In order to investigate -glycosylation of proteins in the fungus QM 9414, a membrane preparation was isolated and used to study the glycosylation of endogenous proteins. Exogenously added GDP-[U-C]mannose was used to mannosylate both endogenous lipid and protein. The kinetics of mannosylation together with pulse-chase experiments with cold GDP-mannose revealed that lipid was labelled before protein. The lipid was identified as mannosyl phosphoryl dolichol (Dol-P-Man) by TLC together with an authentic standard from yeast. Addition of tsushimycin, a specific inhibitor of Dol-P-Man synthesis, completely blocked transfer of mannose from GDP-[U-C]mannose to endogenous lipid. The mannosyl units transferred to endogenous protein could be released by β-elimination, and were shown to consist mainly of tetra-, di- and monomannosyl chains. Mannosylation of endogenous proteins occurred at a lower rate with membranes isolated from glycerol-grown cells. This could be overcome by addition of cold GDP-mannose, suggesting a limitation of endogenous GDP-mannose and/or dolichol phosphate in glycerol-grown (i.e. catabolite-repressed) cells.


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