jumble encodes a 467-amino-acid, single-exon protein of the choanoflagellate Salpingoeca rosetta with a single predicted N-terminal transmembrane segment followed by a large C-terminal region. No known protein domain is detected by InterPro or Pfam, but fold-recognition methods (HHpred, Phyre2) relate it to the catalytic domain of polypeptide N-acetylgalactosaminyltransferases, and it is therefore regarded as a predicted glycosyltransferase; no enzymatic activity, donor or acceptor substrate has been demonstrated. A fluorescently tagged Jumble protein localizes to the apical pole of the cell near the flagellar base, at the position of the Golgi apparatus. A missense allele (L305P, jumble-lw1) that mislocalizes the protein to the endoplasmic reticulum causes cells to clump promiscuously and to fail to form rosette colonies in response to bacterial rosette-inducing factors; mutant cells lose basal-pole staining by the lectin jacalin and fail to upregulate and secrete the rosette-ECM protein Rosetteless. Transgenic wild-type jumble restores rosette formation and basal jacalin staining. Jumble is thus required for normal glycan presentation in the basal extracellular matrix, for suppression of spurious cell-cell adhesion, and for rosette development. Homologs occur across choanoflagellates and in some fungi but not in animals.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005794 Golgi apparatus | IDA PMID:30556809 Predicted glycosyltransferases promote development and preve... | NEW | Summary: A Jumble-mWasabi fusion expressed from the efl promoter in wild-type S. rosetta localized to the apical pole of the cell body near the base of the flagellum, both with and without rosette-inducing factors. By comparison with transmission electron micrographs this position corresponds to the Golgi apparatus, and the authors conclude that Jumble localizes to the Golgi. The functionally dead L305P allele instead is retained in the ER. Reason: Direct imaging of the tagged protein supports a Golgi location, which is also consistent with the predicted type-II-like topology (single N-terminal TM segment) typical of Golgi glycosyltransferases. Caveats: the construct is tagged and driven by a heterologous promoter, and no Golgi marker exists yet in S. rosetta, so organelle identity is assigned by position relative to ultrastructure rather than by co-localization. Proposed at the general Golgi apparatus level; Golgi membrane or a specific cisterna is not established. Supporting Evidence: PMID:30556809 Jumble-mWasabi protein localized to the apical pole of the cell body near the base of the flagellum. PMID:30556809 Jumble-mWasabi localization corresponds to the location of the Golgi apparatus, for which there is not yet a fluorescent marker in S. PMID:30556809 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. |
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Download this section (compressed HTML)Q: Does Jumble have glycosyltransferase activity at all, and if so which donor sugar and acceptor does it use? Its only link to glycosyltransferases is fold recognition to polypeptide GalNAc-transferases, and it lacks any InterPro/Pfam signature.
Q: Is the loss of basal jacalin staining in jumble mutants due to loss of a glycan (a glycosylation step performed by Jumble) or to failed trafficking or secretion of normally glycosylated cargo to the basal pole? Whole-cell jacalin blots showed no difference, which favours a spatial or secretory defect.
Q: Is Rosetteless (or a cadherin or other adhesion protein) a direct glycosylation substrate of Jumble?
Q: Does the CRISPR knockout of jumble (PMID:41037400) phenocopy the jumble-lw1 clumping and rosette-defect phenotype?
Experiment: Express the soluble luminal domain of Jumble (and fungal homologs) and assay glycosyltransferase activity against a panel of nucleotide-sugar donors and peptide/glycan acceptors (e.g. UDP-Glo assays), including Rosetteless-derived peptides.
Hypothesis: Jumble is a catalytically active glycosyltransferase.
Type: biochemical enzyme assay
Experiment: Co-localize tagged Jumble at endogenous expression levels (knock-in) with Golgi markers developed for S. rosetta, or by immuno-EM, to confirm Golgi residence and define the cisternal position.
Hypothesis: Jumble is a Golgi-resident type II membrane protein.
Type: localization / microscopy
Experiment: Compare glycan profiles (glycomics/glycoproteomics, O-glycan release) of wild-type versus jumble knockout cells and of purified rosette ECM, and test whether Rosetteless glycosylation changes.
Hypothesis: Jumble adds specific glycans to ECM glycoproteins such as Rosetteless.
Type: glycomics / mass spectrometry
Experiment: Mutate predicted catalytic residues (identified from a structural model aligned to GalNAc-T catalytic domains) and test the ability of the mutant to rescue rosette formation and basal jacalin staining in jumble knockout cells.
Hypothesis: Catalytic activity, not just protein presence, is required for Jumble function.
Type: genetic complementation
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