Temperature-Sensitive Glycoprotein

A successful tool in the early studies of metabolic pathways was blocking the pathway at some specific point. This could be done by the use of either mutants or inhibitors. Schekman et al have isolated a number of yeast mutants with blocks in their secretion pathway (Schekman, 1982). It is not yet known which proteins these mutations affect, but this is clearly a most promising approach for identifying those components involved in transport. In animal cells there are no cellular mutants with blocks in the intracellular transport of protein from the ER to the cell surface. There are, however, genetic diseases which affect the routing of lysosomal enzymes to the lysosomes (Neufeld et al, 1975 Sly and Fischer, 1982). For viruses it has been possible to isolate temperature-sensitive mutants in which a mutation in the viral glycoprotein arrests... 

In the presence of tunieamycin, cells infected with Rous sarcoma virus produced virus particles that lacked infectivity, and were devoid of the envelope glycoproteins gp85 and gp35. Virus particles lacking envelope glycoproteins had previously been found in a deletion mutant of Rous sarcoma virus and in a temperature-sensitive mutant of... 

Gibson, R., Schlesinger, S., and Kornfeld, S. (1979). The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature sensitive and undergoes intracellular aggregation at elevated temperatures. J. Biol. Chem. 254, 3600-3605. 

In both procedures, a gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomatitis virus (VSV) Is Introduced Into cultured mammalian cells either by transfection or simply by Infecting the cells with the virus. The treated cells, even those that are not specialized for secretion, rapidly synthesize the VSV G protein on the ER like normal cellular secretory proteins. Use of a mutant encoding a temperature-sensitive VSV G protein allows researchers to turn subsequent protein transport on and off. At the restrictive temperature of 40 °C, newly made VSV G protein Is misfolded and therefore retained within the ER by quality control mechanisms discussed in Chapter 16, whereas at the permissive temperature of 32 C, the accumulated... 

Hashimoto, K., and Simizu, B., 1982, A temperature-sensitive mutant of western equine encephalitis virus with an altered envelope protein El and a defect in the transport of envelope glycoproteins. Virology 119 276. 

The carpanone-based hbrary was screened to discover small molecules that perturb a secretory pathway that involves the transport of properly folded proteins from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. Whole-cell fluorescence imaging with temperature-sensitive vesicular stomatitis virus glycoprotein fused to green fluorescent protein (VSVGts-GFP) ... 

Warren, L., Critchley, D., and MacPherson, I., 1972a, Surface glycoproteins and glycolipids of chicken embryo cells transformed by a temperature-sensitive mutant of Rous sarcoma virus. Nature 235 275. 

Crowe JE, Jr, Collins PL, London WT, Chanock RM, Murphy BR. A comparison in chimpanzees of the immunogenicity and efficacy of live attenuated respiratory syncytial virus (RSV) temperature- sensitive mutant vaceines and vaecinia virus recombinants that express the surface glycoproteins of RSV. Vaccine... 

The hydrazone bond can be reduced to stabilize the linkage by the addition of sodium cyanoborohydride to a final concentration of 50mM. React for 30 minutes at room temperature with mixing. All operations with cyanoborohydride should be done in a fume hood. If the glycoprotein being modified is sensitive to disulfide reduction and potential denaturation, then this step should be avoided. 

Frost-resistant (hardy) plants are less sensitive than others to damage by low temperatures that is caused by water loss and intracellular, ice-crystal formation. Production of such highly hydrophilic proteins as glycoproteins would constitute a potential mechanism, through the formation of hydrogen... 

The quantities of antifreeze glycoproteins required for the inhibition of the lectin and depression of the freezing temperature are <0.1 pg and > 1.0 mg, respectively that is, the lectin assay is >104 times more sensitive. The lectin therefore provides a micro method for determining the amounts of the glycoprotein in tissues and fluids of polar fishes and for studying its biosynthesis and metabolism. 

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