ISOLATION OF NEW NONCONDITIONAL SACCHAROMYCES-CEREVISIAE MUTANTS DEFECTIVE IN ASPARAGINE-LINKED GLYCOSYLATION

We describe the isolation and partial characterization of Saccharomyce s cerevisiae nonconditional mutants that show defects in N-glycosylati on of proteins, The selection method is based on the reduction of affi nity for the ion exchanger QAE-Sephadex as a consequence of the decrea se in the negative charge of the cell surface, This characteristic ref lects a decrease in the incorporation of mannosylphosphate units into the N-linked oligosaccharides of the mannoproteins, The mutants exhibi t low affinity for the basic dye alcian blue and for that reason we ha ve called them ldb (low dye binding) mutants, Eight of the complementa tion groups seem to be new as shown by complementation studies with pr eviously isolated mutants of similar phenotype, Four of the groups sho wed a significant reduction in the number and/or size of the N-linked oligosaccharides attached to secreted invertase, We have analyzed the N-linked oligosaccharides of Idb1 and ldb2, the mutants that show the most drastic reduction in the affinity for the alcian blue dye, In bot h cases, the purified endo II-released oligosaccharides from the manno proteins lacked detectable amounts of phosphate groups as shown by ion exchange chromatography and the H-1 NMR spectra. In addition, ldb1 sy nthesizes a truncated and unbranched outer chain lacking any alpha(1,2 ) linked mannoses attached to the alpha(1,6) linear backbone.