Lipid-saccharide intermediates and glycoprotein biosynthesis in a temperature-sensitive Chinese hamster cell mutant

Abstract

The characterization of a temperature‐sensitive Chinese hamster cell mutant has been continued with the aim of localizing the apparent defect in glycoprotein synthesis (Tenner et al., 1977). Although the mutation is lethal, a demonstration of the ability of the mutant cells to support proliferation of Mengo virus at the nonpermissive temperature indicates that the general metabolic processes of the cells remain intact at a time when glycoprotein synthesis is severely depressed. A quantitative study of protein synthesis on membrane‐associated polysomes suggests that the synthesis of the polypeptide portion of the glycoproteins at 40.8°C may be normal. The investigation of lipidsaccharide molecules which have been implicated in the formation and transfer of the oligosaccharide „core„ to polypeptide acceptors shows that mutaant cells at the nonpermissive temperature are capable of synthesizing these lipid saccharides normally, and that the pool of the dolichyl oligosaccharide is maintained at a constant level independent of the temperature. The rate of formation of the lipid‐oligosaccharide, however, is reduced in intact mutant cells at the nonpermissive temperature. Further investigations show this decreased rate to be the result of an increased half life of the lipid‐oligosaccharide at 40.8°C. These data indicate that the temperature‐sensitive step in glycoprotein biosynthesis is the transfer of the oligosaccharide core from the lipid‐oligosaccharide intermediates to the nascent polypeptide chain. The data presented also provide evidence that the lipid‐saccharide intermediates, previously described mainly in in vitro systems, are in fact involved in the glycosylation of a majority, if not all, of the mannose‐containing glycoproteins in intact, growing hamster cells.