| CWH41 is required for beta-1,6-glucan biosynthesis and encodes glucosidase I, an enzyme involved in protein N-chain glucose processing. Therefore, the effects of N-chain glucosylation and processing on beta-1,6-glucan biosynthesis were examined, and it was shown that incomplete N-chain glucose processing results in loss of beta-1,6-glucan. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, the S. cerevisiae KRE5 and CNE1 genes were investigated, which encode homologs of the 'quality control' components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. The essential activity of Kre5p was found to be separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. A ∼30% decrease in beta-1,6-glucan was observed upon disruption of CNE1, a phenotype which is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification.; Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. The fks1Delta mutant was partial K1 killer toxin resistant and showed a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1Delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. FKS2 overexpression suppressed the killer toxin phenotype of fks1Delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1Delta cells. Eight out of twelve fks1tsfks2Delta mutants had altered beta-glucan levels at the permissive temperature: the FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive FKS1T6051 M761T allele increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. beta-1,6-Glucan deficient mutants had reduced in vitro glucan synthase activity and mislocalized Fks1p and Fks2p, possibly contributing to the observed beta-1,6-glucan defects. |
