Rubicon (CG12772, dRubicon) is the single Drosophila member of the Rubicon/Pacer family, with an N-terminal RUN domain, central class III PI3-kinase (VPS34)-binding segments and a C-terminal zinc-containing Rubicon-homology (RH) domain, the same architecture as vertebrate Rubicon (RUBCN). It acts on endolysosomal membranes in two opposite directions, as vertebrate Rubicon does. It restrains canonical autophagy. Its protein level rises with age, and neuronal or systemic knockdown increases autophagosome and autolysosome pools in the brain, extends female lifespan and reduces polyglutamine toxicity. It also promotes LC3-associated phagocytosis (LAP) and LAP-like single-membrane Atg8a conjugation. In glia it localizes to Rab7-positive late endosomes/phagosomes and is required for phagosome-lysosome fusion and degradation of engulfed axon debris after nerve injury. In the fertilized egg, Rubicon on the limiting membrane of maternal multivesicular bodies is required for PtdIns(3)P production, Atg8a recruitment and elimination of the paternal mitochondrial derivative. Direct biochemical partners (VPS34/UVRAG, Rab7) are inferred from vertebrate Rubicon and have not been shown for the fly protein.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005769 early endosome | ISS GO_REF:0000024 | MODIFY | Summary: Early endosome location was transferred by similarity from human RUBCN. In fly glia, tagged Rubicon is mainly on Rab7-positive late endosomes/phagosomes and rarely overlaps with Rab5-positive early endosomes. Reason: Human RUBCN is mainly in late endosomes/lysosomes, and the only direct fly localization data show Rubicon::mRFP puncta almost entirely on Rab7-positive late endosomes/phagosomes, rarely on Rab5-positive early endosomes. Late endosome (GO:0005770) is the better-supported compartment for the fly protein. Its C-terminal RH domain is the Rab7-binding module in human Rubicon. Proposed replacements: late endosome Supporting Evidence: PMID:37248218 Rubicon::mRFP showed a punctate distribution, which rarely overlapped with Rab5-positive early endosomes/phagosomes or Lamp1-marked lysosomes PMID:37248218 Apparently, almost all Rubicon dots colocalized with Rab7-positive late endosomes/phagosomes in uninjured and injured wing nerve glia |
| GO:0006909 phagocytosis | IMP PMID:37248218 LC3-associated phagocytosis promotes glial degradation of ax... | ACCEPT | Summary: Rubicon loss (RNAi, CRISPR frameshift alleles, glia-specific knockout) impairs glial clearance of engulfed axon debris and phagosome-lysosome fusion; overexpression accelerates clearance. This fits the GO definition of phagocytosis, which includes delivery of the engulfed material to the lysosome. Reason: GO:0006909 covers both engulfment and delivery of the phagosome to the lysosome. Rubicon acts at the LAP/phagosome-maturation stage, which is part of this process. A more specific NEW annotation to phagosome maturation is proposed below to capture the step. Supporting Evidence: PMID:37248218 Similar to Rubicon RNAi, Rubiconfs1 hemizygotes and Rubiconfs1/fs2 trans-heterozygotes failed to clear axon debris in injured nerves PMID:37248218 glial Rubicon overexpression decreased the pool of undigested axon fragments, indicating that increasing Rubicon level is sufficient to boost debris clearance |
| GO:0045335 phagocytic vesicle | IDA PMID:37248218 LC3-associated phagocytosis promotes glial degradation of ax... | ACCEPT | Summary: Glial Rubicon::mRFP colocalizes with engulfed, GFP-labelled axon debris in large Rab7-positive vesicles (LAPosomes) after nerve injury. Reason: This is direct imaging evidence that Rubicon sits on phagosomes containing cargo, where it is needed for their maturation. The evidence uses an overexpressed tagged protein, but it matches the vertebrate Rubicon localization during LAP. Supporting Evidence: PMID:37248218 we observed colocalization of glial Rubicon::mRFP and GFP-labeled axon debris at 1 and 2 days after wing nerve injury, indicating that engulfed axon fragments are contained in LAPosomes |
| GO:1901981 phosphatidylinositol phosphate binding | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phosphatidylinositol phosphate binding is inherited from the eukaryote-level root node PTN002915557. Its only experimental seed is human Pacer (RUBCNL). There is no lipid-binding data for fly Rubicon or for human Rubicon. Reason: The fly protein shares the RH and PIKBD modules with Pacer, and it acts on PtdIns(3)P-rich endolysosomal membranes. Fly Rubicon is needed for PtdIns(3)P production on LAP-like vesicles in the egg. Placing the term at the root is therefore plausible. However, the seeding evidence comes only from the vertebrate Pacer paralog, and the PIP-binding region of Pacer is not mapped. Direct lipid binding has not been shown for the fly protein, and fly Rubicon is RUBCN-like. Human Rubicon was not pulled down by PtdIns(3)P beads, and its RH-domain structure lacks the basic residues of a PI(3)P-binding FYVE motif. The term is therefore treated as an over-propagation from the vertebrate Pacer lineage, matching the human and mouse RUBCN reviews. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002915557 SUPPORTS SOURCE BUT NOT TARGET Root node seeded only by vertebrate RUBCNL/Pacer (PMID:28306502); Rubicon-lineage members, including this one, have no lipid-binding evidence. Supporting Evidence: file:DROME/Rubicon/Rubicon-uniprot.txt GO; GO:1901981; F:phosphatidylinositol phosphate binding; IBA:GO_Central. PMID:38977659 Other LAP pathway events, such as Rubicon-dependent production of PtdIns(3)P and association of ROS with the sperm flagellum, also unfold during this process. |
| GO:0010507 negative regulation of autophagy | IMP PMID:30783089 Suppression of autophagic activity by Rubicon is a signature... | NEW | Summary: Systemic knockdown of dRubicon increases autophagosome and autolysosome pools in the brain. Neuronal knockdown extends female lifespan and reduces polyQ inclusions, consistent with Rubicon acting as a conserved negative regulator of autophagy. Reason: This is the defining vertebrate-Rubicon activity, and it is the main fly phenotype reported. Rubicon itself is the regulator, so the participation test is met. The comparator check also holds, because human RUBCN carries GO:0010507 and the Euteleostomi PAINT node PTN002915566 carries the related negative regulation of autophagosome maturation. Fly Rubicon sits outside that vertebrate node, so it does not receive the term by IBA. The evidence is RNAi-based, from one lab. Supporting Evidence: PMID:30783089 Systemic knockdown of dRubicon significantly increased steady-state autophagosome and autolysosome pools in the brain PMID:34189208 Previously, we focused on Rubicon, a negative regulator of autophagy, and demonstrated that knockdown of the Drosophila homolog of Rubicon (dRubicon) suppressed locomotor dysfunction in a fly model of polyQ disease. |
| GO:0090382 phagosome maturation | IMP PMID:37248218 LC3-associated phagocytosis promotes glial degradation of ax... | NEW | Summary: Rubicon on Rab7-positive phagosomes is required for phagosome-lysosome fusion and breakdown of engulfed axon debris in glia (LC3-associated phagocytosis). Reason: The phenotype is specifically a failure of phagosome maturation and fusion with lysosomes, not of engulfment. Rubicon is a component of the LAP-specific PI3KC3 complex on the phagosome, so it does part of the maturation work rather than merely being required for it. This term is a more precise process annotation than phagocytosis and is not an ancestor or descendant of GO:0006909. Supporting Evidence: PMID:37248218 Atg8a and Rubicon co-localize with phagocytosed axon fragments in glia, and glial loss of a subset of autophagy genes and of Rubicon impairs debris elimination and phagosome-lysosome fusion |
| GO:0032585 multivesicular body membrane | IDA PMID:38977659 Egg multivesicular bodies elicit an LC3-associated phagocyto... | NEW | Summary: In the early fertilized egg, maternal Rubicon-eGFP and endogenously tagged Rubicon-tdTomato decorate the limiting membrane of hCD63-positive multivesicular bodies. These MVBs engulf the sperm flagellum. Reason: This is direct imaging of both a rescuing transgene and endogenously tagged Rubicon. It places Rubicon on the MVB limiting membrane, where it drives a LAP-like pathway for paternal mitochondrial elimination. Supporting Evidence: PMID:38977659 Rubicon-eGFP is primarily located at the limiting membrane of the MVBs, while hCD63-tdTomato resides in the ILV compartment PMID:38977659 We demonstrate that mutants and RNAi knockdown of the LAP-specific PI3KC3 component, Rubicon, as well as knockdown of three other main components of the complex, all significantly attenuate degradation of the MD. |
| GO:0035014 phosphatidylinositol 3-kinase regulator activity | ISS GO_REF:0000024 | NEW | Summary: Fly Rubicon keeps the RUN and PIKBD regions that human RUBCN uses to bind and regulate the UVRAG-containing VPS34 complex. Fly genetics shows Rubicon-dependent PtdIns(3)P production on LAP-like vesicles. Reason: Human RUBCN binds VPS34 through its RUN domain and inhibits PIK3C3 lipid kinase activity in canonical autophagy. On LAPosomes, the same Rubicon-containing complex sustains PtdIns(3)P production. Fly Rubicon has the same domain architecture and behaves the same way genetically in both contexts. The direction-neutral parent term is used rather than GO:0141039 inhibitor activity, because the fly protein has not been assayed biochemically and it promotes PI3K output in LAP. Supporting Evidence: PMID:38977659 Other LAP pathway events, such as Rubicon-dependent production of PtdIns(3)P and association of ROS with the sperm flagellum, also unfold during this process. file:worm/Y56A3A.16/Y56A3A.16-bioinformatics/RESULTS.md The fly protein has the full vertebrate-Rubicon architecture (N-terminal RUN, PI3K-binding file:human/RUBCN/RUBCN-uniprot.txt Inhibits PIK3C3 activity |
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Download this section (compressed HTML)Q: Does fly Rubicon bind and inhibit Vps34/UVRAG through its RUN domain and bind Rab7 through its RH domain, as human RUBCN does? If so, GO:0141039 phosphatidylinositol 3-kinase inhibitor activity could replace the broader regulator term.
Q: Should the root PAINT node PTN002915557 carry phosphatidylinositol phosphate binding when the only experimental seed is the vertebrate Pacer paralog and Rubicon-clade members lack lipid-binding data?
Suggested experts: PANTHER/PAINT curators
Experiment: In vitro pull-downs and kinase assays with recombinant fly Rubicon (WT, delta-RUN, RH point mutants) against Vps34-Vps15-Atg6-UVRAG and GTP-Rab7. Then rescue Rubicon-null glia and eggs with these mutants.
Hypothesis: The RUN domain is needed for the anti-autophagy activity and the RH-Rab7 interaction is needed for LAP. If so, the two outputs of fly Rubicon are separable.
Type: biochemistry and genetic rescue
Experiment: Liposome flotation or PIP-strip assays with the purified fly RH and PIKBD regions.
Hypothesis: Fly Rubicon binds phosphoinositides directly, which would test the root-level PAINT phosphatidylinositol phosphate binding inference.
Type: lipid-binding assay
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