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.

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.

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 Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

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.

scholarly journals Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport.

1989 ◽  
Vol 109 (5) ◽  
pp. 2057-2065 ◽  
Author(s):  
D Mack ◽  
B Kluxen ◽  
J Kruppa
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Protein Domain ◽  
Complex Type ◽  
Carbonyl Cyanide ◽  
Vesicular Stomatitis ◽  
High Mannose

G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.

Sign in / Sign up

Export Citation Format

Share Document