B preparations contained such an activity. However, at least some of the activity was due to the presence of an as yet unidentified factor that contaminated the eIF-3 and eIF-4B preparations. A polypeptide of apparent molecular weight 24,000 that can be specifically cross-linked to the oxidized 5'-terminal cap structures of reovirus and other viral mRNAs co-purifies with this new factor, and may be a component of it. This stimulatory activity present in our eIF-4B preparations was found to be greater when lower concentrations of KCl were present in the reaction mixture, possibly explaining why uncapped VSV mRNA is translated relatively more efficiently at low K+ concentrations.

Animals↗
PubMed1979

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.

Animals↗