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

Abstract

We describe the isolation and partial characterization of Saccharomyces cerevisiae nonconditional mutants that show defects in N-glycosylation of proteins. The selection method is based on the reduction of affinity for the ion exchanger QAE-Sephadex as a consequence of the decrease in the negative charge of the cell surface. This characteristic reflects a decrease in the incorporation of mannosyl-phosphate units into the N-linked oligosaccharides of the mannoproteins. The mutants exhibit low affinity for the basic dye alcian blue and for that reason we have called them ldb (low dye binding) mutants. Eight of the complementation groups seem to be new as shown by complementation studies with previously isolated mutants of similar phenotype. Four of the groups showed 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 ldb1 and ldb2, the mutants that show the most drastic reduction in the affinity for the alcian blue dye. In both cases, the purified endo H-released oligosaccharides from the mannoproteins lacked detectable amounts of phosphate groups as shown by ion exchange chromatography and the ¹H NMR spectra. In addition, ldb1 synthesizes a truncated and unbranched outer chain lacking any α(1,2) linked mannoses attached to the α(1,6) linear backbone.