CRH1 (Congo Red Hypersensitive protein 1) encodes a GPI-anchored cell wall transglycosylase of the GH16 family that cross-links chitin to both beta(1-3)- and beta(1-6)-glucan in the S. cerevisiae cell wall. The enzyme uses a retaining double-displacement mechanism: it cleaves a chitin donor chain and transfers it to glucan acceptors, thereby integrating chitin into the glucan network. CRH1 has a secondary weak endochitinase activity (EC 3.2.1.14) but its primary biological role is as a chitin transglycosylase (EC 2.4.-.-). The protein is GPI-anchored at the cell surface and concentrated at chitin-rich areas including the incipient bud site, septum, and bud scars. CRH1 acts redundantly with its paralog CRH2/UTR2; the double mutant completely lacks chitin-glucan cross-links and shows strong sensitivity to Congo Red and Calcofluor White. CRH1 expression is cell-cycle regulated, induced during sporulation, and upregulated by the cell wall integrity pathway (MPK1/RLM1) in response to heat stress and cell wall damage. Present at approximately 44,000 wall-bound copies per cell in log phase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA annotation from phylogenetic inference. CRH1 is a GPI-anchored cell wall protein that is secreted and covalently attached to the outer cell wall. While "extracellular region" is not wrong for a secreted protein, the more specific and accurate term is "fungal-type cell wall" (GO:0009277), which is already annotated with direct experimental evidence (IDA). This IBA adds little information beyond the existing IDA annotations. Reason: Technically correct but too broad. CRH1 is a GPI-CWP covalently incorporated into the cell wall, not a freely secreted extracellular protein. The IDA annotation to fungal-type cell wall is more informative. |
| GO:0006030 chitin metabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation from phylogenetic inference. CRH1 is directly involved in chitin metabolic process as a chitin transglycosylase that transfers chitin chains to glucan acceptors. This is consistent with IMP and IGI evidence from PMID:18694928. Falcon deep research corroborates that loss of CRH1/CRH2 reduces chitin-glucan linkage and the double mutant abolishes it, while overexpression increases it. Reason: Correct and supported by direct experimental evidence. CRH1 transfers chitin to glucan, which is a chitin metabolic process. Supporting Evidence: file:yeast/CRH1/CRH1-deep-research-falcon.md loss of **CRH1** or **CRH2** reduces chitin linked to **Ξ²(1β6)-glucan**, while the **crh1Ξ crh2Ξ** double mutant abolishes detectable chitinβΞ²(1β6)-glucan linkage; overexpression of **CRH1** increases the chitinβΞ²(1β6)-glucan complex |
| GO:0031505 fungal-type cell wall organization | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation from phylogenetic inference. CRH1 is a key enzyme in fungal cell wall organization, cross-linking chitin to the glucan network. This is consistent with IMP and IGI evidence from PMID:18694928. Falcon deep research frames the primary role of Crh1 as strengthening and organizing the wall by creating covalent chitin-beta-glucan cross-links at sites of growth and division. Reason: Correct. CRH1 cross-links chitin to glucan, which is an essential step in cell wall organization. Well-supported by multiple lines of evidence. Supporting Evidence: file:yeast/CRH1/CRH1-deep-research-falcon.md Crh1βs primary role is best described as **strengthening and organizing the wall** by creating **covalent chitinβΞ²-glucan cross-links** at sites of growth and division |
| GO:0009277 fungal-type cell wall | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation from phylogenetic inference for cell wall localization. CRH1 is a GPI-CWP covalently attached to the cell wall, confirmed by mass spectrometry (PMID:15781460) and GFP localization (PMID:10757808). Consistent with IDA evidence. Reason: Correct. CRH1 is covalently linked to the cell wall via its GPI anchor remnant. Multiple IDA annotations confirm this. |
| GO:0016757 glycosyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation from phylogenetic inference. CRH1 is a chitin transglycosylase that transfers chitin chains to glucan acceptors, which is a glycosyltransferase activity. Supported by IDA evidence from PMID:23919454 and IMP/IGI from PMID:18694928. Falcon deep research confirms the family is defined by a GH16-like catalytic motif whose residues are required for in vivo function, and that the enzymes favor transfer (transglycosylation) over net hydrolysis. Reason: Correct. Transglycosylase activity is a form of glycosyltransferase activity. This is the core molecular function of CRH1. Supporting Evidence: file:yeast/CRH1/CRH1-deep-research-falcon.md A catalytic-site mutant allele (Crh1 with substitutions at key acidic residues) fails to complement Congo red sensitivity, implying that the predicted catalytic residues are essential for function in vivo file:yeast/CRH1/CRH1-deep-research-falcon.md The enzyme can join the **reducing end of a chitin donor** to the **non-reducing end of an acceptor** oligosaccharide |
| GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA from InterPro mapping (IPR000757 Beta-glucanase-like domain). CRH1 does have weak endochitinase activity (EC 3.2.1.14) as demonstrated by Mazan et al. (2013), so the annotation is not incorrect. However, the primary function is transglycosylase activity, not hydrolase activity. The hydrolytic activity is secondary and weak. Reason: Technically correct -- CRH1 has documented weak endochitinase activity (PMID:23919454) -- but the primary evolved function is transglycosylation, not hydrolysis. This InterPro-based annotation reflects the ancestral GH16 hydrolase function rather than the derived transglycosylase specialization. Supporting Evidence: file:yeast/CRH1/CRH1-deep-research-bioreason-sft.md [BioReason identifies the GH16 catalytic core as executing a retaining double-displacement reaction, consistent with the transglycosylase being the primary function rather than simple hydrolysis] |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA from InterPro mapping. CRH1 is indeed involved in carbohydrate metabolism (chitin and glucan processing). This is correct but very broad -- the more specific term "chitin metabolic process" (GO:0006030) is already annotated. Reason: Correct but very broad. Subsumed by the more specific GO:0006030 (chitin metabolic process) which is already annotated with experimental evidence. |
| GO:0008843 endochitinase activity | IEA GO_REF:0000003 | KEEP AS NON CORE | Summary: IEA from EC number mapping (EC 3.2.1.14). Mazan et al. (2013) demonstrated weak endochitinase activity for heterologously expressed CRH1. The endo:exo chitinase ratio was approximately 4-fold higher in Crh1 than Crh2. However, the chitinase activity is secondary to the primary transglycosylase function. Reason: Correct. CRH1 has documented endochitinase activity (PMID:23919454), but this is a secondary activity. The primary molecular function is chitin transglycosylase activity. Supporting Evidence: PMID:23919454 Both proteins exhibited a weak chitinolytic activity in different assays whereby the ratio of endo- compared with exo-chitinase activity was approximately 4-fold higher in Crh1 than in Crh2 |
| GO:0009277 fungal-type cell wall | IEA GO_REF:0000117 | ACCEPT | Summary: IEA from ARBA machine learning model. Correct -- CRH1 is covalently linked to the fungal-type cell wall. Redundant with IDA annotations from PMID:10757808 and PMID:15781460. Reason: Correct and consistent with multiple lines of direct experimental evidence. |
| GO:0016020 membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: IEA from UniProtKB subcellular location mapping. CRH1 is GPI-anchored to the outer leaflet of the plasma membrane before being transferred to the cell wall. The annotation is technically correct but imprecise. CRH1 is primarily a cell wall protein, not a membrane protein in the traditional sense. The GPI anchor transiently associates it with the membrane during transport. Reason: Technically correct (GPI-anchored to membrane) but misleading for CRH1, which is primarily a covalently-linked cell wall protein. The GPI anchor is processed during transfer to the cell wall. |
| GO:0016798 hydrolase activity, acting on glycosyl bonds | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA from InterPro mapping (IPR017168 CRH-like family). CRH1 has weak endochitinase activity, so hydrolysis of glycosyl bonds is technically correct. However, this is a parent term of endochitinase activity (GO:0008843) which is already annotated, and the primary function is transglycosylation rather than hydrolysis. Reason: Correct but broad and redundant. The more specific endochitinase activity (GO:0008843) is already annotated. The primary function is transglycosylase, not hydrolase. |
| GO:0031505 fungal-type cell wall organization | IEA GO_REF:0000117 | ACCEPT | Summary: IEA from ARBA machine learning model. Correct -- CRH1 is essential for cell wall organization through chitin-glucan cross-linking. Consistent with IMP/IGI evidence. Reason: Correct and consistent with experimental evidence from PMID:18694928. |
| GO:0071555 cell wall organization | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA from InterPro mapping. Correct but broad -- the more specific term "fungal-type cell wall organization" (GO:0031505) is already annotated with experimental evidence. Reason: Correct but subsumed by the more specific GO:0031505 (fungal-type cell wall organization) which is already annotated. |
| GO:0071944 cell periphery | HDA PMID:26928762 One library to make them all: streamlining the creation of y... | KEEP AS NON CORE | Summary: HDA annotation from the SWAp-Tag high-throughput localization study (Yofe et al. 2016). CRH1 is a GPI-anchored cell wall protein at the cell surface, so cell periphery is correct. However, more specific terms (fungal-type cell wall, incipient cellular bud site) are already annotated with direct experimental evidence. Reason: Correct but broad. The high-throughput dataset correctly places CRH1 at the cell periphery, but more specific localization terms are already available from focused studies. Supporting Evidence: PMID:26928762 we constructed and investigated a library of βΌ1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins |
| GO:0016757 glycosyltransferase activity | IDA PMID:23919454 A novel fluorescence assay and catalytic properties of Crh1 ... | ACCEPT | Summary: IDA annotation based on Mazan et al. (2013). Heterologously expressed CRH1 was shown to transfer chitin chains to glucan oligosaccharide acceptors in a direct in vitro assay using fluorescently-labeled substrates. This is the most direct demonstration of CRH1's transglycosylase activity. Reason: Core molecular function of CRH1 demonstrated by direct biochemical assay. CRH1 uses soluble chitin derivatives as donors and glucan-derived oligosaccharides as acceptors to catalyze transglycosylation. Supporting Evidence: PMID:23919454 Both proteins act as chitin transglycosylases; they use soluble chitin derivatives, such as carboxymethyl chitin, glycol-chitin and/or N-acetyl chito-oligosaccharides of DP (degree of polymerization)β₯5 as the oligoglycosyl donors, and oligosaccharides derived from chitin, Ξ²-(1,3)-glucan (laminarin) and Ξ²-(1,6)-glucan (pustulan), fluorescently labelled with sulforhodamine or FITC as acceptors |
| GO:0000131 incipient cellular bud site | IDA PMID:10757808 A novel family of cell wall-related proteins regulated diffe... | ACCEPT | Summary: IDA annotation based on Rodriguez-Pena et al. (2000). Crh1-GFP was observed at the incipient bud site by confocal fluorescence microscopy. CRH1 localizes to chitin-rich areas where cell wall remodeling occurs during polarized growth. Reason: Correct and specific. CRH1-GFP localization to the incipient bud site is consistent with its function in chitin-glucan cross-linking during bud emergence. Supporting Evidence: PMID:10757808 Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes file:yeast/CRH1/CRH1-deep-research-falcon.md Crh1-GFP localization studies show enrichment at **polarized growth sites**βbud emergence, the **motherβdaughter neck**, and bud scarsβwhere chitin is enriched; Crh1 also appears at mating-related structures and marks **spore envelopes** during sporulation |
| GO:0006030 chitin metabolic process | IMP PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IMP annotation based on Cabib et al. (2008). Mutant phenotype analysis showed that CRH genes are required for chitin transfer to glucan. Fluorescent chitin acceptor labeling in vivo was dependent on CRH genes. This directly demonstrates CRH1 involvement in chitin metabolism. Reason: Core biological process. CRH1 transfers chitin chains to glucan acceptors, directly participating in chitin metabolism. The crh1/crh2 double mutant completely lacks chitin-glucan cross-links. Supporting Evidence: PMID:18694928 the Crh proteins act by transferring chitin chains to beta(1-6)glucan, with a newly observed high activity in the bud scar |
| GO:0006030 chitin metabolic process | IGI PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IGI annotation based on genetic interaction between CRH1 and CRH2 (SGD:S000000766). Cabib et al. (2008) showed that CRH1 and CRH2 are redundantly required for chitin transfer to glucan; the double mutant shows complete loss of chitin-glucan cross-links while single mutants show partial loss. Reason: Correct. The genetic interaction between CRH1 and CRH2 demonstrates redundant function in chitin metabolism. Supporting Evidence: PMID:18694928 In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes |
| GO:0009277 fungal-type cell wall | IDA PMID:10757808 A novel family of cell wall-related proteins regulated diffe... | ACCEPT | Summary: IDA annotation based on Rodriguez-Pena et al. (2000). Crh1-GFP localized to the cell surface, particularly in chitin-rich areas. The protein is a GPI-CWP covalently attached to the cell wall. Reason: Core localization. CRH1 is a GPI-anchored cell wall protein, demonstrated by GFP localization at the cell surface in chitin-rich regions. Supporting Evidence: PMID:10757808 Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas file:yeast/CRH1/CRH1-deep-research-falcon.md it is reported to be covalently incorporated into the wall and releasable by glucanase (laminarinase), consistent with a GPI-anchored wall protein that becomes wall-integrated |
| GO:0009277 fungal-type cell wall | IDA PMID:15781460 Comprehensive proteomic analysis of Saccharomyces cerevisiae... | ACCEPT | Summary: IDA annotation based on Yin et al. (2005). CRH1 was identified as a covalently-bound cell wall protein by mass spectrometry of isolated cell walls. Specifically identified as a GPI-modified CWP. Reason: Direct proteomic identification of CRH1 in isolated cell walls confirms cell wall localization. Supporting Evidence: PMID:15781460 The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs ...seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p, and Scw10p) are classified as glycoside hydrolases |
| GO:0016757 glycosyltransferase activity | IMP PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IMP annotation based on Cabib et al. (2008). Mutant phenotype analysis showed CRH-dependent transfer of chitin to glucan both in vivo (fluorescent acceptor labeling) and in digitonin-permeabilized cells. The linking reaction required CRH genes. Reason: Core molecular function demonstrated by mutant phenotype. Loss of CRH genes eliminates chitin-to-glucan transglycosylation activity. Supporting Evidence: PMID:18694928 The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here |
| GO:0016757 glycosyltransferase activity | IGI PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IGI annotation based on genetic interaction between CRH1 and CRH2. The redundant requirement of both genes for full transglycosylase activity was demonstrated by comparing single and double mutants. Reason: Correct. CRH1 and CRH2 have redundant glycosyltransferase activity for chitin-glucan cross-linking. Supporting Evidence: PMID:18694928 In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes |
| GO:0031505 fungal-type cell wall organization | IMP PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IMP annotation based on Cabib et al. (2008). crh1/crh2 double mutants show altered cell wall architecture with loss of chitin-glucan cross-links and increased alkali-soluble glucan. Deletion aggravated defects of fks1 and gas1 mutants. Reason: Core biological process. CRH1 is essential for proper cell wall organization through chitin-glucan cross-linking. Falcon deep research additionally connects this wall-organizing role to the cell wall integrity (CWI) pathway, with CRH1 induced at 38C in a Slt2-dependent manner. Supporting Evidence: PMID:18694928 The importance of transglycosylation for cell wall assembly is thus firmly established file:yeast/CRH1/CRH1-deep-research-falcon.md shifting cells to **38Β°C** increases CRH1 expression via the **cell integrity pathway**, and this induction is abolished in an **slt2Ξ** mutant, directly linking CRH1 to the CWI MAPK pathway |
| GO:0031505 fungal-type cell wall organization | IGI PMID:18694928 Assembly of the yeast cell wall. Crh1p and Crh2p act as tran... | ACCEPT | Summary: IGI annotation based on genetic interaction between CRH1 and CRH2. Single and double mutant analysis demonstrates their redundant contribution to cell wall organization. Reason: Correct. The genetic interaction confirms both CRH1 and CRH2 contribute to cell wall organization. Supporting Evidence: PMID:18694928 The importance of transglycosylation for cell wall assembly is thus firmly established |
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Download this section (compressed HTML)Q: Is there a more specific GO term for chitin transglycosylase activity that could distinguish the primary transglycosylation function from the secondary endochitinase activity? Currently both activities are annotated under different parent terms (glycosyltransferase vs endochitinase), but there is no specific "chitin transglycosylase activity" term in GO.
Q: What is the relative in vivo contribution of the endochitinase activity versus the transglycosylase activity of CRH1? Is the chitinase activity biologically relevant or simply a side reaction of the GH16 catalytic mechanism?
Experiment: Use compartment-specific fluorescent chitin acceptors with conditional CRH1 expression to determine whether transglycosylation rates differ at different cell surface locations. Compare with CRH2 localization and activity patterns.
Hypothesis: CRH1 chitin transglycosylase activity may have distinct substrate preferences in different cell wall compartments (bud scar vs lateral wall vs septum).
Type: fluorescence microscopy with functional assay
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