Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation

• Published in: Glycobiology (Oxford) Vol. 9; no. 3; pp. 243 - 253
• Main Authors: Berkel, M.A.A. van, Rieger, M, Heesen, S. te, Ram, A.F.J, Ende, H. van den, Aebi, M, Klis, F.M
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.03.1999; Oxford Publishing Limited (England)
• Subjects: Amino Acid Sequence; Calcofluor white hypersensitive; carboxypeptidase Y; cell wall mutant; Dolichol - metabolism; Endoplasmic Reticulum; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes, Fungal; Glycosylation; Hexosyltransferases; hygromycin hypersensitive; Membrane Proteins; Membranes - chemistry; Molecular Sequence Data; Mutation; Oligosaccharides - metabolism; plant biochemistry; plant physiology; Pyrophosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Transferases - metabolism; vanadate resistant
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/9.3.243

The Saccharomyces cerevisiae mutant cwh8 was previously found to have an anomalous cell wall. Here we show that the cwh8 mutant has an N-glycosylation defect. We found that cwh8 cells were resistant to vanadate and sensitive to hygromycin B, and produced glycoforms of invertase and carboxypeptidase Y with a reduced number of N-chains. We have cloned the CWH8 gene. We found that it was nonessential and encoded a putative transmembrane protein of 239 amino acids. Comparison of the in vitro oligosaccharyl transferase activities of membrane preparations from wild type or cwh8Δ cells revealed no differences in enzyme kinetic properties indicating that the oligosaccharyl transferase complex of mutant cells was not affected. cwh8Δ cells also produced normal dolichols and dolichol-linked oligosaccharide intermediates including the full-length form Glc3Man9GlcNAc2. The level of dolichol-linked oligosaccharides in cwh8Δ cells was, however, reduced to about 20% of the wild type. We propose that inefficient N-glycosylation of secretory proteins in cwh8Δ cells is caused by an insufficient supply of dolichol-linked oligosaccharide substrate.