Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae

The Saccharomyces cerevisiae alg3-1 mutant is descilbed as defective in the biosynthesis of dolichol-linked oligosaccharides (Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). Man5GlcNAc2-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. In this study, we describe the cloning of the ALG3 locus by complementation of the temperature sensitive growth defect of the alg3 stt3 double mutant. The isolated ALG3 gene complements both the defect in the biosynthesis of lipidlinked oligosaccharides of the alg3-mutant and the underglycosylation of secretory proteins. The inactivation of the nonessential ALG3 gene results in the accumulation of lipid-linked Man5GlcNAc2 and protein-bound carbohydrates which are completely Endo H resistant. The ALG3 locus encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequenc

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

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 substrat

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae

ISSN:0959-665