Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation.
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 mannosylphosphate 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 Idb (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 Idb1 and Idb2, 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, Ibd1 synthesizes a truncated and unbranched outer chain lacking any alpha (1,2) linked mannoses attached to the alpha (1,6) linear backbone.
Mañas
P
P
Department of Microbiology, University of Extermadura, Badajoz, Spain.
Olivero
I
I
Avalos
M
M
Hernández
L M
LM
D000423:Alcian Blue / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004352:Drug Resistance, Microbial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D043324:beta-Fructofuranosidase
D009844:Oligosaccharides; D014638:Vanadates; D001216:Asparagine; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D000423:Alcian Blue; D008358:Mannose