Rodriguez-Pena et al. (2000) identified CRH1, CRH2, and CRR1 as a novel family of cell wall-related proteins with homology to bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of CRH1 and CRH2 caused additive sensitivity to Congo Red and Calcofluor White (cell wall-perturbing agents). The putative glycosidase domain was critical for function. Crh1-GFP localized to incipient bud site, septum area in late budding, and ascospore envelopes. The alkali-soluble glucan fraction in crh1/crh2 double mutant was almost twice wild-type levels. PMID:10757808
Hamada et al. (1998) identified CRH1 as a GPI-dependent cell wall protein through genome-wide screening. PMID:9613572
Yin et al. (2005) confirmed CRH1 is covalently attached to cell wall via GPI remnants by comprehensive proteomic analysis using tandem mass spectrometry. PMID:15781460
Cabib et al. (2007) showed Crh1p and Crh2p are required for cross-linking chitin to beta(1-6)glucan. In crh1/crh2 double mutants, chitin linked to beta(1-6)glucan was completely absent. Heat stress (38C) increased chitin-beta(1-6)glucan cross-links and CRH1 expression (cell integrity pathway dependent). PMID:17302808
Cabib et al. (2008) demonstrated that Crh1p and Crh2p act as transglycosylases both in vivo and in vitro. Using fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors, they showed CRH-dependent fluorescence at bud scars and cell contour. The cell wall reaction was inhibited by chitooligosaccharides. PMID:18694928
Cabib (2009) developed two novel techniques showing Crh1p and Crh2p transfer chitin to BOTH beta(1-3)- and beta(1-6)glucan. Previous results suggesting residual chitin-beta(1-3)glucan links in crh1/crh2 mutants were due to chitinase contamination in zymolyase. All chitin in crh1/crh2 double mutants is free (unlinked). PMID:19734368
Mazan et al. (2013) heterologously expressed CRH1 and CRH2 in Pichia pastoris and characterized their catalytic properties:
- Both act as chitin transglycosylases
- Donors: soluble chitin derivatives (CM-chitin, glycol-chitin, chitooligosaccharides DP>=5)
- Acceptors: oligosaccharides from chitin, beta(1,3)-glucan (laminarin), beta(1,6)-glucan (pustulan)
- Minimal acceptor: 2 hexopyranose units; effectivity increases with chain length
- Both exhibit weak endochitinase activity (ratio of endo:exo ~4x higher in Crh1 vs Crh2)
- pH optimum: 3.5; temperature optimum: 37C
PMID:23919454
Yin et al. (2007) quantified CRH1 at 44,000 wall-bound copies per cell in log phase YPD. In gas1 mutants (constitutive cell wall integrity pathway), Crh1p levels increased ~3-5 fold. PMID:17617218
CRH1 is positively regulated by MPK1/SLT2 through the cell wall integrity signaling pathway via Rlm1 transcription factor. Expression is upregulated in response to cell wall damage (fks1 disruption) and heat stress. [PMID:10594829, PMID:11016834]
Yofe et al. (2016) SWAp-Tag (SWAT) high-throughput localization study assigned CRH1 to cell periphery. This is a high-throughput dataset (HDA evidence). PMID:26928762
CRH2 (YEL040w, also called UTR2) is the key paralog. CRH1 and CRH2 have redundant transglycosylase activity for chitin-glucan cross-linking. Single mutants show partial phenotypes; double mutants show complete loss of chitin cross-links. CRH1 is cell-cycle regulated and expressed during sporulation; CRH2 expression is constitutive during mitotic cycle.
CRH1 is a dual-function enzyme: primarily a chitin transglycosylase (transfers chitin to glucan acceptors) with secondary weak endochitinase activity. The GH16 catalytic domain uses a retaining mechanism with a catalytic glutamate pair (E134 nucleophile, E138 proton donor, by homology). The enzyme forms a glycosyl-enzyme intermediate from a chitin donor, then transfers the chitin chain to beta(1-3)- or beta(1-6)glucan acceptors. This cross-links chitin into the cell wall glucan network, which is essential for cell wall integrity.