Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation

We describe the isolation and partial characterization of Saccharomycescerevisiae nonconditional mutants that show defects in N-glycosylationof proteins. The selection method is based on the reductionof affinity for the ion exchanger QAE-Sephadex as a consequenceof the decrease in the negative charge of the cell surface.This characteristic reflects a decrease in the incorporationof mannosyl-phosphate units into the N-linked oligosaccharidesof the mannoproteins. The mutants exhibit low affinity for thebasic dye alcian blue and for that reason we have called themldb (low dye binding) mutants. Eight of the complementationgroups seem to be new as shown by complementation studies withpreviously isolated mutants of similar phenotype. Four of thegroups showed a significant reduction in the number and/or sizeof the N-linked oligosaccharides attached to secreted invertase.We have analyzed the N-linked oligosaccharides of ldb1 and ldb2,the mutants that show the most drastic reduction in the affinityfor the alcian blue dye. In both cases, the purified endo H-releasedoligosaccharides from the mannoproteins lacked detectable amountsof phosphate groups as shown by ion exchange chromatographyand the ¹H NMR spectra. In addition, ldb1 synthesizes a truncatedand unbranched outer chain lacking any