Genetic, biochemical, and morphological evidence for the involvement of N-glycosylation in biosynthesis of the cell wall β1,6-glucan of Saccharomyces cerevisiae

Recent evidence indicates that Stt3p plays a central role in the recognition and/or catalytic step in N-glycosylation (asparagine-linked glycosylation) in the lumen of the endoplasmic reticulum. It is known that stt3 mutants exhibit certain phenotypic features that are suggestive of a cell wall defect. To understand the basis of these phenotypes, we devised a genetic screen to isolate strains bearing mutations that lead to synthetic lethality in combination with the stt3-1 mutation. Using this screen, we were surprised to identify two KRE genes (KRE5 and KRE9) that are involved in the biosynthesis of the cell wall beta1,6-glucan. This finding led us to propose that the N-glycosylation process is essential in the biosynthesis of cell wall beta1,6-glucan. This proposal was supported by the observation that several stt3 mutants exhibited a 60-70% reduction in the content of cell wall beta1,6-glucan as compared with WT cells. Transmission electron microscopy revealed that the stt3 mutant strains exhibit a diffused cell wall with loss of the outer mannoprotein layer as compared with the WT cells. Thus, we provide genetic, morphological, and biochemical evidence for the critical involvement of N-glycosylation in some step in assembly of the cell wall beta1,6-glucan in Saccharomyces cerevisiae.