Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta.
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The Jumble mutant forms tightly packed amorphous clumps and never forms rosettes, with or without rosette-inducing factors.
"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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Clumping reflects promiscuous adhesion; mutant cells also adhere to wild-type cells.
"Cell aggregation was not strain-specific, as unlabeled Jumble and Couscous mutant cells adhered to wild type cells identified by their expression of cytoplasmic mWasabi"
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A single point mutation (L305P) in jumble (EGD72416) causes both clumping and rosette defects.
"we infer that the single point mutation at genome position 1:1,919,681 causes both the clumping and rosette defect phenotypes in Jumble mutants."
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jumble is a single-exon gene encoding a 467 aa protein with one predicted transmembrane domain.
"The jumble gene contains a single exon and is predicted to encode a 467 amino acid protein containing a single transmembrane domain."
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Transgenic wild-type jumble (N- or C-terminal mTFP fusions) partially rescues rosette development; the mutant allele does not.
"Importantly, we did not detect any rosettes when we transfected Jumble cells with mTFP alone, jumblelw1-mTFP, or mTFP-jumblelw1."
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No InterPro/Pfam domain; glycosyltransferase relationship predicted only by low-confidence CDD and structure-based homology (HHpred/Phyre2).
"Although Interpro (Finn et al., 2017) and Pfam (Finn et al., 2016) did not reveal any known protein domains in Jumble"
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Homologs are present in other choanoflagellates and fungi but not animals.
"BLAST searches uncovered unannotated jumble homologs in nine other choanoflagellates (Figure 2—figure supplement 2A) and in fungi (Figure 2—figure supplement 3), but none in animals."
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Tagged Jumble localizes apically at the position of the Golgi apparatus; the L305P mutant protein is retained in the ER.
"In contrast, Jumblelw1-mWasabi, was distributed in a tubular pattern throughout the cell and co-localized with an endoplasmic reticulum (ER) marker"
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Jumble mutants lose basal-pole jacalin staining; complementation restores it.
"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"
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In Jumble mutants Rosetteless localizes to the basal pole but is not upregulated or secreted upon 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"
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Glycosyltransferase targets of Jumble are unknown.
"While we have not uncovered the target(s) of the predicted glycotransferases"