id: F2UJ78 gene_symbol: couscous product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:946362 label: Salpingoeca rosetta description: >- couscous (PTSG_07368) encodes an 866-residue predicted glycosyltransferase of the choanoflagellate Salpingoeca rosetta. The protein has an N-terminal hydrophobic signal sequence/anchor, a PAN/Apple domain, and a C-terminal alpha-mannosyltransferase domain (Pfam Mannosyl_trans3) belonging to the fungal MNN2/MNN5 alpha-1,2-mannosyltransferase family, with which it shares 28-35% identity and a conserved DXD motif. Its enzymatic activity, sugar donor and acceptor substrates have not been determined. A frameshift loss-of-function allele (couscous lw1, originally isolated as the "Branched" mutant) abolishes development of clonal multicellular rosettes and instead causes cells to aggregate promiscuously into shear-sensitive clumps, including with wild-type cells; both defects are rescued by transgenic wild-type couscous. Mutant cells lose a basal patch of jacalin-reactive glycans that normally marks the basal extracellular matrix and fills the rosette interior, and they fail to upregulate and secrete the C-type lectin Rosetteless upon rosette induction, suggesting that Couscous-dependent glycosylation contributes to maturation of the basal extracellular matrix. A tagged, overexpressed Couscous fusion localized to cytoplasmic puncta, the collar and the cell membrane rather than to the Golgi apparatus, so its native compartment is unresolved. couscous expression is regulated by the Hippo pathway components Warts and Yorkie. existing_annotations: - term: id: GO:0000026 label: alpha-1,2-mannosyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: enables supporting_entities: - PANTHER:PTN001270341 review: summary: >- TreeGrafter transfer from the PANTHER PTHR31646 (alpha-1,2-mannosyltransferase MNN2) family node. Couscous carries a Mannosyl_trans3 domain that aligns with the alpha-mannosyltransferase domain of yeast and Candida MNN2 at 28-35% identity, including the conserved DXD motif required for nucleotide-sugar/metal binding in GT-A fold glycosyltransferases [PMID:30556809]. No enzymatic assay has been performed, so the activity, linkage and acceptor remain predictions. action: ACCEPT reason: >- The catalytic domain places Couscous within a family whose characterized members (S. cerevisiae Mnn2, Mnn5) are alpha-1,2-mannosyltransferases, and the DXD motif is retained, so there is no sequence evidence of catalytic loss. Loss-of-function mutants show a specific, rescuable glycan (jacalin) defect consistent with a glycosyltransferase [PMID:30556809]. This is the best-supported description of the molecular activity, accepted as a homology-based prediction; the alpha-1,2 linkage specificity in particular has not been tested in a choanoflagellate and the acceptor is unknown. supported_by: - reference_id: PMID:30556809 supporting_text: >- The predicted mannosyltransferase domain shares 28% and 35% amino acid sequence identity to alpha 1–2 mannosyltransferase (MNN2) proteins in Saccharomyces cerevisiae and Candida albicans, respectively, including the conserved DXD motif found in many families of glycosyltransferases - reference_id: PMID:30556809 supporting_text: >- While we have not uncovered the target(s) of the predicted glycotransferases or the exact nature of the interplay between the two phenotypes - term: id: GO:0000139 label: Golgi membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0134 review: summary: >- UniProt subcellular-location mapping (ARBA rule) assigning the family-typical Golgi type II membrane protein topology. The only experimental localization of Couscous (C-terminal mWasabi fusion overexpressed from the efl promoter) found puncta in the cytosol, collar and cell membrane, and the authors state it was clearly not at the Golgi, while cautioning that tagging or overexpression may have mislocalized it [PMID:30556809]. action: UNDECIDED reason: >- The family default (Golgi, as for yeast Mnn2) conflicts with the one direct observation, which placed tagged Couscous outside the Golgi (possibly ER, untested). The authors themselves conclude localization is uncertain, and the N-terminal hydrophobic segment is ambiguous between a cleaved signal peptide and a type II signal anchor. Neither accepting nor removing the Golgi assignment is justified until native localization is determined. supported_by: - reference_id: PMID:30556809 supporting_text: >- While Couscous-mWasabi was clearly not localized to the Golgi, the puncta may co-localize with the ER, where glycosyltransferases are also known to function - reference_id: PMID:30556809 supporting_text: >- Therefore, we are currently uncertain about the subcellular localization of Couscous protein. - term: id: GO:0005794 label: Golgi apparatus evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: located_in supporting_entities: - PANTHER:PTN001270341 review: summary: >- TreeGrafter transfer of the Golgi location of fungal MNN2-family mannosyltransferases. Tagged Couscous did not localize to the Golgi in S. rosetta, in contrast to the other rosette-defect glycosyltransferase Jumble, which did [PMID:30556809]. action: UNDECIDED reason: >- Same conflict as for Golgi membrane: a family-level expectation versus a single overexpressed-fusion observation that the authors regard as possibly artefactual. Kept undecided pending native localization (antibody staining or endogenous tagging with a Golgi marker). supported_by: - reference_id: PMID:30556809 supporting_text: >- Consistent with its role as a glycosyltransferase, Jumble localized to the Golgi apparatus, but Couscous appeared to localize in cytoplasmic puncta and to the cell membrane. - term: id: GO:0009101 label: glycoprotein biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in supporting_entities: - ARBA:ARBA00027560 - InterPro:IPR022751 review: summary: >- Combined ARBA/InterPro inference from the alpha-mannosyltransferase domain. MNN2-family enzymes extend the mannose outer chains of N- and O-linked glycans on glycoproteins. Couscous loss-of-function mutants lose a basal patch of jacalin-reactive glycans in the extracellular matrix, rescued by the wild-type gene [PMID:30556809]. action: ACCEPT reason: >- A glycosyltransferase would do the catalytic work of adding sugars to glycoproteins, so this process term fits the predicted activity. It is general enough to cover the uncertainty about which glycan class (N- or O-linked) and which acceptor proteins are modified. The rescuable glycan defect in the mutant supports a role in glycoconjugate synthesis, although the authors do not exclude an indirect trafficking effect. No more specific child term (e.g. protein N-linked glycosylation) is justified without acceptor identification. supported_by: - reference_id: PMID:30556809 supporting_text: >- The predicted Couscous amino acid sequence contains a specific type of glycosyltransferase domain, an alpha-mannosyltransferase domain, that transfers activated mannose onto the outer chain of core N-linked polysaccharides and O-linked mannotriose - reference_id: PMID:30556809 supporting_text: >- The same was true for Couscous cells, in which transformation with couscous-mTFP rescued both rosette development and the wild type glycosylation pattern - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0162 review: summary: >- UniProt keyword mapping from a predicted single-pass (type II) membrane topology. Tagged Couscous was seen in puncta and at the cell membrane and collar [PMID:30556809]. action: ACCEPT reason: >- Uninformative but consistent with both the predicted N-terminal membrane anchor and the observed membrane/punctate distribution of the tagged protein. It makes no compartment-specific claim, so it is not undermined by the unresolved Golgi versus ER question. supported_by: - reference_id: PMID:30556809 supporting_text: >- In wild type cells transfected with a couscous-mWasabi transgene under the efl promoter, Couscous was found in puncta scattered throughout the cytosol, collar and cell membrane - term: id: GO:0016757 label: glycosyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR022751 review: summary: >- InterPro2GO mapping from IPR022751 (alpha-mannosyltransferase). Couscous is a predicted glycosyltransferase with a conserved DXD motif; activity has not been assayed [PMID:30556809]. action: ACCEPT reason: >- Correct at the general level and consistent with the more specific alpha-1,2-mannosyltransferase prediction. Retained as a companion IEA; it is the safest statement of the activity given that sugar and linkage specificity have not been measured. supported_by: - reference_id: PMID:30556809 supporting_text: >- Couscous maps to a predicted mannosyltransferase with a PAN/Apple domain. - term: id: GO:0046354 label: mannan biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN001270341 review: summary: >- TreeGrafter transfer from the fungal MNN2 family, where Mnn2 builds the branched mannose outer chains of cell-wall mannoproteins. The GO term is defined as synthesis of mannan, "the main hemicellulose of soft (coniferous) wood, made up of D-mannose, D-glucose and D-galactose". S. rosetta has no fungal-type mannan cell wall, and the only glycan change detected in couscous mutants is loss of a jacalin-reactive glycan at the basal pole, not a mannan [PMID:30556809]. action: REMOVE reason: >- The term describes polysaccharide (hemicellulose/cell-wall mannan) biosynthesis, a fungal/plant-lineage function that the TreeGrafter node carries over from yeast Mnn2. There is no evidence that choanoflagellates synthesize a mannan polysaccharide, and Couscous substrates are unknown. The appropriate process for a glycoprotein-modifying mannosyltransferase is already captured by glycoprotein biosynthetic process. This is an over-propagated electronic inference that can be argued against on biological grounds. supported_by: - reference_id: PMID:30556809 supporting_text: >- MNN2 proteins catalyze the addition of the first branch of mannose-containing oligosaccharides found on cell wall proteins - reference_id: PMID:30556809 supporting_text: >- In contrast with wild type cells, the basal patch of jacalin staining was absent or significantly diminished in Couscous and Jumble mutants propagation_review: root_cause: PROPAGATION_BAD failure_modes: - LINEAGE_OR_TAXON_MISMATCH - ROLE_CONFLATION source_entities: - source_id: PANTHER:PTN001270341 source_label: MNN2-family alpha-1,2-mannosyltransferase node (PTHR31646) source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET comment: >- The GO:0046354 IBD on this node is seeded only by Candida albicans MNN2-family genes (all 56 IBA rows from the node list the same six CGD seeds; QuickGO 2026-10-01), yet it reaches oomycetes, green algae and choanoflagellates. A fungal cell-wall mannan process does not transfer to a choanoflagellate enzyme acting on the rosette basal matrix. core_functions: - description: >- Predicted alpha-1,2-mannosyltransferase (MNN2-family, GT-A fold with conserved DXD motif) that is required for a basal-pole, jacalin-reactive glycosylation pattern of the extracellular matrix. The activity, donor and acceptor are inferred by homology and from the rescuable glycan defect of the loss-of-function mutant; they have not been measured biochemically. molecular_function: id: GO:0000026 label: alpha-1,2-mannosyltransferase activity directly_involved_in: - id: GO:0009101 label: glycoprotein biosynthetic process supported_by: - reference_id: PMID:30556809 supporting_text: >- The predicted mannosyltransferase domain shares 28% and 35% amino acid sequence identity to alpha 1–2 mannosyltransferase (MNN2) proteins in Saccharomyces cerevisiae and Candida albicans, respectively, including the conserved DXD motif found in many families of glycosyltransferases - reference_id: PMID:30556809 supporting_text: >- In contrast with wild type cells, the basal patch of jacalin staining was absent or significantly diminished in Couscous and Jumble mutants, both in the presence and absence of RIFs - reference_id: PMID:30556809 supporting_text: >- Thus, the glycosylation defects in Jumble and Couscous mutant cells were directly linked to the genetic lesions in jumble and couscous, respectively. suggested_questions: - question: >- Does Couscous have mannosyltransferase activity in vitro, and what are its donor (GDP-mannose?), linkage and acceptor specificities? - question: >- Where does native Couscous reside - Golgi, ER, or a post-Golgi/secretory compartment - and is its N-terminal hydrophobic segment a cleaved signal peptide or a type II signal anchor? - question: >- Which glycoproteins carry the Couscous-dependent basal jacalin epitope, and is Rosetteless (with its mucin-like Ser/Thr repeats) or a cadherin among them? - question: >- Is the loss of basal glycans in couscous mutants caused by missing glycosylation or by defective trafficking of glycoconjugates to the basal pole? - question: >- What does the PAN/Apple domain contribute - substrate recognition, dimerization or binding to extracellular partners? - question: >- How does Hippo pathway (Warts/Yorkie) regulation of couscous expression relate to rosette size control? suggested_experiments: - hypothesis: Couscous is an active alpha-1,2-mannosyltransferase. description: >- Express the soluble luminal domain (with and without the PAN domain, and a DXD-motif mutant) and assay transfer from GDP-mannose to mannoside acceptors (e.g. alpha-1,6-mannobiose, Man-alpha-O-Ser/Thr peptides), with linkage determined by NMR or exoglycosidase digestion. experiment_type: biochemical enzyme assay - hypothesis: Native Couscous resides in an early secretory compartment. description: >- Endogenously tag couscous at its locus by CRISPR knock-in (avoiding overexpression) and co-image with ER (HDEL) and Golgi markers; complement with immunostaining using an anti-Couscous antibody. experiment_type: localization / imaging - hypothesis: Couscous glycosylates specific ECM or adhesion glycoproteins, including Rosetteless. description: >- Jacalin affinity enrichment and comparative glycoproteomics of wild-type versus couscous knockout cells, with site-level glycan mapping on Rosetteless and cadherins. experiment_type: glycoproteomics - hypothesis: Couscous catalytic activity, not only protein presence, is required for rosette development. description: >- Complement couscous knockout cells with wild-type couscous versus a DXD-motif catalytic mutant and score rosette formation, clumping and basal jacalin staining. experiment_type: genetic complementation references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:30556809 title: Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Primary paper for couscous; full text cached. Names the gene, gives GenBank EGD77026 / XM_004990809 (matching UniProt F2UJ78), and reports mapping, rescue, localization and glycosylation data. findings: - statement: >- couscous loss-of-function mutants fail to form rosettes and aggregate into amorphous, shear-sensitive clumps. supporting_text: >- For this study, we focused on four Class C mutants — Seafoam, Soapsuds, Jumble, and Couscous (previously named Branched in Levin et al., 2014) — that form amorphous, tightly packed clumps of cells, both in the presence and absence of RIFs, but never develop into rosettes - statement: >- The causative lesion is a single-nucleotide deletion producing a frameshift and early stop codon in couscous (EGD77026). supporting_text: >- The mutation causes a predicted frameshift leading to an early stop codon in the mutant protein, Couscouslw1 - statement: Transgenic wild-type couscous rescues rosette development. supporting_text: >- Rosette formation in Couscous mutant cells can be rescued by transgenic expression of couscous-mTFP or mTFP-couscous, but not couscouslw1-mTFP, mTFP-couscouslw1, or mTFP alone. - statement: >- Couscous contains an alpha-mannosyltransferase domain related to fungal MNN2 with a conserved DXD motif, plus a PAN/Apple domain. supporting_text: >- The predicted mannosyltransferase domain shares 28% and 35% amino acid sequence identity to alpha 1–2 mannosyltransferase (MNN2) proteins in Saccharomyces cerevisiae and Candida albicans, respectively, including the conserved DXD motif found in many families of glycosyltransferases - statement: Tagged Couscous did not localize to the Golgi; native localization is unresolved. supporting_text: >- While Couscous-mWasabi was clearly not localized to the Golgi, the puncta may co-localize with the ER, where glycosyltransferases are also known to function - statement: >- Mutants lose basal-pole jacalin staining; rescue restores the glycosylation pattern. supporting_text: >- The same was true for Couscous cells, in which transformation with couscous-mTFP rescued both rosette development and the wild type glycosylation pattern - statement: >- In couscous mutants Rosetteless localizes basally but is not upregulated or secreted after rosette induction. supporting_text: >- In both Jumble and Couscous cells, Rosetteless protein properly localized to the basal pole, but its expression did not increase nor was it secreted upon treatment with RIFs, as normally occurs in wild type cells - id: PMID:25299189 title: The Rosetteless gene controls development in the choanoflagellate S. rosetta. reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Original forward-genetic screen in which the couscous mutant was isolated under the name Branched; the gene was not mapped in this paper. findings: - statement: >- The Branched (later Couscous) mutant class forms branched chain colonies and no rosettes. supporting_text: >- In contrast, classes D–F developed into highly branched chain colonies - id: PMID:31896587 title: Biophysical principles of choanoflagellate self-organization. reference_review: relevance: LOW correctness: VERIFIED review_notes: Background citation framing couscous as an ECM regulator; no new couscous data. findings: - statement: couscous is one of three genes known to be required for rosette development. supporting_text: >- Interestingly, all 3 genes known to be required for rosette development are regulators of the extracellular matrix (ECM): a C-type lectin called rosetteless (18) and 2 predicted glycosyltransferases called jumble and couscous (19). - id: PMID:41037400 title: A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling. full_text_unavailable: true reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Only the abstract is cached. Reports CRISPR knockout of couscous (phenotype details not in the abstract) and Warts/Yorkie-dependent regulation of couscous expression. findings: - statement: couscous was inactivated with a CRISPR-Cas9 selection-based knockout method. supporting_text: >- We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble) - statement: Warts and Yorkie regulate couscous expression. supporting_text: >- RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous)