scholarly journals Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain.

1978 ◽  
Vol 253 (3) ◽  
pp. 716-722 ◽  
Author(s):  
I. Tabas ◽  
S. Schlesinger ◽  
S. Kornfeld
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Heavy Chain ◽  
Complex Type ◽  
Form Complex ◽  
Vesicular Stomatitis ◽  
High Mannose

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.

scholarly journals Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization.

1985 ◽  
Vol 101 (2) ◽  
pp. 460-469 ◽  
Author(s):  
C A Gabel ◽  
J E Bergmann
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Complex Type ◽  
Major Class ◽  
Vesicular Stomatitis ◽  
High Mannose

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.

scholarly journals Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

1986 ◽  
Vol 102 (6) ◽  
pp. 2147-2157 ◽  
Author(s):  
L Puddington ◽  
C E Machamer ◽  
J K Rose
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Heavy Chain ◽  
Cytoplasmic Domain ◽  
Integral Membrane Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Hybrid Proteins ◽  
Vesicular Stomatitis ◽  
Immunoglobulin Mu

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

scholarly journals Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

1987 ◽  
Vol 104 (3) ◽  
pp. 749-760 ◽  
Author(s):  
W E Balch ◽  
K R Wagner ◽  
D S Keller
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Free System ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

scholarly journals Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

1989 ◽  
Vol 108 (3) ◽  
pp. 811-819 ◽  
Author(s):  
K Suh ◽  
J E Bergmann ◽  
C A Gabel
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Plasmid Vector ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
High Mannose

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

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