Dark is the Drosophila Apaf-1/CED-4 family apoptosome adaptor that recruits the initiator caspase Dronc through CARD-dependent contacts and promotes Dronc autoprocessing in the cytosolic apoptosome. Its NB-ARC nucleotide-binding core and WD40 repeats support oligomerization and Dronc recruitment, placing Dark upstream of effector caspase activation in most developmental and stress-induced fly apoptosis. Dark/Dronc signaling is also reused in nonapoptotic caspase outputs such as spermatid individualization and sensory organ precursor patterning.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0002921 negative regulation of humoral immune response | IMP PMID:24746817 Necrosis-driven systemic immune response alters SAM metaboli... | MARK AS OVER ANNOTATED | Summary: negative regulation of humoral immune response is a systemic downstream phenotype of apoptosis-deficient mutants, not a direct Dark function. Reason: The PMID:24746817 abstract describes apoptosis-deficient mutants that spontaneously develop necrosis-driven systemic inflammation, altered hemolymph SAM metabolism, and FOXO/Gnmt-linked energy effects. That evidence places Dark upstream by preventing failed apoptosis and secondary necrosis, not as a direct executor of this distal immune or metabolic process. Supporting Evidence: PMID:24746817 apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila PMID:24746817 Gnmt was cell-autonomously upregulated by dFoxO in the fat body |
| GO:0005198 structural molecule activity | IDA PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | ACCEPT | Summary: Dark has structural molecule activity as the oligomeric apoptosome scaffold that recruits Dronc. Reason: Purified full-length Dark assembles into the apoptosome platform and into Dark/Dronc complexes, so the structural-molecule row captures Dark's direct adaptor/scaffold function. Supporting Evidence: PMID:21220123 Dark-Dronc complex cleaves DrICE PMID:38381783 the auto-inhibited Dark monomer recruits Dronc zymogen |
| GO:0005515 protein binding | IPI PMID:10984473 An essential role for the caspase dronc in developmentally p... | MODIFY | Summary: The Dark-Dronc interaction is a CARD-mediated recruitment interaction rather than generic protein binding. Reason: The original abstract establishes genetic and biochemical interaction between Dronc and Dark, and later cryo-EM work shows that Dark binds Dronc through CARD/CARD plus CARD/WD1 interfaces. Replace generic protein binding with CARD domain binding. Proposed replacements: CARD domain binding Supporting Evidence: PMID:10984473 interacts, both genetically and biochemically PMID:25644603 nearby Dark protomer are indispensable for Dronc activation |
| GO:0005515 protein binding | IPI PMID:20700104 Metabolic regulation of Drosophila apoptosis through inhibit... | MODIFY | Summary: The Dronc phosphorylation study detects Dark-CARD association, best represented as CARD domain binding. Reason: The co-immunoprecipitation experiment used the Dark CARD-containing N terminus and showed that the Dronc S130 phosphomutant associated more strongly with Dark; the informative function is the CARD-mediated Dark/Dronc docking step. Proposed replacements: CARD domain binding Supporting Evidence: PMID:20700104 Dronc phosphomutant (C318A/S130A) displayed a stronger interaction with Dark |
| GO:0005515 protein binding | IPI PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | MODIFY | Summary: The structural Dark-Dronc row is a CARD-mediated apoptosome recruitment interaction. Reason: Structural reconstitution shows Dronc-CARD binding to Dark-CARD and the Dark WD1 region during apoptosome assembly, so the generic protein-binding row should be narrowed to CARD domain binding. Proposed replacements: CARD domain binding Supporting Evidence: PMID:21220123 Dark-Dronc complex cleaves DrICE PMID:25644603 nearby Dark protomer are indispensable for Dronc activation |
| GO:0005524 ATP binding | IDA PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | ACCEPT | Summary: The Dark nucleotide-binding domain binds ATP/dATP in the assembled apoptosome. Reason: Dark contains the conserved apoptosome nucleotide-binding fold, the 4.0 Angstrom cryo-EM structure resolved ATP bound between NBD and HD1, and nucleotide binding promotes in vitro oligomerization of the structural platform. Supporting Evidence: PMID:25644603 ATP is bound at the interface between the NBD and HD1 PMID:21220123 Dark complex assembly |
| GO:0005829 cytosol | IEA GO_REF:0000117 | ACCEPT | Summary: Cytosol is the expected compartment for Dark apoptosome assembly. Reason: ARBA imports the same cytosolic location used by the canonical Drosophila apoptosome pathway: Dark assembles with cytosolic Dronc after DIAP control is relieved. No contrary organelle-specific localization is supported by the reviewed evidence. Supporting Evidence: PMID:38381783 Dark exists mainly as a relatively unstable, auto-inhibited monomer in the cytoplasm |
| GO:0006915 apoptotic process | IDA PMID:10619022 HAC-1, a Drosophila homolog of APAF-1 and CED-4 functions in... | MODIFY | Summary: The first HAC-1/Dark apoptosis row is too broad for Dark's upstream Dronc-activation role. Reason: Dark promotes caspase activation in developmental and radiation-induced apoptosis rather than catalyzing execution itself, so GO:0006915 should be tightened to positive regulation of apoptotic signaling pathway. Proposed replacements: positive regulation of apoptotic signaling pathway Supporting Evidence: PMID:10619022 Loss of hac-1 function causes reduced cell death PMID:38381783 auto-inhibited Dark monomer recruits Dronc zymogen |
| GO:0006915 apoptotic process | IEA GO_REF:0000117 | MODIFY | Summary: The ARBA apoptotic-process import is real but mapped only to the root apoptosis term. Reason: Dark is not a generic executor of all apoptosis; it is the Dronc-recruiting scaffold for the upstream apoptotic signaling platform. Replace the top-level term with positive regulation of apoptotic signaling pathway. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: ARBA:ARBA00027755 SUPPORTS TRANSFER The ARBA model captures Dark as apoptosome machinery but maps the assertion only to root apoptotic process instead of the signaling step that Dark performs. Proposed replacements: positive regulation of apoptotic signaling pathway Supporting Evidence: PMID:38381783 directly binds Dronc and mediates its activation through the formation of an unsealed single-ring |
| GO:0006915 apoptotic process | IGI PMID:16645639 ARK, the Apaf-1 related killer in Drosophila, requires diver... | MODIFY | Summary: The ark/Hid genetic interaction supports Dark in apoptotic signaling but GO:0006915 is too broad. Reason: Loss-of-function ark alleles suppress Hid-induced cell death because Dark is the Dronc-activation scaffold downstream of RHG/DIAP control. This row should use the direct positive apoptotic-signaling term rather than the root apoptosis process. Proposed replacements: positive regulation of apoptotic signaling pathway Supporting Evidence: PMID:16645639 ark alleles isolated in this study represent loss-of-function alleles PMID:38381783 directly binds Dronc and mediates its activation through the formation of an unsealed single-ring |
| GO:0140608 cysteine-type endopeptidase activator activity | IDA PMID:25644603 Structure of the apoptosome: mechanistic insights into activ... | NEW | Summary: Dark allosterically activates Dronc zymogen in the apoptosome. Reason: Structural reconstitution showed that Dark-Dronc apoptosome contacts promote Dronc activation, so Dark should be annotated to cysteine-type endopeptidase activator activity rather than only to ATP binding and CARD/domain scaffold terms. Supporting Evidence: PMID:25644603 nearby Dark protomer are indispensable for Dronc activation PMID:38381783 the auto-inhibited Dark monomer recruits Dronc zymogen |
| GO:0007291 sperm individualization | IMP PMID:14737191 Multiple apoptotic caspase cascades are required in nonapopt... | KEEP AS NON CORE | Summary: Dark is reused directly for the nonapoptotic caspase cascade that drives spermatid individualization. Reason: Germline Ark RNAi blocks the local Dronc activation needed for sperm individualization; this is a bona fide but specialized, nonlethal use of the Dark/Dronc module. Supporting Evidence: PMID:14737191 inhibition of ARK |
| GO:0007291 sperm individualization | IMP PMID:16362035 The two Drosophila cytochrome C proteins can function in bot... | KEEP AS NON CORE | Summary: A second sperm-individualization paper independently supports Ark in bulk cytoplasm removal. Reason: Ark and Dronc are required for the caspase-linked removal of bulk cytoplasm during spermatogenesis, a direct but highly tissue-specific function rather than Dark's core apoptotic role. Supporting Evidence: PMID:16362035 proper removal of bulk cytoplasm during spermatogenesis |
| GO:0010332 response to gamma radiation | IDA PMID:16533943 The Drosophila melanogaster Apaf-1 homologue ARK is required... | KEEP AS NON CORE | Summary: Dark is needed for radiation-induced apoptosis, but response to gamma radiation is a stimulus context. Reason: ark mutant larval brain, eye-disc, and wing-disc tissues do not increase AO staining after gamma irradiation. The annotation is true, but the direct Dark activity remains Dronc activation in apoptotic signaling. Supporting Evidence: PMID:16533943 Radiation-induced cell death is inhibited in ark mutants |
| GO:0012501 programmed cell death | IDA PMID:16533943 The Drosophila melanogaster Apaf-1 homologue ARK is required... | MODIFY | Summary: The ARK programmed-cell-death row is correct but less precise than apoptotic signaling. Reason: The evidence shows that Ark/Dark is essential for most developmental and stress-induced cell death by facilitating Dronc activation; this should be represented as positive regulation of apoptotic signaling pathway rather than broad programmed cell death. Proposed replacements: positive regulation of apoptotic signaling pathway Supporting Evidence: PMID:16533943 ARK is essential for most PCD in the embryo and larval tissues |
| GO:0021556 central nervous system formation | IMP PMID:16533943 The Drosophila melanogaster Apaf-1 homologue ARK is required... | MARK AS OVER ANNOTATED | Summary: The CNS phenotype is a downstream consequence of failed Dark-dependent apoptosis. Reason: ark mutant embryos retain extra neurons and show CNS morphology defects because normal developmental apoptosis is reduced. The experiment supports Dark-dependent apoptotic signaling in neural tissues, not a direct Dark role in constructing the central nervous system. Supporting Evidence: PMID:16533943 extra neurons in chordotonal cell clusters in ark mutant embryos |
| GO:0022416 chaeta development | IMP PMID:16222340 Drosophila caspase transduces Shaggy/GSK-3beta kinase activi... | KEEP AS NON CORE | Summary: Dark has a direct nonapoptotic caspase-signaling role in chaeta/SOP patterning. Reason: Dark-dependent caspase activity cleaves Shaggy/GSK-3beta to tune Wingless signaling during sensory organ precursor selection; this is a real but non-core differentiation function. Supporting Evidence: PMID:16222340 is cleaved by the Dark-dependent caspase |
| GO:0033353 L-methionine cycle | IMP PMID:24746817 Necrosis-driven systemic immune response alters SAM metaboli... | MARK AS OVER ANNOTATED | Summary: L-methionine cycle is a systemic downstream phenotype of apoptosis-deficient mutants, not a direct Dark function. Reason: The PMID:24746817 abstract describes apoptosis-deficient mutants that spontaneously develop necrosis-driven systemic inflammation, altered hemolymph SAM metabolism, and FOXO/Gnmt-linked energy effects. That evidence places Dark upstream by preventing failed apoptosis and secondary necrosis, not as a direct executor of this distal immune or metabolic process. Supporting Evidence: PMID:24746817 apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila PMID:24746817 Gnmt was cell-autonomously upregulated by dFoxO in the fat body |
| GO:0034352 positive regulation of glial cell apoptotic process | IMP PMID:16645639 ARK, the Apaf-1 related killer in Drosophila, requires diver... | KEEP AS NON CORE | Summary: Dark promotes developmentally programmed midline glial apoptosis. Reason: Midline glial survival increases in ark mutants, which fits a direct Dark requirement in apoptosome signaling during that glial death program. The cell-type-specific term is valid but contextual. Supporting Evidence: PMID:16645639 ark H16 mutant embryos contain on average 10.7 MG per segment |
| GO:0035006 melanization defense response | IMP PMID:24746817 Necrosis-driven systemic immune response alters SAM metaboli... | MARK AS OVER ANNOTATED | Summary: melanization defense response is a systemic downstream phenotype of apoptosis-deficient mutants, not a direct Dark function. Reason: The PMID:24746817 abstract describes apoptosis-deficient mutants that spontaneously develop necrosis-driven systemic inflammation, altered hemolymph SAM metabolism, and FOXO/Gnmt-linked energy effects. That evidence places Dark upstream by preventing failed apoptosis and secondary necrosis, not as a direct executor of this distal immune or metabolic process. Supporting Evidence: PMID:24746817 apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila PMID:24746817 Gnmt was cell-autonomously upregulated by dFoxO in the fat body |
| GO:0035070 salivary gland histolysis | IMP PMID:16485033 Systematic in vivo RNAi analysis of putative components of t... | UNDECIDED | Summary: The RNAi screen is abstract-only locally and does not expose salivary-gland histolysis assays. Reason: The abstract supports Dark/Dronc as rate-limiting for in vivo apoptosis paradigms but does not verify this exact tissue-specific histolysis row. |
| GO:0035070 salivary gland histolysis | IMP PMID:16540507 Autophagy occurs upstream or parallel to the apoptosome duri... | KEEP AS NON CORE | Summary: Dark is required for histolytic removal of larval salivary glands. Reason: The salivary-gland histolysis row is supported: Dark is needed for histolysis while the autophagic program itself can still be induced. This is a tissue-specific programmed-death output, not the core molecular role. Supporting Evidence: PMID:16540507 dark was essential for histolysis |
| GO:0042594 response to starvation | IMP PMID:24746817 Necrosis-driven systemic immune response alters SAM metaboli... | MARK AS OVER ANNOTATED | Summary: response to starvation is a systemic downstream phenotype of apoptosis-deficient mutants, not a direct Dark function. Reason: The PMID:24746817 abstract describes apoptosis-deficient mutants that spontaneously develop necrosis-driven systemic inflammation, altered hemolymph SAM metabolism, and FOXO/Gnmt-linked energy effects. That evidence places Dark upstream by preventing failed apoptosis and secondary necrosis, not as a direct executor of this distal immune or metabolic process. Supporting Evidence: PMID:24746817 apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila PMID:24746817 Gnmt was cell-autonomously upregulated by dFoxO in the fat body |
| GO:0042802 identical protein binding | IPI PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | KEEP AS NON CORE | Summary: Dark self-association is part of apoptosome scaffold assembly. Reason: Dark protomers assemble into single- and double-ring apoptosome complexes, so identical protein binding is mechanistically true. Keep it as a structural subfunction rather than a standalone core function because the informative activity is assembly of the Dronc-activating apoptosome. Supporting Evidence: PMID:21220123 Dark-Dronc complexes formed a mixture of single- and double-rings PMID:38381783 the auto-inhibited Dark monomer recruits Dronc zymogen |
| GO:0043293 apoptosome | IDA PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | ACCEPT | Summary: Dark is the scaffold subunit of the Drosophila apoptosome. Reason: The 2011 structural reconstitution directly purified and imaged full-length Dark apoptosome complexes and showed their Dronc-dependent DrICE-cleavage activity. Supporting Evidence: PMID:21220123 Dark-Dronc complex cleaves DrICE |
| GO:0043293 apoptosome | IPI PMID:25644603 Structure of the apoptosome: mechanistic insights into activ... | ACCEPT | Summary: Dark remains in the Dronc-CARD-bound apoptosome/post-activation complex. Reason: The 2015 cryo-EM structure directly resolved a Dark apoptosome bound to Dronc-CARD and identified CARD and WD-repeat Dark surfaces required for Dronc activation. Supporting Evidence: PMID:25644603 nearby Dark protomer are indispensable for Dronc activation |
| GO:0043531 ADP binding | IDA PMID:38381783 Dark and Dronc activation in Drosophila melanogaster. | ACCEPT | Summary: Dark monomers bind ADP in their autoinhibited state. Reason: The 2024 cryo-EM and LC-MS/MS study identified ADP in the nucleotide pocket of autoinhibited Dark monomer, matching the InterPro NBD-family prediction. Supporting Evidence: PMID:38381783 mostly ADP after SEC |
| GO:0043531 ADP binding | IEA GO_REF:0000002 | ACCEPT | Summary: The InterPro NB-ARC family import correctly infers nucleotide binding by Dark. Reason: Direct Dark structural work confirms ADP in the monomeric nucleotide pocket and ATP in assembled structures, supporting retention of this computationally inferred ADP-binding row. Propagation Review Root cause: NO FAILURE CORE Sources checked: InterPro:IPR002182 SUPPORTS TRANSFER The NB-ARC domain signature maps to the experimentally confirmed Dark nucleotide-binding pocket. Supporting Evidence: PMID:38381783 mostly ADP after SEC PMID:25644603 ATP is bound at the interface between the NBD and HD1 |
| GO:0046667 compound eye retinal cell programmed cell death | IMP PMID:16485033 Systematic in vivo RNAi analysis of putative components of t... | UNDECIDED | Summary: The RNAi screen is abstract-only locally and does not expose the retinal-cell programmed-death assay. Reason: The abstract supports Dronc/Dark as rate-limiting components in several apoptosis paradigms but not this exact compound-eye retinal cell term. |
| GO:0048102 autophagic cell death | IMP PMID:16533943 The Drosophila melanogaster Apaf-1 homologue ARK is required... | MODIFY | Summary: The autophagic-cell-death row conflates salivary-gland histolysis with the upstream autophagy program. Reason: Dark is needed for salivary-gland histolysis, but Akdemir et al. explicitly separate that requirement from the autophagic features themselves. Replace the autophagic-cell-death term with salivary gland histolysis. Proposed replacements: salivary gland histolysis Supporting Evidence: PMID:16533943 Larval salivary gland removal was markedly delayed PMID:16540507 dark was essential for histolysis |
| GO:0048813 dendrite morphogenesis | IMP PMID:16980964 Local caspase activity directs engulfment of dendrites durin... | UNDECIDED | Summary: The dendrite-pruning paper is abstract-only locally and does not expose Dark-specific dendrite evidence. Reason: The abstract establishes local caspase activity during dendrite pruning but does not name Dark, so this IMP row cannot be fully checked without the full text. |
| GO:0050700 CARD domain binding | ISM PMID:21220123 Structure of the Drosophila apoptosome at 6.9 å resolution. | ACCEPT | Summary: Dark binds Dronc through CARD-dependent apoptosome-recruitment interfaces. Reason: Structural studies show Dronc-CARD contacts with Dark-CARD and Dark WD1 during Dark/Dronc assembly; this is the right specific term for Dark recruitment of Dronc. Supporting Evidence: PMID:25644603 nearby Dark protomer are indispensable for Dronc activation PMID:21220123 Dark-Dronc complex cleaves DrICE |
| GO:0070050 neuron cellular homeostasis | IGI PMID:22153373 Glial-derived prodegenerative signaling in the Drosophila ne... | MARK AS OVER ANNOTATED | Summary: Dark participates in ank2 stress-induced motoneuron degeneration rather than ordinary neuron homeostasis. Reason: The paper shows that dark loss suppresses ank2-dependent neuromuscular degeneration, consistent with Dark amplifying local caspase activity in a pathological stress model. GO:0070050 makes this look like a homeostatic maintenance function and overstates the biology. Supporting Evidence: PMID:22153373 dark significantly suppresses ank2-dependent NMJ degeneration |
| GO:0070328 triglyceride homeostasis | IMP PMID:24746817 Necrosis-driven systemic immune response alters SAM metaboli... | MARK AS OVER ANNOTATED | Summary: triglyceride homeostasis is a systemic downstream phenotype of apoptosis-deficient mutants, not a direct Dark function. Reason: The PMID:24746817 abstract describes apoptosis-deficient mutants that spontaneously develop necrosis-driven systemic inflammation, altered hemolymph SAM metabolism, and FOXO/Gnmt-linked energy effects. That evidence places Dark upstream by preventing failed apoptosis and secondary necrosis, not as a direct executor of this distal immune or metabolic process. Supporting Evidence: PMID:24746817 apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila PMID:24746817 Gnmt was cell-autonomously upregulated by dFoxO in the fat body |
| GO:0160108 animal gross anatomical part developmental process | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: The ARBA developmental-process row is too broad for Dark's apoptosome role. Reason: Dark is directly reused in several developmental contexts through apoptosome-mediated Dronc activation, but animal gross anatomical part developmental process is an uninformative parent and should not be the propagated label for the protein. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: ARBA:ARBA00098139 SUPPORTS TRANSFER The rule recognizes a real developmental apoptosis protein but transfers an overly broad developmental-process parent. Supporting Evidence: PMID:16533943 ARK is essential for most PCD in the embryo and larval tissues |
| GO:1904747 positive regulation of apoptotic process involved in development | IMP PMID:10984473 An essential role for the caspase dronc in developmentally p... | MODIFY | Summary: The development-qualified apoptosis row is better represented as Dark-dependent apoptotic signaling. Reason: The source establishes that Dark is required for Dronc processing and embryonic programmed cell death, but the direct mechanistic step is Dronc activation by the Dark apoptosome. Use the proximal positive regulation of apoptotic signaling pathway term shared by the broad apoptosis rows. Proposed replacements: positive regulation of apoptotic signaling pathway Supporting Evidence: PMID:10984473 Dark is required for Dronc processing PMID:10619022 where cells undergo natural death |
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Download this section (compressed HTML)Q: Does the physiologic Dark/Dronc activation complex in vivo resemble the unsealed single ring, the post-activation Dark/Dronc-CARD double ring, or a regulated mixture of assemblies?
Q: Which Dark-dependent Dronc substrates distinguish lethal apoptosis from nonapoptotic outputs in spermatid individualization and chaeta patterning?
Q: How should GO separate salivary-gland histolysis, autophagy, and apoptosome-dependent killing so that Dark is not annotated to autophagy when autophagic features proceed upstream or in parallel?
Experiment: Introduce endogenous Dark WD1 interface alleles and Dronc CARD loop alleles, then compare apoptosis, Dronc processing, spermatid individualization, Dark/Dronc complex geometry, and DrICE cleavage in the same genetic backgrounds.
Hypothesis: Dronc-CARD binding to Dark WD1 gates assembly of the active single-ring apoptosome rather than merely stabilizing post-activation complexes.
Type: structure-guided endogenous rescue
Experiment: Measure endogenous Dark/Dronc assembly, Dronc cleavage, and effector-caspase substrates with live reporters in spermatids and proneural clusters, and compare them with matched cells undergoing lethal developmental apoptosis.
Hypothesis: Nonapoptotic Dark/Dronc outputs use spatially restricted Dronc activation below the threshold for effector-caspase cascade amplification.
Type: live caspase-activity imaging with separation-of-function alleles
Death-associated APAF1-related killer (Dark) encodes an essential component of the apoptosome. The products of Dark and Dronc form a pivotal holoenzyme required for apoptotic cell death with the products of Dark acting as an adaptor of the multimeric complex.
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