Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

scholarly journals A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

1985 ◽  
Vol 5 (11) ◽  
pp. 3074-3083 ◽  
Author(s):  
C E Machamer ◽  
R Z Florkiewicz ◽  
J K Rose
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Glycosylation Sites ◽  
Vesicular Stomatitis ◽  
The Individual

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

scholarly journals Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells.

1985 ◽  
Vol 101 (6) ◽  
pp. 2036-2046 ◽  
Author(s):  
C Featherstone ◽  
G Griffiths ◽  
G Warren
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Independent Method ◽  
Transport Pathway ◽  
Post Translational Modifications ◽  
Vesicular Stomatitis ◽  
G1 Cells ◽  
Mitotic Cells

Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post-translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle-mediated step on the pathway.

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

1983 ◽  
Vol 97 (5) ◽  
pp. 1381-1388 ◽  
Author(s):  
R Z Florkiewicz ◽  
A Smith ◽  
J E Bergmann ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Rough Endoplasmic Reticulum ◽  
Mouse Cell ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Truncated Form ◽  
Vesicular Stomatitis

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.

A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface

1985 ◽  
Vol 5 (11) ◽  
pp. 3074-3083
Author(s):  
C E Machamer ◽  
R Z Florkiewicz ◽  
J K Rose
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Glycosylation Sites ◽  
Vesicular Stomatitis ◽  
The Individual

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

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

1980 ◽  
Vol 86 (1) ◽  
pp. 162-171 ◽  
Author(s):  
J E Rothman ◽  
H Bursztyn-Pettegrew ◽  
R E Fine
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Coated Vesicles ◽  
Transmembrane Glycoprotein ◽  
Vesicular Stomatitis ◽  
Two Stages

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.

scholarly journals Newly synthesized G protein of vesicular stomatitis virus is not transported to the cell surface during mitosis.

1983 ◽  
Vol 97 (5) ◽  
pp. 1623-1628 ◽  
Author(s):  
G Warren ◽  
C Featherstone ◽  
G Griffiths ◽  
B Burke
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Viral Protein ◽  
Intracellular Transport ◽  
Mitotic Cell ◽  
Interphase Cells ◽  
Vesicular Stomatitis ◽  
Simultaneous Inhibition

Indirect immunofluorescence, immunoelectron microscopy, and digestion by protease were used to study intracellular transport of the G protein of vesicular stomatitis virus in mitotic and interphase cells. Quantitation showed that the appearance of G protein on the surface of mitotic cells was inhibited at least 10-fold when compared with that on interphase cells, even though similar amounts of viral protein were being synthesized. This dramatic inhibition, taken together with the simultaneous inhibition of endocytosis (Berlin, R. D., and J. M. Oliver, 1980, J. Cell Biol. 85: 660-671), points to a general cessation of membrane traffic in the mitotic cell.

The glycoprotein of VSV accumulates in a distal Golgi compartment in the presence of CCCP

1989 ◽  
Vol 92 (4) ◽  
pp. 643-654
Author(s):  
J.K. Burkhardt ◽  
S. Hester ◽  
Y. Argon
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Preceding Paper ◽  
Marker Enzyme ◽  
Perinuclear Region ◽  
Post Translational Modifications ◽  
Golgi Stack ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Dose Dependent

The post-translational modifications of the G protein of vesicular stomatitis virus, described in the preceding paper, indicate that its transport is arrested by carbonylcyanide m-chlorophenylhydrazone (CCCP) in or near the trans-Golgi. Immunofluorescence microscopy of BHK-21 cells infected with vesicular stomatitis virus and treated with CCCP shows an accumulation of G protein in the Golgi area. In the same cells, the morphology of wheat germ agglutinin (WGA)-staining structures in the perinuclear region is aberrant. Using anti-BiP antibody, there is no obvious change in the structure of the endoplasmic reticulum. Electron microscopy reveals that the aberrant structures in the perinuclear region result from dilation of Golgi cisternae and accumulation of large vacuoles near the Golgi stack. The appearance of these aberrant structures is dose-dependent and they disappear after the protonophore is removed. The vast majority of the vacuoles accumulate on the trans side of the Golgi stack. A small fraction of them contain the marker enzyme thiamine pyrophosphatase (TPPase). By immunoelectron microscopy, most of the vacuoles contain G protein. We conclude that most of the Golgi-associated vacuoles are derived from a distal Golgi transport compartment, possibly the trans-Golgi reticulum, and that CCCP reversibly inhibits the transport of newly synthesized G protein through this distal compartment.

scholarly journals Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography.

1982 ◽  
Vol 94 (1) ◽  
pp. 36-41 ◽  
Author(s):  
J J Bergeron ◽  
G J Kotwal ◽  
G Levine ◽  
P Bilan ◽  
R Rachubinski ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Transmembrane Glycoprotein ◽  
Glycoprotein G ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Endoglycosidase H

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins

1988 ◽  
Vol 8 (7) ◽  
pp. 2869-2874
Author(s):  
J L Guan ◽  
A Ruusala ◽  
H Cao ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Cytoplasmic Domain ◽  
Secretory Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

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