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

Abstract

SummaryIn budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to b(1-3)glucan, and in the lateral wall, attached in part to b(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to b(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 b(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1D and gas1D mutants, which are impaired in cell wall synthesis. A temperature shift from 30°C to 38°C increased the proportion of chitin attached to b(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-b(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.

The GPI‐anchored Gas and Crh families are fungal antigens

“…Saccharomyces cerevisiae (Rodriguez-Pena et al, 2000Cabib et al, 2007) but the enzymatic activity remains unknown in vitro. All members of the Gas and Crh families have extremely well conserved sequences in Saccharomyces, Candida and Aspergillus spp.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Cross-linkage of chitin to b1,6-and b1,3-glucan Related Crh1 and Crh2 generate cross-linkages between the reducing ends of chitin chains and both the nonreducing end of b1,3-glucose side branches on b1,6-glucan and the nonreducing ends of b1,3-glucan chains (Cabib et al 2007;Cabib 2009), and their homolog Crr1 likely does so during ascospore wall assembly. These proteins are in GH family 16, and Crh2 and Crr1 also have a chitin-binding module (RodriguezPena et al 2000;Cabib et al 2008).…”

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

“…Chitin can also be attached to b-1,6-glucan chains associated with GPI-CWPs (Cabib and Duran 2005), particularly in response to cell wall stress (Figure 1), when cell wall chitin levels can rise to as high as 20% of total wall polymer (Popolo et al 1997;García-Rodriguez et al 2000;Valdivieso et al 2000; see Chitin synthase 3: the chitin emergency response). Chitin is attached to both b-1,3-glucan and b-1,6-glucan by the redundant Crh1 and Crh2 transglycosylases (Cabib et al 2007(Cabib et al , 2008Cabib 2009). …”