Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall

Abstract

ABSTRACT Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique, crh1 Δ crh2 Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1 Δ crh2 Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.