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Total cell mannoprotein was isolated from Saccharomyces cerevisiaeX2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem.255, 5986–5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9mutation eliminates all but two mannoses of the outer chain, whereas the mnn8and mnn10strains produce outer chains of variable but similar lengths. The isolate designated mnn7was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9or mnn9 mnn10had the mnn9phenotype, which established that the mnn9defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8and mnn10. Analysis of the mnn1 mnn2 mnn10oligosaccharides revealed that the heterogeneous outer chain contained 6–16 α1→6-linked mannose units and each was terminated by a single α1→2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A.84, 8824–8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain.

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnnmutant mannoproteins is enlarged by the addition of the outer chain to the α1→3-linked mannose in the side chain that is attached to the β1→4-linked mannose rather than by addition to the terminal α1→6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.

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.