Phagophore assembly site (PAS)Cellular Componentphagophore_assembly_site
A phase-separated condensate of Atg proteins, anchored at the vacuolar membrane, at which the autophagy machinery is concentrated and the phagophore is nucleated. Modeled as nucleation, composition, positioning, and turnover.
Context
Saccharomyces cerevisiae
Connections
Scaffold-driven phase separation creates the condensate onto which the membrane component and the downstream machinery are loaded.
Atg17 is the most upstream organizer; the Atg1 complex assembles onto it and phase-separates.
Vac8 tethering both localizes the condensate and, in the selective route, nucleates it by concentrating cargo and early Atg factors.
Atg8 lipidated by the machinery at the site activates Atg1, which then dismantles the Atg1 complex.
Atg8-PE produced on the growing phagophore activates Atg1, closing the negative-feedback loop that disassembles the site.
Atg13 phosphorylation is the off-switch for the condensate: it dissociates the complex and is the same modification that blocks phase separation in vitro.
Part 1: scaffold-driven nucleation of the condensate
Nucleation of the PAS condensateBiological Processpas_nucleation
Autophagy induction dephosphorylates Atg13, letting it bridge Atg1 to the Atg17-Atg31-Atg29 scaffold. The assembled Atg1 complex undergoes liquid-liquid phase separation, and the resulting droplet is the PAS. Mutations or phosphorylation events that block phase separation in vitro block PAS formation in vivo, so the condensate is not incidental to the site - it is the site.
Annotons
Atg17-Atg31-Atg29 scaffold complex
atg17_atg31_atg29_scaffold
Participant: Protein Complex: Atg17-Atg31-Atg29 ternary complex
Protein Complex:
Atg17-Atg31-Atg29 ternary complex Constitutive, dimeric, equimolar ternary complex forming a double-crescent scaffold. No GO cellular-component term exists for it. Fungal-specific; the mammalian ULK1 complex uses FIP200/RB1CC1 in this structural role.
Active units:
Atg17 subunit
Participant: Gene Product: Atg17 (S. cerevisiae)
Role: crescent-shaped scaffold; the most upstream PAS organizer
Atg31 subunit
Participant: Gene Product: Atg31 (S. cerevisiae)
Role: scaffold subunit; phosphorylated form is part of the ternary complex
Atg29 subunit
Participant: Gene Product: Atg29 (S. cerevisiae)
Role: scaffold subunit
Function
molecular condensate scaffold activityGO:0140693 Scaffolding role in organizing the Atg proteins into the PAS condensate. Atg17 targeted ectopically to the plasma membrane recruits the other Atg proteins there. Processes
Locations
PMID:17295840Atg17 is the most upstream PAS organizer and acts as a scaffold.
This analysis suggests that Atg17p is the most basic protein in PAS organization: when it is specifically targeted to the plasma membrane, other Atg proteins are recruited to that location, suggesting that Atg17p acts as a scaffold protein to organize Atg proteins to the PAS.
PMID:19755117Atg17, Atg29, and Atg31 form a constitutive equimolar ternary complex that is a functional unit for autophagosome formation.
Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included.
PMID:23219485Atg17-Atg31-Atg29 assembles with Atg1 and Atg13 on autophagy induction to initiate phagophore formation.
Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore.
Atg1 complex phase separation into the PAS droplet
atg1_complex_phase_separation
Participant: Protein Complex: Atg1/ULK1 kinase complex
Protein Complex:
Atg1/ULK1 kinase complexGO:1990316
Active units:
Atg1 kinase subunit
Participant: Gene Product: Atg1 (S. cerevisiae)
Role: initiating serine/threonine kinase
Function:
protein serine/threonine kinase activityGO:0004674
Atg13 subunit
Participant: Gene Product: Atg13 (S. cerevisiae)
Role: intrinsically disordered bridge between Atg1 and the Atg17 scaffold; its phosphorylation state is the switch for both condensate formation and condensate dissolution
Function
molecular condensate scaffold activityGO:0140693 The Atg1 complex is the phase-separating component; the droplet it forms is the PAS. Locations
PMID:32025038The PAS is a liquid-like condensate of Atg proteins.
Here we show that the PAS is in fact a liquid-like condensate of Atg proteins.
PMID:32025038The Atg1 complex phase-separates in vitro, and mutations or phosphorylation that block phase separation block PAS formation in vivo.
The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo.
Part 2: composition of the assembled site
Composition of the PASCellular Componentpas_composition
Three layers occupy the site: the protein condensate itself, a small amount of vesicular membrane clustered within it, and the downstream machinery recruited onto it in a defined hierarchy. Separating these is the point of the module - conflating the second and third layers is what makes GO:0034045 unusable.
Part 1: membrane component clustered within the site
Atg9 vesicle cluster at the PASCellular Componentpas_membrane_component
A small number of Atg9-containing vesicles assemble individually into the PAS and are ultimately incorporated into the outer autophagosomal membrane. This is the membrane that is genuinely resident at the site, as distinct from the phagophore membrane that grows out of it and from the nearby ER. GO has no term for the Atg9 vesicle or its membrane.
Annotons
Atg9 vesicles as the membrane seed of the site
atg9_vesicle_membrane_seed
Participant: Gene Product: Atg9 (S. cerevisiae)
Gene Product:
Atg9 (S. cerevisiae)UniProtKB:Q12142 The sole transmembrane protein of the core autophagy machinery; its scramblase activity is modeled in MODULE:phagophore_organelle_contact_site, where it acts at the phagophore rim.
Processes
Locations
Supplies the vesicular membrane resident at the PAS. Only a few vesicles are consumed per autophagosome, so this seeds rather than builds the phagophore.
PMID:22826123Atg9 vesicles assemble individually into the PAS and end up in the outer autophagosomal membrane.
During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane.
PMID:22826123Only a few Atg9 vesicles are needed per round of autophagosome formation.
only a few Atg9 vesicles were required for a single round of autophagosome formation
Atg1 EAT-domain tethering of Atg9 vesicles
atg1_eat_vesicle_tethering
Participant: Gene Product: Atg1 (S. cerevisiae)
Function
membrane-curvature-sensing vesicle tethering by the Atg1 EAT domain The C-terminal EAT domain senses membrane curvature, dimerizes, and tethers lipid vesicles, organizing Atg9 vesicles at the site. No GO molecular-function term is asserted for this tethering role.
Locations
PMID:23219485The Atg1 EAT domain senses curvature, dimerizes, and tethers vesicles, suggesting a mechanism for organizing Atg9 vesicles into the early phagophore.
The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles.
Part 2: downstream machinery recruited onto the condensate
Machinery recruited to the PASCellular Componentpas_recruited_machinery
Atg proteins arrive at the site in a hierarchy downstream of Atg17. The class III PI3K complex I writes PI3P there; the Atg2-Atg18 complex reads it and builds the bridge to the ER; and the Atg8 conjugation system lipidates Atg8 on the emerging phagophore. These are listed here as PAS residents - their molecular mechanics belong to other modules.
Annotons
Class III PI3K complex I at the PAS
pas_pi3k_complex_i
Participant: Protein Complex: phosphatidylinositol 3-kinase complex, class III, type I
Protein Complex:
phosphatidylinositol 3-kinase complex, class III, type IGO:0034271
Active units:
Vps34 catalytic subunit
Participant: Gene Product: Vps34 (S. cerevisiae)
Role: PI3P-generating lipid kinase
Vps15 subunit
Participant: Gene Product: Vps15 (S. cerevisiae)
Role: membrane-anchoring regulatory subunit
Vps30/Atg6 subunit
Participant: Gene Product: Vps30/Atg6 (S. cerevisiae)
Role: scaffold subunit (Beclin-1 counterpart)
Atg14 subunit
Participant: Gene Product: Atg14 (S. cerevisiae)
Role: autophagy-specific targeting subunit
Atg38 subunit
Participant: Gene Product: Atg38 (S. cerevisiae)
Role: complex I-specific subunit stabilizing the Atg14-Vps30 arm
Function
1-phosphatidylinositol-3-kinase activityGO:0016303
Products:
1-phosphatidyl-1D-myo-inositol 3-phosphate (PI3P)
Locations
Recruited downstream of the Atg1 complex; supplies the PI3P that the Atg18/WIPI propellers read.
PMID:17295840PI3K complex components occupy a defined position in the PAS recruitment hierarchy downstream of Atg17.
Here, we performed a systematic and quantitative analysis by fluorescence microscopy to develop a hierarchy map of Atg proteins involved in PAS organization.
Atg2-Atg18 complex at the PAS
pas_atg2_atg18
Participant: Protein Complex: Atg2-Atg18 complex
Protein Complex:
Atg2-Atg18 complex PI3P-dependent lipid transfer bridge. Present at the PAS, but its tethering and transfer activities are modeled in MODULE:phagophore_organelle_contact_site.
Active units:
Atg2 subunit
Participant: Gene Product: Atg2 (S. cerevisiae)
Role: ER-phagophore lipid transfer rod
Atg18 subunit
Participant: Gene Product: Atg18 (S. cerevisiae)
Role: PI3P-binding beta-propeller
Function:
phosphatidylinositol-3-phosphate bindingGO:0032266
Locations
The interface between this module and the contact-site module: recruited to the PAS, it builds the bridge to the ER.
PMID:37465355Atg2/ATG2 and Atg18/WIPI4 are among the proteins localized at the phagophore-ER membrane contact sites that form at this site.
In this review, we will focus on the proteins localized at these MCSs, more specifically on Atg9/ATG9A, Atg2/ATG2A/ATG2B and Atg18/WIPI4, and their function during phagophore expansion.
Atg8 conjugation machinery at the PAS
pas_atg8_conjugation
Participant: Protein Complex: Atg12-Atg5-Atg16 complex
Protein Complex:
Atg12-Atg5-Atg16 complexGO:0034274
Active units:
Atg5 subunit
Participant: Gene Product: Atg5 (S. cerevisiae)
Role: Atg12-conjugated subunit carrying the E3-like activity
Atg16 subunit
Participant: Gene Product: Atg16 (S. cerevisiae)
Role: dimerizing subunit that targets the E3-like complex to the membrane
Function
Locations
Lipidates Atg8 on the emerging phagophore. Relevant here because Atg8-PE feeds back on the site: it activates Atg1 and thereby drives PAS turnover.
PMID:34798055Lipidated Atg8 activates Atg1 on the growing autophagosomal membrane.
we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane.
Part 3: positioning and membrane anchoring of the site
Positioning of the PAS at the vacuolar membraneRegulatory Steppas_positioning
The PAS condensate is not free-floating. Vac8 tethers it to the vacuolar membrane through Atg13 and holds it there for the whole of autophagosome biogenesis, creating a confined space between the ER and the vacuole. Tethering is by specific protein-protein interaction with the droplet, which is what explains the vacuolar localization of the site. The vacuolar membrane is not intrinsically special: ectopic Vac8 redirects autophagosome formation to the nuclear membrane.
Annotons
Vac8 anchoring of the PAS to the vacuolar membrane
vac8_pas_anchor
Participant: Gene Product: Vac8 (S. cerevisiae)
Function
vacuolar membrane tether and nucleation hub for the PAS Armadillo-repeat tether that anchors the PAS via Atg13 and, in selective autophagy, recruits the cargo complex via Atg11 and the PI3K complex. No exact GO molecular-function term is asserted; GO's tether-activity terms are ER-centric. Locations
PMID:31649143Vac8 is a vacuolar tether that anchors the PAS via Atg13 throughout autophagosome biogenesis.
We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13.
PMID:34893607Vac8 is a central hub that nucleates the PAS at the vacuolar membrane in selective autophagy, recruiting cargo via Atg11 and the PI3K complex.
Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy.
PMID:34893607The vacuolar membrane is not specifically required - ectopic Vac8 redirects autophagosome formation to the nuclear membrane.
Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required.
Protein-protein tethering of the Atg1 droplet to membrane
atg1_droplet_membrane_tethering
Participant: Protein Complex: Atg1/ULK1 kinase complex
Reconstituted Atg1-complex droplets are tethered to membranes by specific protein-protein interactions, which accounts for the vacuolar localization of the PAS in cells.
PMID:32025038Atg1-complex droplets tether to membranes via specific protein-protein interactions, explaining PAS vacuolar localization.
In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo.
Part 4: turnover and disassembly of the site
Turnover and disassembly of the PASRegulatory Steppas_turnover
The site takes itself apart. Atg1, activated by Atg8-PE on the growing phagophore, phosphorylates Atg13; that phosphorylation dissociates the Atg1 complex and drives rapid turnover of its subunits at the PAS. The same phosphorylation that blocks Atg1-complex phase separation in vitro is therefore the physiological off-switch for the condensate, closing a negative-feedback loop from the product membrane back onto the site that made it.
Annotons
Atg1-dependent Atg13 phosphorylation and Atg1 complex dissociation
atg1_atg13_phosphorylation_dissolution
Participant: Gene Product: Atg1 (S. cerevisiae)
Function
protein serine/threonine kinase activityGO:0004674 Processes
Locations
Disassembly mechanism of the site: phosphorylated Atg13 no longer holds the complex together, so subunits exchange rapidly rather than remaining as a stable condensate.
PMID:34798055Atg1-dependent Atg13 phosphorylation dissociates the Atg1 complex and drives rapid subunit turnover at the PAS.
Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS).
PMID:32025038Phosphorylation that inhibits Atg1-complex phase separation impairs PAS formation, linking the same modification to condensate dissolution.
point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo
Variant set: Scaffolding route that nucleates the site by induction route / cargo selectivity (Exactly One)
The two routes build the same kind of condensate at the same place but use different upstream scaffolds, and the Atg17-Atg31-Atg29 complex is specifically required for the starvation-induced route.
Starvation-induced bulk autophagy (Atg17-scaffolded)Cellular Componentbulk_starvation_route
Nitrogen starvation and TORC1 inactivation dephosphorylate Atg13, which then bridges Atg1 to the Atg17-Atg31-Atg29 scaffold. Cargo is non-selective.
Annotons
Atg17-Atg31-Atg29 requirement for the starvation route
bulk_route_atg17_requirement
Participant: Gene Product: Atg17 (S. cerevisiae)
Locations
Scaffold whose requirement is specific to starvation-induced autophagy; selective autophagy can proceed without it.
PMID:19755117Atg17, Atg29, and Atg31 are specifically required for starvation-induced autophagosome formation and for PAS organization.
Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization.
Selective autophagy and the Cvt pathway (Atg11-scaffolded)Cellular Componentselective_cargo_route
Cargo-bound receptors recruit the Atg11 scaffold, which Vac8 holds at the vacuolar membrane; cargo-dependent clustering plus Vac8-dependent sequestering of early Atg factors, with local Atg1 activation, builds the site. Operates constitutively rather than on starvation.
cytoplasm to vacuole targeting by the Cvt pathwayGO:0032258
Annotons
Atg11 cargo-linked scaffold
atg11_cargo_scaffold
Participant: Gene Product: Atg11 (S. cerevisiae)
Function
protein-macromolecule adaptor activityGO:0030674 Links cargo-receptor complexes to Vac8 and to the Atg1 machinery, taking the structural role that Atg17 plays in the starvation route. Processes
cytoplasm to vacuole targeting by the Cvt pathwayGO:0032258 Locations
PMID:34893607Vac8 recruits the cargo complex via the Atg11 scaffold to nucleate the PAS in selective autophagy.
Vac8 directly recruits the cargo complex via the Atg11 scaffold.
PMID:34893607Cargo clustering plus Vac8-dependent sequestering of early Atg factors and local Atg1 activation promote PAS assembly.
Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole.