Structure of the Phosphorylated N-linked Oligosaccharides from the mnn9and mnn10Mutants of Saccharomyces cerevisiae

The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester. The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnnmutants (Hernandez, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849–11856). The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation. The mnn1 mnn2 mnn10acidic oligosaccharide fraction contained a mono- and a diphosphate ester. The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where xmay range from 2 to 12. The diphosphate had a mannosyl phosphate in this position as well as one on the terminal α1→6-linked mannose in the core. The presence in the mnn1 mnn9or mnn1 mnn2 mnn10background of the mnn4or mnn6mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it.