Effects of Mutations in Three Domains of the Vesicular Stomatitis Viral Glycoprotein on Its Lateral Diffusion in the Plasma Membrane

The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded...

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Published inThe Journal of cell biology Vol. 105; no. 1; pp. 69 - 75
Main Authors Scullion, Brian F., Hou, Yu, Puddington, Lynn, Rose, John K., Jacobson, Ken
Format Journal Article
LanguageEnglish
Published New York, NY Rockefeller University Press 01.07.1987
The Rockefeller University Press
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Abstract The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded a mean diffusion coefficient (D) of 8.5 (± 1.3) × 10-11cm2/ s and a mean mobile fraction of 75% (± 3%). Eight mutant proteins were also examined: dTM14, lacking six amino acids from the transmembrane domain; TA2, lacking an oligosaccharide in the extracellular domain; QN2, possessing an extra N-linked oligosaccharide in the extracellular domain; CS2, possessing a serine instead of a cysteine at residue 489 in the cytoplasmic domain, preventing palmitate addition to the glycoprotein; TMR-stop, lacking the entire cytoplasmic domain except an arginine at residue 483; and three chimeric proteins, Gμ, G23, and GHA, containing in place of the 29 amino acid wild type cytoplasmic domain the cytoplasmic domains from the surface IgM from the spike protein of the infectious bronchitis virus or from the hemagglutinin protein of the influenza virus, respectively. The mean D for the mutant proteins varied over a relatively small range, with the slowest mutant, G23, exhibiting a value of 11.3 (± 1.4) × 10-11cm2/ s and the fastest mutant, GHA, having a D of 28.6 (± 4.5) × 10-11cm2/ s. The mean mobile fraction similarly varied over a small range, extending from 55 to 68%. None of the mutations resulted in the more rapid diffusion characteristic of membrane proteins embedded in artificial bilayers. Therefore, it appears that the cytoplasmic and transmembrane domains themselves contribute little to restraining the lateral mobility of this integral membrane protein when expressed in transfected cells.
AbstractList The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded a mean diffusion coefficient (D) of 8.5 (± 1.3) × 10-11cm2/ s and a mean mobile fraction of 75% (± 3%). Eight mutant proteins were also examined: dTM14, lacking six amino acids from the transmembrane domain; TA2, lacking an oligosaccharide in the extracellular domain; QN2, possessing an extra N-linked oligosaccharide in the extracellular domain; CS2, possessing a serine instead of a cysteine at residue 489 in the cytoplasmic domain, preventing palmitate addition to the glycoprotein; TMR-stop, lacking the entire cytoplasmic domain except an arginine at residue 483; and three chimeric proteins, Gμ, G23, and GHA, containing in place of the 29 amino acid wild type cytoplasmic domain the cytoplasmic domains from the surface IgM from the spike protein of the infectious bronchitis virus or from the hemagglutinin protein of the influenza virus, respectively. The mean D for the mutant proteins varied over a relatively small range, with the slowest mutant, G23, exhibiting a value of 11.3 (± 1.4) × 10-11cm2/ s and the fastest mutant, GHA, having a D of 28.6 (± 4.5) × 10-11cm2/ s. The mean mobile fraction similarly varied over a small range, extending from 55 to 68%. None of the mutations resulted in the more rapid diffusion characteristic of membrane proteins embedded in artificial bilayers. Therefore, it appears that the cytoplasmic and transmembrane domains themselves contribute little to restraining the lateral mobility of this integral membrane protein when expressed in transfected cells.
The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site- directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded a mean diffusion coefficient (D) of 8.5 (+/- 1.3) X 10(-11) cm2/s and a mean mobile fraction of 75% (+/- 3%). Eight mutant proteins were also examined: dTM14, lacking six amino acids from the transmembrane domain; TA2, lacking an oligosaccharide in the extracellular domain; QN2, possessing an extra N-linked oligosaccharide in the extracellular domain; CS2, possessing a serine instead of a cysteine at residue 489 in the cytoplasmic domain, preventing palmitate addition to the glycoprotein; TMR-stop, lacking the entire cytoplasmic domain except an arginine at residue 483; and three chimeric proteins, G mu, G23, and GHA, containing in place of the 29 amino acid wild type cytoplasmic domain the cytoplasmic domains from the surface IgM from the spike protein of the infectious bronchitis virus or from the hemagglutinin protein of the influenza virus, respectively. The mean D for the mutant proteins varied over a relatively small range, with the slowest mutant, G23, exhibiting a value of 11.3 (+/- 1.4) X 10(-11) cm2/s and the fastest mutant, GHA, having a D of 28.6 (+/- 4.5) X 10(-11) cm2/s. The mean mobile fraction similarly varied over a small range, extending from 55 to 68%. None of the mutations resulted in the more rapid diffusion characteristic of membrane proteins embedded in artificial bilayers. Therefore, it appears that the cytoplasmic and transmembrane domains themselves contribute little to restraining the lateral mobility of this integral membrane protein when expressed in transfected cells.
Author Hou, Yu
Scullion, Brian F.
Jacobson, Ken
Puddington, Lynn
Rose, John K.
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Issue 1
Keywords Virus
Cell culture
Vesicular stomatitis virus
Vesiculovirus
Transfection
Rhabdoviridae
Lateral diffusion
Plasma membrane
Site directed mutagenesis
Glycoproteins
Diffusion coefficient
Language English
License CC BY 4.0
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
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PublicationTitle The Journal of cell biology
PublicationTitleAlternate J Cell Biol
PublicationYear 1987
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References J Cell Biol 1988 Jan;106(1):325
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Snippet The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of...
The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site- directed mutagenesis of...
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StartPage 69
SubjectTerms Amino Acid Sequence
Amino acids
Animals
Biological and medical sciences
Cell coat. Cell surface
Cell Line
Cell membranes
Cell structures and functions
Cells
Cercopithecus aethiops
COS cells
Diffusion
Diffusion coefficient
DNA - genetics
DNA, Viral - genetics
Fibroblasts - metabolism
Fibroblasts - ultrastructure
Fundamental and applied biological sciences. Psychology
Glycoproteins
L cells
Lymphocytes
Membrane Glycoproteins
Membrane proteins
Membrane Proteins - genetics
Membrane Proteins - metabolism
Molecular and cellular biology
Plasma diffusion
Protein Conformation
Protein Processing, Post-Translational
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Vesicular stomatitis Indiana virus - genetics
Vesicular stomatitis Indiana virus - metabolism
Viral Envelope Proteins
Viral Proteins - genetics
Viral Proteins - metabolism
Title Effects of Mutations in Three Domains of the Vesicular Stomatitis Viral Glycoprotein on Its Lateral Diffusion in the Plasma Membrane
URI https://www.jstor.org/stable/1612520
https://www.ncbi.nlm.nih.gov/pubmed/3038931
https://pubmed.ncbi.nlm.nih.gov/PMC2114925
Volume 105
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