A Refined Method for the Determination of Saccharomyces cerevisiae Cell Wall Composition and β-1,6-Glucan Fine Structure

✍ Scribed by Paula Magnelli; John F. Cipollo; Claudia Abeijon

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 452 KB
• Volume: 301
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5473

✦ Synopsis

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between ␤1,3and ␤1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50 -100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant ␤-1,3-glucanase is initially used, releasing all of the chitin and 60 -65% of the ␤1,3-glucan from the cell walls. Next, recombinant endo-␤-1,6-glucanase from Trichoderma harzianum is utilized to release all the ␤-1,6-glucan present in the wall. The chromathographic pattern of endoglucanase digested ␤-1,6-glucan provides a characteristic "fingerprint" of ␤-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1 H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and ␤-glucosidase to release the remaining ␤-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.