Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [14C]glucosamine catalyzes the transfer of N-acetylglucosamine to an endogenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimuated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine. The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose. The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[14C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.