Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta.
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couscous loss-of-function mutants fail to form rosettes and aggregate into amorphous, shear-sensitive clumps.
"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"
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The causative lesion is a single-nucleotide deletion producing a frameshift and early stop codon in couscous (EGD77026).
"The mutation causes a predicted frameshift leading to an early stop codon in the mutant protein, Couscouslw1"
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Transgenic wild-type couscous rescues rosette development.
"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."
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Couscous contains an alpha-mannosyltransferase domain related to fungal MNN2 with a conserved DXD motif, plus a PAN/Apple domain.
"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"
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Tagged Couscous did not localize to the Golgi; native localization is unresolved.
"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"
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Mutants lose basal-pole jacalin staining; rescue restores the glycosylation pattern.
"The same was true for Couscous cells, in which transformation with couscous-mTFP rescued both rosette development and the wild type glycosylation pattern"
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In couscous mutants Rosetteless localizes basally but is not upregulated or secreted after rosette induction.
"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"