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

• Published in: The 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
• Language: English
• Published: New York, NY Rockefeller University Press 01.07.1987; The Rockefeller University Press
• Subjects: 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; Virus; Cell culture; Vesicular stomatitis virus; Vesiculovirus; Transfection; Rhabdoviridae; Lateral diffusion; Plasma membrane; Site directed mutagenesis; Glycoproteins; Diffusion coefficient
• DOI: 10.1083/jcb.105.1.69
• ISSN: 0021-9525

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.