Crh1p and Crh2p are required for the cross-linking of chitin to β(1-6)glucan in the Saccharomyces cerevisiae cell wall

Abstract

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to β(1-3)glucan, and in the lateral wall, attached in part to β(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to β(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to β(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Δ and gas1Δ mutants, which are impaired in cell wall synthesis. A temperature shift from 30°C to 38°C increased the proportion of chitin attached to β(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38°C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-β(1-6)glucan complex is found, was greatly enhanced at 38°C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.