SrSeptin2 is one of three septin GTPases of the colonial choanoflagellate Salpingoeca rosetta. Phylogenetic analysis places it in septin Group 4, the group containing yeast Cdc12, rather than with mammalian SEPT2 despite its name. The 412-residue protein has an N-terminal septin-type G domain with the G1, G3 and G4 GTP-binding motifs, followed by a C-terminal coiled-coil region that in other septins mediates higher-order assembly of heteromeric filaments. When expressed as a fluorescent fusion, SrSeptin2 is distributed through the cytosol and enriched at the basal pole of single cells; in rosette colonies it concentrates at the basal poles where neighbouring cells meet. At the basal pole it forms filaments that lie between cortical microtubules, and its basal enrichment depends on the coiled-coil region. It colocalises with the paralogue SrSeptin6, consistent with assembly into heteromeric septin filaments. This basal localisation resembles septin localisation in animal epithelial cells. No loss-of-function or biochemical data exist, so its proposed contribution to rosette development and intercellular contacts is untested.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005525 GTP binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro/UniRule inference from the septin family (IPR016491) and septin-type G domain (IPR030379) signatures. SrSeptin2 has a complete septin-type G domain with G1, G3 and G4 motifs and PIRSR-predicted GTP contact residues. GTP binding has not been measured for the protein. Reason: Septin G domains bind guanine nucleotide, and the motifs needed for this are present. The term is a biochemical activity of the whole septin family, so the electronic transfer to a choanoflagellate septin is sound. It is the most specific molecular function supported for SrSeptin2; whether it hydrolyses GTP is unknown. Supporting Evidence: file:SALRS/SrSeptin2/SrSeptin2-uniprot.txt PROSITE; PS51719; G_SEPTIN; 1. PMID:30281390 a septin with the diagnostic G-domain and coiled-coil domain that typify human and fungal septins |
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning rule. Consistent with the experimental data: an mTFP1-SrSeptin2 fusion is distributed throughout the cytosol and enriched at the basal pole of the cell. Reason: Septins are cytoplasmic cytoskeletal proteins and the tagged protein is cytosolic in S. rosetta. The term is broad but correct; the more informative locations (basal part of cell, septin cytoskeleton) are added as separate IDA rows. Supporting Evidence: PMID:30281390 Cotransfecting cells with mTFP1-SrSeptin2 and a plasma membrane marker revealed SrSeptin2 distributed throughout the cytosol and enriched at the basal pole in confocal slices through the center of the cell. |
| GO:0005856 cytoskeleton | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning rule. Septins are cytoskeletal filament-forming GTPases, and tagged SrSeptin2 forms filaments at the basal pole that intercalate between cortical microtubules. Reason: Correct but general. The observed filaments support the more specific GO:0032156 septin cytoskeleton, which is added as a NEW IDA row; this electronic row can stay as a correct parent. Supporting Evidence: PMID:30281390 Fluorescence microscopy showed that septin filaments intercalate between cortical microtubules at the basal pole of the cell (Figure 4G). |
| GO:0031105 septin complex | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule transfer from the septin family. Septins from different groups assemble into heteromeric complexes through their G domains. SrSeptin2 (a Group 4 septin) colocalises at the basal pole with SrSeptin6 (a Group 1B septin), which the authors take as evidence that the two co-assemble; no biochemical isolation of an S. rosetta septin complex has been reported. Reason: Membership of a heteromeric septin complex is a defining property of the family. S. rosetta has septins from several groups, and the colocalisation data are consistent with co-assembly. The electronic annotation is appropriate; it should not be upgraded to experimental evidence until a complex is purified or an interaction is shown. Supporting Evidence: PMID:30281390 Septin paralogues interact directly through their G-domains to form heteromeric filaments PMID:30281390 strongly suggest that SrSeptin2 and SrSeptin6 assemble together at the basal pole. |
| GO:0045178 basal part of cell | IDA PMID:30281390 Transfection of choanoflagellates illuminates their cell bio... | NEW | Summary: Live imaging of a transfected mTFP1-SrSeptin2 fusion shows enrichment at the basal pole of single cells and of rosette cells, and at contacts between adjacent rosette cells, which correspond to the basal poles. Basal enrichment is lost when the coiled-coil region is deleted. Reason: GOA has no experimental annotation for this protein. Basal part of cell is the main finding of the only experimental study and is stated directly for both unicellular and colonial cells. The evidence is a tagged, transiently expressed fusion rather than staining of the endogenous protein, which is acceptable for IDA. The cell-cell contact signal in rosettes is described by the authors as the basal pole, so no separate cell-cell contact term is proposed. Supporting Evidence: PMID:30281390 Strikingly, mTFP1-SrSeptin2 was enriched at the basal poles of single and rosettes cells (Figure 4, B and E) and at points of contact between adjacent cells in rosettes (Figure 4E). PMID:30281390 In rosettes, mTFP1-SrSeptin2 localized to points of cell PMID:30281390 We further found that the basal localization of SrSeptin2 requires the coiled-coil domain |
| GO:0032156 septin cytoskeleton | IDA PMID:30281390 Transfection of choanoflagellates illuminates their cell bio... | NEW | Summary: Co-imaging of mTFP1-SrSeptin2 with an alpha-tubulin marker shows SrSeptin2 filaments at the basal pole, intercalated between cortical microtubules. A coiled-coil deletion forms ectopic filamentous rings around vacuoles. Reason: The fusion protein is seen in filaments, which places it in a septin-based cytoskeletal structure. This is more specific than the electronic cytoskeleton row. Microtubule association is reported as intercalation between microtubules, not binding, so no microtubule-binding function is proposed. Supporting Evidence: PMID:30281390 Fluorescence microscopy showed that septin filaments intercalate between cortical microtubules at the basal pole of the cell (Figure 4G). |
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Download this section (compressed HTML)Q: Is SrSeptin2 required for rosette development, cell division or the integrity of rosette cell contacts? Septin knockouts have not been reported in the published S. rosetta genome-editing studies.
Q: Does SrSeptin2 co-assemble with SrSeptin6 and PTSG_04106 into a heteromeric filament, and in what order? The colocalisation data predict this but do not test it.
Q: The name SrSeptin2 does not reflect orthology: the protein groups with yeast Cdc12 (Group 4). Should database names follow the phylogenetic group instead?
Experiment: Inactivate PTSG_07215 with the selection-based CRISPR pipeline and score rosette development, cell division and the basal localisation of SrSeptin6 and Rosetteless.
Type: Gene knockout by genome editing
Experiment: Express the three S. rosetta septins together and test complex formation and filament assembly, including nucleotide binding and GTPase activity.
Type: Co-immunoprecipitation or in vitro reconstitution
Experiment: Confirm the basal localisation with an antibody or knock-in tag at the endogenous locus, since the published data use a plasmid-expressed fusion.
Type: Endogenous tagging or immunofluorescence
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