Analysis of beta-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography.
We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1-->3)-beta-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a 'knr4 disrupted' tetrad demonstrated that mutant cell walls contained much less (1-->3)-beta-glucan and (1-->6)-beta-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.
Hong
Z
Z
Chemotherapy and Molecular Genetics, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539.
Mann
P
P
Shaw
K J
KJ
Didomenico
B
B
D002473:Cell Wall / Q000737:chemistry* / Q000235:genetics; D002686:Chitin / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000235:genetics*; D005697:Galactosides / Q000378:metabolism; D005936:Glucans / Q000032:analysis*; D007211:Indoles / Q000378:metabolism; D009154:Mutation; D010539:Permeability; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D047071:beta-Glucans
D005656:Fungal Proteins; D005697:Galactosides; D005936:Glucans; D007211:Indoles; C085451:SMI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D047071:beta-Glucans; D002686:Chitin; C064197:beta-1,6-glucan; C033363:beta-1,3-glucan; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside