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ATG101

Autophagy-related protein 101 · UniProt Q9BSB4

Length
218 aa
Mass
25.0 kDa
Annotated
2026-04-28
23 papers in source corpus 14 papers cited in narrative 14 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ATG101 is a HORMA domain protein that functions as an essential component of the autophagy initiation machinery by forming a stable heterodimer with the HORMA domain of ATG13 within the ULK1-ATG13-FIP200 complex, stabilizing ATG13 against proteasomal degradation and coupling the ULK1 kinase complex to downstream autophagy effectors (PMID:19597335, PMID:19287211, PMID:26299944). Its C-terminal region undergoes a conformational change upon ATG13 binding and bridges the ULK1 complex to the class III PI3K complex, while its conserved WF finger motif inserts into membranes upon engagement with PI3P-binding WIPI2/WIPI3 proteins, positioning ULK1 for phosphorylation of ATG16L1 (PMID:30081750, PMID:26299944). ATG101 also interacts with ATG9A independently of ULK1, forming an ATG9A-ATG13-ATG101 complex required for proper ATG9A trafficking and autophagosome nucleation (PMID:34369648, PMID:40931865). The protein is subject to HUWE1-mediated ubiquitination and proteasomal turnover at its C-terminal region, providing a regulatory checkpoint for autophagy flux (PMID:34502089).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2009 High

    Identification of ATG101 as a novel autophagy-essential factor that directly binds ATG13, associates with the ULK1-FIP200 complex, and stabilizes ATG13 against proteasomal degradation resolved the question of how ATG13 protein levels are maintained and established ATG101 as a core initiation complex subunit.

    Evidence Reciprocal co-immunoprecipitation, siRNA knockdown with autophagy readouts, proteasome inhibitor rescue, and GFP-ATG101 live imaging in mammalian cells, independently reported by two laboratories

    PMID:19287211 PMID:19597335

    Open questions at the time
    • No structural information on ATG101 fold or binding mode with ATG13
    • No reconstitution of the complex with purified components
    • Mechanism by which ATG101 stabilizes ATG13 not defined
  2. 2012 High

    Demonstration that the C. elegans ortholog EPG-9 directly interacts with EPG-1/ATG13 and phenocopies loss of the Atg1 kinase established evolutionary conservation of ATG101 function across metazoa.

    Evidence Genetic screen, loss-of-function mutant analysis, direct protein interaction assay, and epistasis in C. elegans

    PMID:22885670

    Open questions at the time
    • Conservation in fungi and non-metazoan lineages not yet addressed
    • Specific structural basis of EPG-9–EPG-1 interaction unknown
  3. 2014 Medium

    Prediction that ATG101 adopts a HORMA domain fold and binds the N-terminal HORMA domain of ATG13, combined with Drosophila loss-of-function phenotypes, framed the mechanistic question of how two HORMA domains heterodimerize to regulate autophagy.

    Evidence HORMA domain prediction, co-immunoprecipitation mapping, and genetic loss-of-function in Drosophila

    PMID:24895579

    Open questions at the time
    • HORMA domain fold was predicted, not experimentally determined
    • Functional significance of ATG101 homodimerization not resolved
  4. 2015 High

    The crystal structure of the ATG13HORMA–ATG101 heterodimer confirmed a Mad2-like HORMA dimer architecture and revealed that the conserved WF finger motif is sequestered in a hydrophobic pocket, suggesting regulated exposure as a functional switch.

    Evidence X-ray crystallography of human ATG13 HORMA–ATG101 complex with functional analysis of the WF finger

    PMID:26299944

    Open questions at the time
    • What triggers WF finger exposure was unknown
    • Binding partners recognized by the WF finger were not identified
    • No membrane interaction data
  5. 2018 High

    Structural and functional analysis showed that ATG101's C-terminal region undergoes a secondary-structure switch upon ATG13 binding and mediates bridging to the class III PI3K complex, establishing ATG101 as the physical link between the two core autophagy initiation complexes.

    Evidence Crystal structure of ATG101–ATG13HORMA, SEC-SAXS, C-terminal deletion mutants, co-immunoprecipitation, and autophagosome formation assay in mammalian cells

    PMID:30081750

    Open questions at the time
    • Specific PI3K complex subunit contacted by ATG101 C-terminus not identified
    • Regulation of the conformational switch not determined
  6. 2018 Medium

    The finding that PTCH1 physically interacts with ATG101 to block basal autophagic flux independently of Hedgehog/SMO signaling revealed a non-canonical regulatory input into the ULK1 complex through ATG101.

    Evidence Co-immunoprecipitation, SMO-deficient cells, autophagic flux assay

    PMID:29453315

    Open questions at the time
    • Binding interface on ATG101 not mapped
    • Physiological contexts where PTCH1-ATG101 axis is dominant are unclear
    • Not independently replicated
  7. 2019 Medium

    Drosophila Atg101 knockout demonstrated that ATG101 is required for both developmental and starvation-induced autophagy in vivo, with accumulation of ubiquitin-positive aggregates and impaired stem cell differentiation, expanding the functional scope beyond starvation responses.

    Evidence Drosophila loss-of-function mutant with multiple tissue-specific rescue experiments

    PMID:30760524

    Open questions at the time
    • Mechanistic basis for stem cell differentiation defect not defined
    • Whether phenotypes reflect direct autophagy loss or secondary effects unclear
  8. 2021 High

    Two key advances established ATG101 turnover and ULK1-independent functions: HUWE1 was identified as the E3 ligase targeting the ATG101 C-terminus for proteasomal degradation, and ATG9A was shown to interact with the ATG13-ATG101 dimer independently of ULK1, with loss of ATG101 causing ATG9A mis-accumulation at stalled p62/ubiquitin clusters.

    Evidence HUWE1 CRISPR-KO, ubiquitination assay, C-terminal mutants (HUWE1 study); BioID proteomics, KO/reconstitution, split-mVenus dimer capture, ATG9A trafficking assay (ATG9A study)

    PMID:34369648 PMID:34502089

    Open questions at the time
    • Specific ubiquitination sites on ATG101 not mapped
    • How ATG13-ATG101 directs ATG9A vesicle trafficking mechanistically is unresolved
    • Whether HUWE1-mediated degradation is regulated by autophagy-inducing signals unknown
  9. 2025 Medium

    Reconstitution and phylogenetic studies together established that ATG101 is required for ATG13-ATG9A complex formation, that its WF finger inserts into membranes upon WIPI2/3 binding to position ULK1 for substrate phosphorylation, and that a ULK1-phosphorylation-triggered conformational switch in ATG101 drives autocatalytic positive-feedback assembly of the initiation complex.

    Evidence Biochemical reconstitution, molecular dynamics, mutagenesis of WF finger and PVP motif (preprint); interaction kinetics and phosphorylation-driven homo-dimerization assays (preprint); phylogenetic BLAST analysis and yeast genetic epistasis in K. phaffii

    PMID:40931865 PMID:bio_10.1101_2025.06.27.661946 PMID:bio_10.1101_2025.11.07.687251

    Open questions at the time
    • WF finger membrane insertion and autocatalytic feedback are from preprints not yet peer-reviewed
    • In vivo validation of the fold-switch and positive-feedback model is lacking
    • Structural basis of ATG101 homo-dimerization at atomic resolution not determined

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the identity of the specific PI3K complex subunit contacted by ATG101, the structural basis of phosphorylation-triggered ATG101 homo-dimerization and fold switching, and whether ATG101 has functions beyond canonical autophagy initiation in developmental or selective autophagy contexts.
  • No high-resolution structure of ATG101 in the context of the full ULK1 supercomplex on membranes
  • Specific phosphorylation sites on ATG101 driving the conformational switch not mapped in vivo
  • Role of ATG101 in selective autophagy pathways beyond p62-mediated aggrephagy not characterized

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0008289 lipid binding 1
Localization
GO:0005829 cytosol 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-9612973 Autophagy 6
Partners
ATG13ATG9AFIP200HUWE1PTCH1ULK1WIPI2WIPI3
Complex memberships
ATG13HORMA-ATG101 heterodimerATG9A-ATG13-ATG101 complexULK1-ATG13-FIP200-ATG101 complex

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 ATG101 is a novel mammalian protein that directly interacts with ATG13 and associates with the ULK1-ATG13-FIP200 complex. ATG101 is required for autophagy, localizes to the isolation membrane/phagophore, and is important for the stability and basal phosphorylation of ATG13 and ULK1. Co-immunoprecipitation, siRNA knockdown, GFP-ATG101 live imaging, western blot for phosphorylation Autophagy High 19597335
2009 ATG101 interacts with ULK1 in an ATG13-dependent manner and stabilizes ATG13 expression by protecting it from proteasomal degradation. Co-immunoprecipitation, siRNA knockdown, proteasome inhibitor treatment, western blot Autophagy High 19287211
2012 C. elegans ATG101 ortholog EPG-9 directly interacts with EPG-1/ATG13 and is essential for autophagic degradation of protein aggregates; loss of epg-9 phenocopies loss of unc-51/Atg1 and epg-1/Atg13. Genetic screen, loss-of-function mutant analysis, direct protein interaction assay, epistasis Autophagy High 22885670
2014 Drosophila ATG101 dimerizes, is predicted to fold into a HORMA domain, binds the N-terminal HORMA domain of ATG13, and may also interact with two unstructured regions of ATG1. Loss of ATG101 impairs both starvation-induced and basal autophagy. Genetic loss-of-function, mapping/binding assays, HORMA domain prediction, co-immunoprecipitation BioMed research international Medium 24895579
2015 Crystal structure of human ATG13 HORMA domain in complex with full-length ATG101 HORMA domain reveals a heterodimer architecture conserved with the Mad2 conformational heterodimer. The WF finger motif essential for ATG101 function is sequestered in a hydrophobic pocket, suggesting its exposure is regulated, and two conserved hydrophobic pockets suggest additional protein interaction sites. X-ray crystallography, structural comparison, functional analysis of WF finger motif Structure High 26299944
2018 The C-terminal region of ATG101, which adopts a β-strand in free ATG101 but an α-helix or random coil in the ATG101-ATG13HORMA complex, is responsible for binding PtdIns3K complex components. C-terminal deletion of ATG101 impairs interaction with PtdIns3K and autophagosome formation, establishing ATG101 as a bridge between the ULK1 and PtdIns3K complexes. Crystal structure of ATG101-ATG13HORMA complex, SEC-SAXS, C-terminal deletion mutants, co-immunoprecipitation, autophagosome formation assay Autophagy High 30081750
2018 ATG101 physically interacts with the C-terminal domain (CTD) of PTCH1 (Patched1) and connects PTCH1 to the ULK complex; this interaction results in blockade of basal autophagic flux and accumulation of autophagosomes with undegraded cargo, independently of PTCH1's repressive activity on SMO. Co-immunoprecipitation, SMO-deficient cells, SMO inhibitor treatment, autophagic flux assay Molecular cancer research Medium 29453315
2019 Drosophila Atg101 loss-of-function is semi-lethal; both developmental and starvation-induced autophagy are defective in Atg101 mutants, with accumulation of ubiquitin-positive aggregates in brains and impaired intestinal stem cell differentiation in midguts. Drosophila loss-of-function mutant generation, autophagy assays, cell-type specific rescue experiments Journal of biological chemistry Medium 30760524
2021 HUWE1 is an E3 ubiquitin ligase that ubiquitinates ATG101 at its C-terminal region, promoting its proteasomal degradation and thereby suppressing autophagy. HUWE1 depletion stabilizes ATG101 and increases autophagosome formation. Co-immunoprecipitation, CRISPR knockout, siRNA knockdown, ATG101 C-terminal mutant transfection, autophagy flux assay International journal of molecular sciences Medium 34502089
2021 ATG9A interacts with the ATG13-ATG101 dimer independently of ULK1. Deletion of ATG13 or ATG101 causes aberrant accumulation of ATG9A at stalled p62/SQSTM1-ubiquitin clusters, which can be rescued by a ULK1 binding-deficient ATG13 mutant. BioID quantitative proteomics, knockout/reconstitution, split-mVenus ATG13-ATG101 dimer capture, ATG9A distribution assay EMBO reports High 34369648
2025 ATG13:ATG101 HORMA dimer forms a tight complex with the PI3P-binding proteins WIPI3 and WIPI2; when bound to WIPIs, the ATG101 WF finger inserts into the membrane. Molecular dynamics simulations show cooperative stabilization of the complex on membranes. The ULK1 IDR PVP motif docks onto the ATG13:ATG101 HORMA dimer surface, bringing the ULK1 kinase domain close to the membrane. WIPI3:ATG13 engagement is required for ULK1-mediated ATG16L1 phosphorylation, ATG13 puncta formation, and autophagic flux. Biochemical reconstitution, molecular dynamics simulations, cell-based assays, mutagenesis of WF finger and PVP motif, ATG16L1 phosphorylation assay bioRxivpreprint High bio_10.1101_2025.11.07.687251
2025 ATG101 forms an essential autophagy initiation complex with ATG9A and ATG13 (ATG9A-ATG13-ATG101). ATG101 interaction with ATG13 and ATG9A is exceptionally slow, but is dramatically accelerated by a conformational change (fold switch) induced by ATG101 transient homo-dimerization triggered by ULK1-mediated phosphorylation. ATG101 dimers create an auto-catalytic positive feedback that propagates activation to further ATG101 molecules, and the memory of activation persists for hours after dephosphorylation. Interaction kinetics assays, phosphorylation assays, homo-dimerization assays, in vitro reconstitution of complex assembly bioRxivpreprint Medium bio_10.1101_2025.06.27.661946
2025 ATG101 is required for ATG13-ATG9 interaction in mammals; in yeast species that retained ATG101 (K. phaffii), ATG101 participates in Atg1 complex assembly with an additive effect with Atg31 in starvation-induced autophagy. Loss of ATG101 in some Holomycota was enabled by the acquisition of a cap structure in Atg13 that shifted the Atg9 binding site. BLAST phylogenetic analysis, yeast two-hybrid assays, atg101/atg31 double deletion analysis in K. phaffii Autophagy Medium 40931865
2025 ATG101 can be recruited by a hydrophobic-tag-based small molecule degrader (AZ-9) to initiate the autophagy-lysosome pathway, leading to LC3 recruitment, autophagosome formation, and degradation of CDK9 and Cyclin T1. Small molecule degrader design, autophagy pathway inhibitors, western blot, LC3 puncta assay, in vitro and in vivo degradation assays Acta pharmaceutica Sinica. B Medium 40487652

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 45 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 17 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
2018 Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED1 and ATG101. Molecular cancer research : MCR 11 29453315
2025 First ATG101-recruiting small molecule degrader for selective CDK9 degradation via autophagy-lysosome pathway. Acta pharmaceutica Sinica. B 10 40487652
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 5 37567770
2023 miR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy. Heliyon 4 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