Affinage

ATG101

Autophagy-related protein 101 · UniProt Q9BSB4

Length
218 aa
Mass
25.0 kDa
Annotated
2026-06-09
23 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ATG101 is a HORMA-domain protein essential for autophagy initiation in metazoans, functioning as an integral subunit of the ULK1-Atg13-FIP200 complex that nucleates phagophore formation (PMID:19597335, PMID:19287211). It associates with the complex through a constitutive heterodimer with the Atg13 HORMA domain, an interaction conserved from C. elegans (EPG-9/EPG-1) and Drosophila to humans, and assembles into an architecture analogous to the Mad2 conformational heterodimer (PMID:22885670, PMID:26299944). A central function of ATG101 is to stabilize Atg13: it protects Atg13 from proteasomal degradation and caps the otherwise exposed Atg13 HORMA domain, and its own levels are controlled by HUWE1-mediated ubiquitination of its C-terminal region (PMID:19287211, PMID:26754330, PMID:34502089). ATG101 acts as an interaction hub that couples the ULK1 complex to downstream autophagy machinery: its WF (Trp-Phe) finger, normally sequestered in a hydrophobic pocket, becomes exposed to recruit downstream factors and, together with PI3P-binding WIPI2/WIPI3 proteins, inserts into the membrane to position the ULK1 kinase for ATG16L1 phosphorylation [PMID:26299944, PMID:26754330, PMID:bio_10.1101_2025.11.07.687251]. Its C-terminal region bridges the ULK1 complex to the PtdIns3K complex (VPS34, VPS15, BECN1, UVRAG) (PMID:30081750). Independently of ULK1, the ATG13-ATG101 dimer binds ATG9A to govern ATG9A distribution, and loss of ATG101 causes aberrant ATG9A accumulation at stalled cargo clusters (PMID:34369648). Loss of ATG101 across model organisms impairs basal and starvation-induced autophagy, causing accumulation of ubiquitin/p62-positive aggregates and tissue-specific developmental defects (PMID:24895579, PMID:30760524). ATG101 additionally links autophagy to other pathways through physical interaction with the Hedgehog receptor PTCH1, which blocks autophagic flux independently of SMO (PMID:29453315).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2009 High

    Established ATG101 as a previously unknown core component of the mammalian autophagy initiation complex, answering whether the ULK1-Atg13-FIP200 machinery had additional essential subunits.

    Evidence Reciprocal Co-IP, siRNA knockdown with LC3 readout, GFP-localization to the phagophore, and proteasome-inhibitor rescue in mammalian cells (two concurrent papers)

    PMID:19287211 PMID:19597335

    Open questions at the time
    • Structural basis of the Atg13-ATG101 interaction not defined
    • Mechanism by which ATG101 stabilizes Atg13 against degradation unresolved
  2. 2012 Medium

    Showed the Atg13-binding and autophagy function of ATG101 is conserved across metazoans, generalizing the mammalian finding beyond a single system.

    Evidence Genetic loss-of-function and direct interaction assays of EPG-9/EPG-1 in C. elegans

    PMID:22885670

    Open questions at the time
    • Single-organism genetic data
    • No structural or biochemical mapping of the interaction interface
  3. 2014 Medium

    Identified ATG101 as a HORMA-domain protein that dimerizes and maps binding to the Atg13 HORMA domain, framing the molecular nature of the subcomplex.

    Evidence Drosophila loss-of-function genetics, domain-mapping pulldowns, and dimerization assays

    PMID:24895579

    Open questions at the time
    • HORMA fold inferred by prediction, not solved
    • Functional significance of putative Atg1 interactions unclear
  4. 2015 High

    Solved the human Atg13HORMA-ATG101 heterodimer structure, revealing a Mad2-like architecture and a sequestered WF finger whose regulated exposure underlies downstream recruitment.

    Evidence X-ray crystallography with functional mapping of the WF finger motif

    PMID:26299944

    Open questions at the time
    • Trigger for WF finger exposure not defined
    • Identity of downstream partners docking at the unique hydrophobic pockets unknown
  5. 2016 Medium

    Synthesized the structural data into a model explaining why ATG101 is required in most eukaryotes (to cap an exposed Atg13 HORMA) but dispensable in budding yeast, defining the evolutionary logic of its essentiality.

    Evidence Structural and cell-biological synthesis of prior work

    PMID:26754330

    Open questions at the time
    • Review synthesis rather than new primary data
    • Direct test of the capping model in diverse species not provided
  6. 2018 High

    Defined how ATG101 physically couples autophagy initiation to lipid-kinase activation by mapping its C-terminal region to PtdIns3K binding.

    Evidence Crystal structure, SEC-SAXS, Co-IP with deletion mutants, and autophagosome formation assays with KO/reconstitution

    PMID:30081750

    Open questions at the time
    • Conformational switch driving C-terminal availability not kinetically resolved
    • Stoichiometry of the ULK1-PtdIns3K bridge unknown
  7. 2018 Medium

    Extended ATG101 function beyond core autophagy by showing it physically links the Hedgehog receptor PTCH1 to the ULK complex to restrain autophagic flux.

    Evidence Co-IP and autophagic flux assays with SMO-deficient cells and SMO inhibitor controls

    PMID:29453315

    Open questions at the time
    • Interaction interface on ATG101 not mapped
    • Mechanism by which PTCH1 binding blocks flux unresolved
  8. 2019 Medium

    Demonstrated physiological consequences of ATG101 loss in a whole organism, including neuronal aggregate accumulation and cell-type-specific roles in tissue homeostasis.

    Evidence Drosophila loss-of-function genetics with cell-type-specific rescue and phenotypic assays

    PMID:30760524

    Open questions at the time
    • Molecular basis of tissue-specific requirements not defined
    • Mammalian in vivo phenotype not addressed
  9. 2021 Medium

    Identified HUWE1 as the E3 ligase controlling ATG101 turnover via its C-terminal region, establishing a ubiquitin-dependent layer of autophagy regulation acting through ATG101 levels.

    Evidence CRISPR knockout, Co-IP, ubiquitination assays with C-terminal deletion mutants, and epistatic siRNA rescue

    PMID:34502089

    Open questions at the time
    • Signals regulating HUWE1-ATG101 activity unknown
    • Single-lab data
  10. 2021 High

    Uncovered a ULK1-independent function of the ATG13-ATG101 dimer in binding ATG9A and controlling its distribution, separating ATG101's roles within and outside ULK1 kinase signaling.

    Evidence BioID proteomics, KO/reconstitution, split-mVenus complementation, and rescue with a ULK1-binding-deficient ATG13 mutant

    PMID:34369648

    Open questions at the time
    • Structural basis of ATG9A engagement not defined at this stage
    • How ATG9A trafficking is mechanistically corrected unclear
  11. 2025 High

    Reconstituted how the ATG13:ATG101 dimer engages PI3P-binding WIPI2/3 and inserts its WF finger into membranes to position ULK1 for ATG16L1 phosphorylation, linking membrane targeting to downstream conjugation machinery.

    Evidence Biochemical reconstitution, molecular dynamics, in vitro kinase assay, and cell-based flux assays (preprint)

    PMID:bio_10.1101_2025.11.07.687251

    Open questions at the time
    • Peer review pending
    • In vivo relevance of the membrane-insertion model not yet tested
  12. 2025 Medium

    Proposed an autocatalytic mechanism in which ULK1-phosphorylation-driven ATG101 homodimerization accelerates assembly of an ATG9A-ATG13-ATG101 initiation complex, providing a kinetic switch and memory for autophagy onset.

    Evidence Interaction kinetics, phosphorylation and homodimerization assays, and complex formation rate measurements (preprint)

    PMID:bio_10.1101_2025.06.27.661946

    Open questions at the time
    • Preprint awaiting peer review
    • Physiological role of the proposed activation memory not established
    • Structural basis of the metamorphic HORMA change not solved
  13. 2025 Medium

    Defined the species-specific basis for ATG101's requirement in the ATG13-ATG9 interaction, clarifying why ATG101 was retained in metazoans but lost in some fungal lineages.

    Evidence Yeast two-hybrid assays, evolutionary BLAST analysis, and genetic deletions in K. phaffii

    PMID:40931865

    Open questions at the time
    • Mechanism of the Atg13 binding-site shift not structurally resolved
    • Selective pressures driving ATG101 loss inferred, not demonstrated

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the regulated exposure of the WF finger, the C-terminal conformational switch, and ATG101 homodimerization are coordinated in time to control autophagy initiation in vivo remains unresolved.
  • No integrated kinetic model linking phosphorylation, dimerization, and membrane engagement
  • Mammalian in vivo loss-of-function phenotype not characterized in the corpus
  • Disease relevance not established in the corpus

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 3 GO:0140313 molecular sequestering activity 2 GO:0008289 lipid binding 1
Localization
GO:0005829 cytosol 1
Pathway
R-HSA-9612973 Autophagy 4
Partners
ATG13ATG9AHUWE1PIK3C3PTCH1ULK1WIPI2WIPI3
Complex memberships
ATG13-ATG101 HORMA heterodimerATG9A-ATG13-ATG101 complexULK1-ATG13-FIP200 complex

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 ATG101 (FLJ11773) is a novel mammalian Atg13-binding protein that associates with the ULK1-Atg13-FIP200 complex, most likely through direct interaction with Atg13. In Atg13 siRNA-treated cells, ATG101 is present solely as a monomer, indicating that complex incorporation depends on Atg13. ATG101 is important for the stability and basal phosphorylation of Atg13 and ULK1. GFP-ATG101 localizes to the isolation membrane/phagophore, and ATG101 knockdown suppresses GFP-LC3 dot formation and causes accumulation of LC3-I, establishing ATG101 as essential for autophagy initiation. Co-immunoprecipitation, siRNA knockdown, GFP-localization (live imaging), immunoblot for LC3 processing Autophagy High 19597335
2009 ATG101 (FLJ11773) interacts with ULK1 in an Atg13-dependent manner, stabilizes Atg13 expression by protecting it from proteasomal degradation, and is essential for macroautophagy. Intracellular localization of the ULK1 complex is regulated by nutrient conditions. Co-immunoprecipitation, siRNA knockdown, proteasome inhibitor rescue, autophagy flux assays Autophagy High 19287211
2012 The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagic degradation of protein aggregates and starvation survival, placing ATG101 function in the Atg1/Atg13 pathway across metazoans. Genetic loss-of-function screen, direct protein interaction assay, autophagy flux assays in C. elegans Autophagy Medium 22885670
2014 Drosophila Atg101 dimerizes and is predicted to fold into a HORMA domain. Loss of Atg101 impairs both starvation-induced and basal autophagy, leading to accumulation of ref(2)P/p62-positive aggregates. Mapping experiments show Atg101 binds the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Atg101 also interacts with ref(2)P. Genetic loss-of-function (Drosophila mutant), domain-mapping pulldowns, dimerization assay, immunofluorescence for selective autophagy cargo BioMed research international Medium 24895579
2015 Crystal structure of the human Atg13 HORMA domain in complex with full-length ATG101 HORMA domain was determined. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer. The WF finger motif essential for ATG101 function is sequestered in a hydrophobic pocket, suggesting its exposure is regulated. Two benzamidine-marked hydrophobic pockets unique to animals suggest additional protein interaction sites, identifying the Atg13-ATG101 subcomplex as an interaction hub. X-ray crystallography, structural comparison, functional mapping of WF finger motif Structure High 26299944
2016 Structural and cell biological analysis established that ATG101 is required for stabilization of 'uncapped' Atg13 in most eukaryotes (because Atg13 HORMA domain is exposed/uncapped without ATG101), and ATG101 recruits downstream Atg proteins through its WF motif. By contrast, S. cerevisiae Atg13 is stably 'capped' and does not require Atg101. Structural analysis, cell biology (reviewed synthesis of prior structural/functional studies) Cell structure and function Medium 26754330
2018 The C-terminal region of ATG101, which adopts a β-strand conformation in free ATG101 but a different conformation in the ATG101-ATG13HORMA complex, is responsible for binding PtdIns3K complex components (PIK3C3/VPS34, PIK3R4/VPS15, BECN1, UVRAG). C-terminal deletion of ATG101 shows significant defects in PtdIns3K interaction and impairs autophagosome formation, establishing ATG101 as a bridge between the ULK1 and PtdIns3K complexes. Crystal structure, SEC-SAXS, co-immunoprecipitation with deletion mutants, autophagosome formation assay, KO/reconstitution Autophagy High 30081750
2018 ATG101 physically interacts with the C-terminal domain (CTD) of the Hedgehog receptor PATCHED1 (PTCH1), connecting PTCH1 to the ULK complex. This interaction results in a blockade of basal autophagic flux and accumulation of autophagosomes with undegraded cargo, independent of PTCH1's repressive activity on SMO. Co-immunoprecipitation, autophagic flux assays, SMO-deficient cells and SMO inhibitor controls Molecular cancer research Medium 29453315
2019 Drosophila Atg101 loss-of-function mutants are semi-lethal, with defective developmental and starvation-induced autophagy, accumulation of ubiquitin-positive aggregates in brains (neuronal defect), shortened/thickened midguts with enlarged enterocytes, and impaired differentiation of intestinal stem cells to enterocytes. Cell type-specific rescue showed ATG101 functions in enterocytes to limit their growth. Drosophila loss-of-function genetics, cell-type-specific rescue, immunofluorescence, lifespan and mobility assays Journal of biological chemistry Medium 30760524
2021 HUWE1 is the major E3 ubiquitin ligase targeting ATG101 for ubiquitination and proteasomal degradation, with the C-terminal region of ATG101 identified as the key ubiquitination domain. HUWE1 depletion stabilizes ATG101 and increases autophagy activity; this enhanced autophagy is reversed by siRNA-mediated ATG101 knockdown, placing ATG101 downstream of HUWE1 in autophagy regulation. CRISPR knockout, co-immunoprecipitation, siRNA knockdown, ubiquitination assays with C-terminal deletion mutants, autophagy flux assays International journal of molecular sciences Medium 34502089
2021 ATG9A interacts specifically with the ATG13-ATG101 dimer independently of ULK1, as demonstrated by knockout/reconstitution and split-mVenus approaches. Deletion of ATG13 or ATG101 causes aberrant accumulation of ATG9A at stalled p62/SQSTM1-ubiquitin clusters, rescuable by a ULK1 binding-deficient ATG13 mutant, establishing a ULK1-independent ATG13-ATG101 complex function in regulating ATG9A distribution. BioID quantitative proteomics, knockout/reconstitution, split-mVenus bimolecular complementation, immunofluorescence EMBO reports High 34369648
2025 ATG101 HORMA domain forms a tight complex with PI3P-binding proteins WIPI3 and WIPI2. Bound to WIPI2/3, the ATG13:ATG101 dimer aligns with membranes to insert its WF (Trp-Phe) finger into the membrane. Molecular dynamics simulations show cooperative stabilization of the complex on membranes by WIPIs and the ATG101 WF finger. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement is required for ATG16L1 phosphorylation by ULK1, ATG13 puncta formation, and bulk autophagic flux. A PVP motif in the ULK1 IDR docks onto the ATG13:ATG101 HORMA dimer surface, bringing the ULK1 kinase domain close to the membrane. Biochemical reconstitution, molecular dynamics simulation, in vitro kinase assay (ATG16L1 phosphorylation), cell-based autophagic flux assay, structural modeling bioRxivpreprint High bio_10.1101_2025.11.07.687251
2025 ATG101 HORMA domain undergoes a conformational change (fold change/metamorphosis) that enables interaction with ATG9A and ATG13 to form the essential ATG9A-ATG13-ATG101 complex. ATG101 homo-dimerization, initiated by ULK1 phosphorylation, dramatically accelerates complex formation. This creates an auto-catalytic positive feedback where ATG101 dimers propagate activation to further ATG101 molecules. Memory of ATG101 activation persists for many hours after dephosphorylation and continues to accelerate ATG9A-ATG13-ATG101 assembly. Interaction kinetics assays, phosphorylation assays, homodimerization assays, complex formation rate measurements bioRxivpreprint Medium bio_10.1101_2025.06.27.661946
2025 ATG101 is required for the ATG13-ATG9 interaction in mammals but is dispensable for this interaction in Aspergillus oryzae, due to a shift in the AoAtg9-binding site in AoAtg13. Yeast two-hybrid assays established this species-specific dependency, and evolutionary analysis showed that ATG101 was lost in some Holomycota lineages after acquisition of Atg29/Atg31 and a cap structure in Atg13. Yeast two-hybrid assay, evolutionary BLAST analysis, genetic deletion (atg101 and atg31 in K. phaffii) Autophagy Medium 40931865

Source papers

Stage 0 corpus · 23 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 397 19597335
2009 A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy 338 19287211
2015 Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex. Structure (London, England : 1993) 81 26299944
2018 The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation. Autophagy 51 30081750
2012 The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy. Autophagy 46 22885670
2021 BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters. EMBO reports 42 34369648
2019 The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis. The Journal of biological chemistry 38 30760524
2014 The putative HORMA domain protein Atg101 dimerizes and is required for starvation-induced and selective autophagy in Drosophila. BioMed research international 38 24895579
2020 Circ-HIPK2 Accelerates Cell Apoptosis and Autophagy in Myocardial Oxidative Injury by Sponging miR-485-5p and Targeting ATG101. Journal of cardiovascular pharmacology 28 33027196
2016 Atg101: Not Just an Accessory Subunit in the Autophagy-initiation Complex. Cell structure and function 26 26754330
2021 ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells. International journal of molecular sciences 19 34502089
2024 ATG-101 Is a Tetravalent PD-L1×4-1BB Bispecific Antibody That Stimulates Antitumor Immunity through PD-L1 Blockade and PD-L1-Directed 4-1BB Activation. Cancer research 18 38501978
2017 ATG101 Single-Stranded Antisense RNA-Loaded Triangular DNA Nanoparticles Control Human Pulmonary Endothelial Growth via Regulation of Cell Macroautophagy. ACS applied materials & interfaces 17 29154530
2025 First ATG101-recruiting small molecule degrader for selective CDK9 degradation via autophagy-lysosome pathway. Acta pharmaceutica Sinica. B 12 40487652
2018 Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED1 and ATG101. Molecular cancer research : MCR 12 29453315
2024 In vitro study of the expression of autophagy genes ATG101, mTOR and AMPK in breast cancer with treatment of lactoferrin and in silico study of their communication networks and protein interactions. Progress in biophysics and molecular biology 8 38782098
2023 The role of the HORMA domain proteins ATG13 and ATG101 in initiating autophagosome biogenesis. FEBS letters 6 37567770
2023 miR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy. Heliyon 5 37539299
2022 Comprehensive Analysis of the Potential Immune-Related Biomarker ATG101 that Regulates Apoptosis of Cholangiocarcinoma Cells After Photodynamic Therapy. Frontiers in pharmacology 3 35592424
2023 Metamorphosis by ATG13 and ATG101 in human autophagy initiation. Autophagy 2 37394799
2025 Caloric restriction mimetics chlorogenic acid and fisetin as potential autophagy inducers targeting ATG101. Biochemistry and biophysics reports 0 40641740
2025 Evolutionary diversification of the autophagy initiation complex: reduced Atg101 dependency and changes in Atg9 binding to Atg13. Autophagy 0 40931865
2025 RAN Promotes Autophagy and Malignant Progression of Lung Adenocarcinoma through ATG101. Advanced biology 0 41263115

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