{"gene":"YKT6","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2001,"finding":"Mammalian YKT6 forms a SNARE complex with syntaxin 5, GS28, and Bet1, localizes primarily to Golgi membranes, and functions at a late stage in ER-to-Golgi transport; antibodies against YKT6 inhibit in vitro ER-Golgi transport of VSVG before the EGTA-sensitive stage, and microinjection of YKT6 antibodies fragments the Golgi apparatus.","method":"Co-immunoprecipitation, in vitro ER-Golgi transport assay, antibody inhibition, microinjection, immunofluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus in vitro transport assay with antibody inhibition, two orthogonal methods in a single study","pmids":["11323436"],"is_preprint":false},{"year":2001,"finding":"In yeast, YKT6 (R-SNARE) acts as a multicopy and low-copy suppressor of vti1-2 defects, functionally interacting with VTI1 in transport to the prevacuole and vacuole; YKT6 participates in SNARE complexes containing Pep12p and Vam3p/Vam7p. Mutation of the zero ionic layer arginine (ykt6-R165Q) renders these complexes nonfunctional, establishing that arginine in the 0-layer is essential.","method":"Genetic suppressor screen, double-mutant analysis, in vivo transport assays, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis combined with mutagenesis of the catalytic zero-layer residue, replicated across multiple transport steps","pmids":["11445562"],"is_preprint":false},{"year":2002,"finding":"GS15 forms a distinct SNARE complex with syntaxin 5, GS28, and Ykt6 in the medial-cisternae of the Golgi; co-immunoprecipitation of COPI coat components with GS15 from Golgi extracts links this complex to early Golgi trafficking.","method":"Co-immunoprecipitation, immuno-electron microscopy, siRNA knockdown, dominant-negative overexpression","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, immuno-EM localization, and functional siRNA knockdown, multiple orthogonal methods in one study","pmids":["12388752"],"is_preprint":false},{"year":2003,"finding":"The yeast SNARE Ykt6 mediates palmitoylation of the vacuolar fusion factor Vac8 via its N-terminal longin domain, which presents palmitoyl-CoA (Pal-CoA) to Vac8; transfer to Vac8's SH4 domain occurs spontaneously rather than enzymatically. This acyltransferase activity operates during a Sec17-independent subreaction of vacuole fusion controlled by Sec18.","method":"In vitro vacuole fusion assay, palmitoylation assay, domain mutagenesis, biochemical reconstitution","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of palmitoylation activity with domain mutagenesis, single lab but multiple orthogonal methods","pmids":["14685280"],"is_preprint":false},{"year":2003,"finding":"Rat neuronal Ykt6 localizes to a specialized punctate compartment distinct from conventional endomembrane markers; targeting to this compartment is directed by the profilin-like longin domain even in the absence of prenylation. Cytosolic Ykt6 is conformationally inactive for SNARE complex assembly, suggesting autoinhibition.","method":"Immunofluorescence microscopy, subcellular fractionation, domain deletion/mutagenesis, SNARE complex assembly assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with domain mutagenesis and SNARE assembly assays, single lab","pmids":["12589064"],"is_preprint":false},{"year":2004,"finding":"Both cytosolic and membrane-bound mammalian Ykt6 are farnesylated at the C-terminal CCAIM cysteine; farnesylation is a prerequisite for subsequent palmitoylation of the upstream cysteine, enabling stable membrane association. The double lipid modification (farnesyl + palmitoyl) is essential for intra-Golgi transport in vitro and cell survival in vivo. The N-terminal longin domain interacts with the SNARE motif, maintaining Ykt6 in an inactive closed conformation that controls membrane recruitment and palmitoylation.","method":"Metabolic labeling, in vitro intra-Golgi transport assay, site-directed mutagenesis of CAAX cysteines, cell viability assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of Golgi transport combined with lipid modification biochemistry and mutagenesis, multiple orthogonal methods","pmids":["15044687"],"is_preprint":false},{"year":2004,"finding":"Human Ykt6 has intrinsic self-palmitoylating activity: incubation of recombinant hYkt6 with [3H]Pal-CoA leads to covalent attachment of palmitate to C-terminal cysteine residues. The N-terminal longin domain contains a Pal-CoA binding site and is required for the reaction.","method":"In vitro palmitoylation assay with [3H]palmitoyl-CoA and recombinant protein, domain deletion","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic assay with radiolabeled substrate and domain mutagenesis, single lab","pmids":["15479160"],"is_preprint":false},{"year":2004,"finding":"In mammalian neuronal Ykt6, the longin domain controls conformation and subcellular targeting through intramolecular protein-protein interactions with the SNARE motif and protein-lipid interactions with C-terminal lipid groups. Two hydrophobic pockets on opposite faces of the longin domain participate; one suppresses palmitoylation-dependent mislocalization to the plasma membrane. Both protein-protein and protein-lipid intramolecular interactions are required for a tightly closed, autoinhibited conformation.","method":"Site-directed mutagenesis of longin domain surface residues, immunofluorescence localization, cell fractionation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-guided mutagenesis combined with direct localization experiments, single lab","pmids":["15331663"],"is_preprint":false},{"year":2004,"finding":"Syntaxin 5, GS28, Ykt6, and GS15 function as a SNARE complex mediating transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN); antibodies to each of these four SNAREs specifically inhibit STxB transport in vitro. GS15 and Ykt6 redistribute from the Golgi to endosomes when the recycling endosome is perturbed, indicating they cycle between these compartments.","method":"In vitro EE/RE-to-TGN transport assay with STxB, antibody inhibition, siRNA knockdown of GS15, overexpression of SNX3 to perturb recycling endosomes, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro transport assay with antibody inhibition validated by siRNA knockdown and morphological redistribution, multiple orthogonal methods","pmids":["15215310"],"is_preprint":false},{"year":2005,"finding":"Ykt6 is released from yeast vacuolar membranes during an early stage of homotypic vacuole fusion in a manner dependent on SNARE disassembly (priming by Sec18). Yeast Ykt6 undergoes palmitoylation at its C-terminal CAAX motif in vitro; mutagenesis of the palmitoylation site prevents stable membrane association and is lethal, indicating depalmitoylation drives Ykt6 cycling between membranes and cytosol.","method":"In vitro vacuole fusion assay, [3H]palmitate labeling, site-directed mutagenesis of palmitoylation site, cell viability assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of vacuole fusion combined with palmitoylation biochemistry and lethality of mutagenesis, multiple methods","pmids":["15723044"],"is_preprint":false},{"year":2008,"finding":"The Ykt6 longin domain–SNARE domain intramolecular interaction controls cycling between cytosol and membranes; a mutant deficient in this interaction accumulates on membranes and is not released from vacuoles. Ykt6 is a substrate of the DHHC acyltransferase network; overexpression of the vacuolar acyltransferase Pfa3 drives a constitutively membrane-associated Ykt6 mutant into the vacuolar lumen via the MVB pathway, showing that depalmitoylation and release are required to prevent entry into the MVB pathway.","method":"Site-directed mutagenesis of longin-SNARE interface, overexpression of Pfa3 acyltransferase, vacuole isolation and fractionation, fluorescence microscopy","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis and acyltransferase overexpression with organelle fractionation, single lab, two orthogonal approaches","pmids":["18541004"],"is_preprint":false},{"year":2008,"finding":"In vitro farnesylation of the C-terminal CAAX box of recombinant Ykt6 stabilizes the native protein, increases helical content, and promotes a more compact structure. The farnesyl moiety folds onto a hydrophobic groove in the longin domain, consistent with a closed autoinhibited conformation; the crystal structure of the yeast Ykt6 longin domain (residues 1–140) was determined at 2.5 Å resolution.","method":"In vitro farnesylation assay, size exclusion chromatography, limited proteolysis, circular dichroism spectroscopy, surface plasmon resonance, X-ray crystallography","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with multiple biophysical assays (CD, SPR, proteolysis) and in vitro enzymatic modification in one study","pmids":["18329045"],"is_preprint":false},{"year":2016,"finding":"Single-molecule FRET and fluorescence cross-correlation spectroscopy reveal that rat Ykt6 undergoes intramolecular conformational dynamics between its longin domain and SNARE core at a timescale of ~200 μs. The presence of the lipid dodecylphosphocholine (DPC) regulates and can eliminate these dynamics, locking Ykt6 in a closed conformation; molecular dynamics simulations show that the SNARE core is flexible while the longin domain is relatively stable in the apo state.","method":"Single-molecule FRET, fluorescence cross-correlation spectroscopy (FCCS), molecular dynamics simulation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — single-molecule biophysical measurements combined with MD simulation, single lab, two orthogonal methods","pmids":["27493064"],"is_preprint":false},{"year":2017,"finding":"RNAi depletion of YKT6 in Drosophila cells blocks constitutive secretory carrier fusion with the plasma membrane; YKT6 participates in at least two SNARE complexes mediating Golgi-to-PM transport (STX1/SNAP24-29/YKT6 and STX4/SNAP24/Syb). RNAi depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion, establishing an evolutionarily conserved role.","method":"Quantitative secretion assay, combinatorial RNAi in Drosophila cells, RNAi in mammalian cells","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative loss-of-function assay replicated across Drosophila and mammalian cells, single lab","pmids":["28403141"],"is_preprint":false},{"year":2018,"finding":"In mammalian cells, YKT6 is an autophagosomal SNARE that mediates autophagosome-lysosome fusion independently of STX17. YKT6 forms a SNARE complex with SNAP29 and lysosomal STX7. Recruitment of YKT6 to autophagosomes requires its N-terminal longin domain but not C-terminal palmitoylation/farnesylation (which are required for Golgi localization). YKT6 depletion completely blocks autophagosome-lysosome fusion in STX17 KO cells, indicating two independent SNARE complexes mediate this fusion.","method":"SNARE screen by siRNA, STX17 CRISPR KO, YKT6 depletion, co-immunoprecipitation of SNARE complex (YKT6/SNAP29/STX7), domain mutant analysis, autophagy flux assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, CRISPR KO epistasis, domain mutagenesis, and functional autophagy flux measurements, multiple orthogonal methods","pmids":["29789439"],"is_preprint":false},{"year":2018,"finding":"In Drosophila, Ykt6 is required for autophagosome-lysosome fusion and localizes to lysosomes and autolysosomes. Ykt6 forms a SNARE complex with Syx17 and Snap29 but can be outcompeted by Vamp7; Vamp7 overexpression rescues the fusion defect of ykt6 loss-of-function cells. A zero-ionic-layer mutation (R→Q) in Ykt6 does not impair autophagic activity, whereas palmitoylation/farnesylation site mutations do, supporting a non-canonical regulatory (non-fusogenic) role for Ykt6 in this complex.","method":"Drosophila genetics (loss-of-function mutants, transgenic rescue), Co-immunoprecipitation, immunofluorescence localization, Vamp7 overexpression rescue, site-directed mutagenesis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with rescue experiments, Co-IP, localization, and mutagenesis across multiple orthogonal methods in vivo","pmids":["29694367"],"is_preprint":false},{"year":2018,"finding":"In yeast, Ykt6 is the autophagosomal SNARE required for autophagosome-vacuole fusion. A novel in vitro fusion assay using intact autophagosomes and vacuoles demonstrated that Ykt6 localizes to the autophagosome side of the fusion machinery, and that this process requires ATP, physiological temperature, HOPS tethering complex, Ypt7 GTPase, and Mon1-Ccz1 GEF.","method":"Novel in vitro autophagosome-vacuole fusion assay, SNARE localization by biochemical fractionation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of fusion with intact organelles, direct assignment of Ykt6 as autophagosomal SNARE, single lab rigorous study","pmids":["30097515"],"is_preprint":false},{"year":2019,"finding":"During lysosomal stress, cytosolic ykt6 (normally autoinhibited by a farnesyl-mediated regulatory mechanism) activates and redistributes to membranes to promote lysosomal hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, disabling this lysosomal stress response. Farnesyltransferase inhibitors restore ykt6 activity and reduce α-synuclein in patient-derived neurons and mice.","method":"Membrane fractionation, co-immunoprecipitation of α-synuclein with ykt6, patient-derived iPSC neurons, farnesyltransferase inhibitor treatment, lysosomal activity assays, mouse in vivo experiments","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of α-syn/ykt6, farnesylation-dependent mechanism validated in patient neurons and in vivo mouse models, multiple orthogonal methods","pmids":["31648898"],"is_preprint":false},{"year":2020,"finding":"In yeast, Ykt6 is recruited to autophagosomes at an early stage of their formation via a mechanism dependent on the ER-resident Dsl1 complex and COPII-coated vesicles. The Atg1 kinase complex directly phosphorylates Ykt6 on autophagosomes to keep it inactive; dephosphorylation of Ykt6 allows its engagement in autophagosome-vacuole fusion.","method":"In vitro kinase assay (Atg1 phosphorylation of Ykt6), genetic epistasis with Dsl1 complex and COPII mutants, fluorescence microscopy, autophagy flux assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay combined with genetic epistasis and imaging, single lab, multiple orthogonal methods","pmids":["33025734"],"is_preprint":false},{"year":2020,"finding":"In Drosophila wing epithelium, most Ykt6 is cytosolic but is recruited to de-acidified endosomal compartments where it recycles Wnt/Wingless to the plasma membrane via Rab4-positive recycling endosomes; this recycling is required for proper Wnt secretion. Proximity-dependent proteomics and biochemical analyses confirmed Ykt6 interactions within endosomal compartments.","method":"In vivo Drosophila genetics, proximity-dependent proteomics (BioID), immunofluorescence co-localization with Rab4, membrane fractionation, Wnt trafficking assays","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetics and proximity proteomics with Rab4 co-localization and functional Wnt trafficking readout, single lab","pmids":["32611603"],"is_preprint":false},{"year":2020,"finding":"Phosphorylation of Ykt6 SNARE domain serine residues drives the conformational switch from a closed cytosolic form to an open membrane-bound form; phosphorylation mediates Ykt6 recruitment to several organelle membranes and functionally regulates Wnt protein trafficking and extracellular vesicle secretion.","method":"Phosphomimetic and phospho-dead mutagenesis, proximity-dependent labeling (BioID), membrane fractionation, Wnt trafficking assay, extracellular vesicle quantification","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphomimetic mutagenesis combined with proximity labeling and functional trafficking assays, single lab","pmids":["33207719"],"is_preprint":false},{"year":2021,"finding":"In mammalian cells, Ykt6 is phosphorylated at an evolutionarily conserved site regulated by Ca2+ signaling; this phosphorylation triggers a conformational change from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways and enhanced toxicity in Parkinson's disease models.","method":"Mass spectrometry identification of phosphorylation site, NMR structural analysis, phosphomimetic/phospho-dead mutagenesis, Ca2+ signaling modulation, Co-IP of interaction partners, autophagy and secretory pathway assays, PD model toxicity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure combined with phosphorylation site identification, mutagenesis, and functional pathway assays, multiple orthogonal methods in one rigorous study","pmids":["33723042"],"is_preprint":false},{"year":2021,"finding":"Double prenylation of Ykt6 (farnesylation of Cys195 by farnesyltransferase followed by geranylgeranylation of Cys194 by a novel GGTase-III consisting of PTAR1/Bet2) is required for efficient trafficking of lysosomal hydrolases (cathepsin D and β-hexosaminidase) from the trans-Golgi network to lysosomes. In PTAR1 KO cells (singly farnesylated Ykt6), hydrolases are missorted and secreted extracellularly, their maturation is impaired, and LC3B accumulates indicating autophagic defects.","method":"PTAR1 knockout cells, lysosomal hydrolase secretion assay, hydrolase maturation analysis, LC3B accumulation by immunoblot, in vitro prenylation assays","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO combined with biochemical prenylation analysis and functional hydrolase trafficking assays, replicated across multiple readouts","pmids":["33035318"],"is_preprint":false},{"year":2023,"finding":"ULK1 (mammalian Atg1 ortholog) phosphorylates YKT6 on autophagosomes, preventing premature SNARE complex assembly with lysosomal SNAREs and inhibiting autophagosome-lysosome fusion; this regulation is conserved in yeast, mammalian cells, and C. elegans. Alterations in YKT6 phosphorylation status produce both early and late autophagy defects and reduce cellular survival.","method":"In vitro ULK1 kinase assay, phosphomimetic/phospho-dead YKT6 mutants, autophagy flux assays in mammalian cells and C. elegans, survival assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay confirmed with phospho-mutant phenotypes replicated across three organisms (yeast, mammals, nematodes)","pmids":["36644903"],"is_preprint":false},{"year":2023,"finding":"In Parkinson's disease patient iPSC-derived midbrain neurons, chronic endogenous α-synuclein accumulation directly impairs autophagosome-lysosome fusion by blocking ykt6-SNAP-29 complex formation. ykt6 depletion causes near-complete block of autophagic flux in human neurons. Increased farnesyltransferase activity in PD suppresses active (membrane-associated) ykt6; farnesyltransferase inhibitors restore autophagic flux by promoting active ykt6.","method":"iPSC-derived midbrain neuron culture from PD patients, Co-immunoprecipitation of ykt6-SNAP29 complex, autophagic flux assays, farnesyltransferase activity measurement, FTase inhibitor treatment in neurons and mice","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of endogenous complex in patient-derived neurons, loss-of-function with defined phenotype, pharmacological intervention replicated in mice","pmids":["36788031"],"is_preprint":false},{"year":2024,"finding":"YKT6 forms a priming SNARE complex with STX17 and SNAP29 on autophagosomes via its SNARE domain, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex to form the fusogenic STX17-SNAP29-VAMP8 complex. The YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, indicating YKT6 plays a priming (not direct fusogenic) role for efficient autophagosome-lysosome fusion.","method":"Co-immunoprecipitation of YKT6/STX17/SNAP29 complex, VAMP8 displacement assay, in vitro lipid mixing and content mixing fusion assays, domain mutagenesis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro lipid and content mixing reconstitution assays combined with Co-IP and competitive displacement experiments, single lab, multiple orthogonal methods","pmids":["38340317"],"is_preprint":false},{"year":2024,"finding":"In yeast, Ykt6 and Nyv1 are functionally redundant R-SNAREs in homotypic vacuole fusion; a ykt6-104 nyv1Δ double mutant exhibits highly fragmented vacuoles while neither single mutant does. Ykt6 can also substitute for exocytic R-SNAREs Snc1/Snc2 when those lose the ability to assemble into exocytic SNARE complexes, indicating Ykt6 can function as a backup R-SNARE maintaining robustness of the vesicular transport network.","method":"Yeast genetic double-mutant analysis, vacuole morphology by fluorescence microscopy, SNARE complex assembly assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with double mutants and SNARE complex assembly assays, single lab","pmids":["38588809"],"is_preprint":false},{"year":2025,"finding":"In budding yeast, the uncharacterized protein Ecm9 is the functional α subunit of yeast GGTase-III; Ecm9 forms a complex with Bet2 and transfers a geranylgeranyl group to mono-farnesylated Ykt6. MALDI-TOF/TOF mass spectrometry confirmed double prenylation (farnesyl + geranylgeranyl) of Ykt6 in wild-type but not ecm9Δ cells. Loss of Ecm9 impairs Ykt6 localization to organelle membranes including autophagosomes, reduces autophagic activity, and causes Golgi mannosyltransferase mislocalization and cell wall fragility.","method":"Structural prediction, in vitro prenylation assay with recombinant Ecm9/Bet2 complex, MALDI-TOF/TOF mass spectrometry of Ykt6 prenylation state, ecm9Δ yeast phenotyping, immunofluorescence of Ykt6 localization and mannosyltransferases, autophagy flux assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — MS confirmation of double prenylation, in vitro enzymatic reconstitution, and genetic KO phenotypes across multiple readouts in one study","pmids":["40049413"],"is_preprint":false},{"year":2025,"finding":"Ykt6 is highly expressed in the mammalian hippocampus, localizes to synaptic spines, and is required for LTP-dependent insertion of GluA1 and GluA2 AMPA receptor subunits at the postsynaptic membrane. Loss of Ykt6 function alters synaptic vesicle pool dynamics and the amplitude and frequency of miniature excitatory postsynaptic currents, modulates spine morphology, and impairs LTP.","method":"Immunofluorescence localization in hippocampal neurons, loss-of-function with defined synaptic phenotype, surface GluA1/GluA2 expression assay, mEPSC electrophysiology, spine morphology analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct synaptic localization and loss-of-function with multiple electrophysiological and biochemical readouts, single lab","pmids":["40840626"],"is_preprint":false},{"year":2025,"finding":"Elevated α-synuclein reduces membrane-associated YKT6, impairing YKT6-mediated extracellular vesicle (exosome) secretion in H4 cells and iPSC-derived dopaminergic neurons. Pharmacological farnesyltransferase inhibition (FTI) decreases both membrane-associated YKT6 and EV secretion, establishing that farnesylation-dependent membrane association of YKT6 is required for EV secretion and that α-synuclein disrupts this by reducing membrane-associated YKT6.","method":"α-Synuclein-inducible H4 cells and iPSC-derived dopaminergic neurons, nanoparticle tracking analysis of EVs, membrane fractionation of YKT6, farnesyltransferase inhibitor treatment","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic link of α-syn to YKT6 membrane localization and EV secretion confirmed in two cell models with pharmacological intervention, single lab","pmids":["39794126"],"is_preprint":false},{"year":2018,"finding":"In mammalian prostate epithelial cells, Ykt6 acts as a negative regulator of cell migration and invasion by upregulating microRNA-145, which decreases Junctional Adhesion Molecule A (JAM-A) expression, thereby reducing Rap1 and Rac1 GTPase activity and attenuating cell spreading and motility.","method":"Ykt6 overexpression/knockdown in prostate epithelial cells, migration and invasion assays, miR-145 quantification, JAM-A protein level measurement, Rap1/Rac1 activity assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect pathway (via miRNA upregulation) with functional readouts but no direct biochemical link between Ykt6 and miR-145 induction mechanism","pmids":["30010460"],"is_preprint":false}],"current_model":"YKT6 is an evolutionarily conserved, lipid-anchored R-SNARE that lacks a transmembrane domain and instead cycles between an autoinhibited closed cytosolic form (farnesylated only) and an active open membrane-bound form (doubly prenylated by farnesyltransferase and GGTase-III, plus palmitoylated); this conformational switch is regulated by intramolecular longin domain–SNARE motif interactions, double prenylation state, and phosphorylation by Atg1/ULK1 kinases. YKT6 participates in multiple distinct SNARE complexes mediating: (1) late ER-to-Golgi and intra-Golgi transport (with STX5/GS28/Bet1 or GS15); (2) endosome-to-TGN recycling (with STX5/GS28/GS15); (3) constitutive secretory carrier fusion with the plasma membrane; (4) autophagosome-lysosome fusion, where it first forms a priming complex with STX17/SNAP29 on autophagosomes before being displaced by VAMP8 for the fusogenic step, and also independently forms a fusogenic complex with SNAP29/STX7; YKT6 additionally serves as a palmitoyl-CoA-presenting acyltransferase for Vac8/palmitoylation events at the vacuole. Farnesylation-dependent membrane association of YKT6 is also essential for lysosomal hydrolase trafficking, exosome secretion, and hippocampal AMPA receptor insertion during LTP, while α-synuclein pathologically inhibits YKT6 activity to impair autophagosomal–lysosomal fusion and cellular clearance in Parkinson's disease models."},"narrative":{"mechanistic_narrative":"YKT6 is an evolutionarily conserved R-SNARE that, uniquely among SNAREs, lacks a transmembrane domain and instead cycles between an autoinhibited cytosolic pool and an active membrane-bound pool to drive fusion at multiple steps of the secretory and degradative pathways [PMID:11323436, PMID:12589064, PMID:15044687]. Membrane recruitment is governed by C-terminal lipidation: farnesylation of the CAAX cysteine is a prerequisite for palmitoylation of the upstream cysteine, and this double lipid modification, together with a longin domain–SNARE motif intramolecular interaction that maintains a closed conformation, controls stable membrane association and activity [PMID:15044687, PMID:18329045]. Efficient targeting further requires double prenylation, in which the singly farnesylated protein is geranylgeranylated by a GGTase-III (PTAR1/Bet2 in mammals; Ecm9/Bet2 in yeast), a step needed for lysosomal hydrolase trafficking from the TGN and for organelle membrane localization including autophagosomes [PMID:33035318, PMID:40049413]. The closed–open conformational switch is additionally driven by phosphorylation of SNARE-domain serines, including a Ca2+-regulated site, which redistributes YKT6 to membranes and reshapes its interactome [PMID:33207719, PMID:33723042]. In its open form YKT6 assembles into distinct SNARE complexes mediating late ER-to-Golgi and intra-Golgi transport (with syntaxin 5, GS28, Bet1 or GS15), endosome-to-TGN recycling, and constitutive secretory carrier fusion at the plasma membrane [PMID:11323436, PMID:12388752, PMID:15215310, PMID:28403141]. At the autophagosome, YKT6 forms a priming complex with STX17 and SNAP29 that is subsequently displaced by VAMP8 for the fusogenic step, and it can also act in a STX17-independent complex with SNAP29 and STX7, with its activity gated by ULK1/Atg1 phosphorylation that prevents premature complex assembly [PMID:29789439, PMID:36644903, PMID:38340317]. Beyond fusion, the yeast longin domain presents palmitoyl-CoA to the vacuolar factor Vac8, conferring a non-enzymatic acyltransferase-like activity [PMID:14685280, PMID:15479160]. YKT6 also supports exosome secretion and hippocampal AMPA receptor insertion during LTP [PMID:40840626, PMID:39794126], and pathological α-synuclein binds and deactivates YKT6 to block YKT6–SNAP29 complex formation and impair autophagosome-lysosome fusion in Parkinson's disease neuron models, a defect reversible by farnesyltransferase inhibitors [PMID:31648898, PMID:36788031].","teleology":[{"year":2001,"claim":"Established YKT6 as a functional SNARE in the early secretory pathway, answering where this R-SNARE acts and with which partners.","evidence":"Co-IP and in vitro ER-Golgi transport assay with antibody inhibition in mammalian cells, plus genetic suppressor analysis in yeast","pmids":["11323436","11445562"],"confidence":"High","gaps":["Did not resolve how YKT6 associates with membranes without a transmembrane domain","Mechanism of cycling between compartments unaddressed"]},{"year":2002,"claim":"Defined a distinct GS15-containing SNARE complex, showing YKT6 participates in more than one Golgi fusion complex.","evidence":"Reciprocal Co-IP, immuno-EM, and siRNA knockdown in Golgi extracts","pmids":["12388752"],"confidence":"High","gaps":["Functional discrimination between GS15 and Bet1 complexes not fully resolved"]},{"year":2003,"claim":"Revealed a non-fusion biochemical activity, with the longin domain presenting palmitoyl-CoA to Vac8, broadening YKT6's molecular repertoire.","evidence":"In vitro vacuole fusion and palmitoylation assays with domain mutagenesis in yeast","pmids":["14685280"],"confidence":"High","gaps":["Whether this acyltransferase-like activity operates on substrates beyond Vac8 unknown","Relevance in mammalian cells not established"]},{"year":2004,"claim":"Established the lipidation hierarchy and autoinhibition model, explaining how a TM-less SNARE achieves regulated membrane association.","evidence":"Metabolic labeling, in vitro intra-Golgi transport, recombinant self-palmitoylation assays, and longin-domain surface mutagenesis in mammalian/neuronal cells","pmids":["15044687","15479160","15331663","12589064"],"confidence":"High","gaps":["Trigger for opening the closed conformation in vivo not identified","Identity of palmitoyltransferase in vivo unresolved"]},{"year":2005,"claim":"Demonstrated that depalmitoylation drives YKT6 cycling off membranes, linking lipid turnover to its trafficking life cycle.","evidence":"In vitro vacuole fusion, [3H]palmitate labeling, and lethal palmitoylation-site mutagenesis in yeast","pmids":["15723044"],"confidence":"High","gaps":["Depalmitoylating enzyme not identified","Coupling of disassembly to depalmitoylation mechanistically incomplete"]},{"year":2008,"claim":"Provided structural and biophysical proof of the closed autoinhibited conformation, with the farnesyl group folding into the longin domain.","evidence":"X-ray crystallography of the yeast longin domain plus CD, SPR, limited proteolysis, and acyltransferase overexpression in yeast","pmids":["18329045","18541004"],"confidence":"High","gaps":["No structure of the full-length open membrane-bound form","How conformational opening is triggered physiologically unresolved"]},{"year":2016,"claim":"Quantified the conformational dynamics of the longin–SNARE switch and showed lipid environment locks the closed state.","evidence":"Single-molecule FRET, FCCS, and MD simulation on rat YKT6","pmids":["27493064"],"confidence":"Medium","gaps":["Dynamics measured in detergent/lipid mimics, not native membranes","Link between observed microsecond dynamics and SNARE engagement not directly demonstrated"]},{"year":2017,"claim":"Extended YKT6 function to constitutive secretory fusion at the plasma membrane, showing a conserved exocytic role.","evidence":"Quantitative secretion assays with combinatorial RNAi in Drosophila and mammalian cells","pmids":["28403141"],"confidence":"Medium","gaps":["Direct demonstration of the proposed PM SNARE complexes biochemically incomplete","Regulation of YKT6 recruitment to secretory carriers unaddressed"]},{"year":2018,"claim":"Identified YKT6 as an autophagosomal SNARE acting independently of STX17, redefining the autophagosome-lysosome fusion machinery.","evidence":"siRNA SNARE screen, STX17 CRISPR KO epistasis, Co-IP, and autophagy flux assays in mammalian cells; genetics and in vitro fusion in Drosophila and yeast","pmids":["29789439","29694367","30097515"],"confidence":"High","gaps":["Whether YKT6 acts fusogenically or regulatorily in autophagy not yet settled at this stage","Relationship between STX7- and STX17-containing complexes unclear"]},{"year":2019,"claim":"Linked YKT6 to a lysosomal stress response and to Parkinson's disease, showing α-synuclein deactivates YKT6 and FTase inhibitors rescue it.","evidence":"Membrane fractionation, Co-IP of α-synuclein, patient iPSC neurons, and FTase inhibitor treatment in neurons and mice","pmids":["31648898"],"confidence":"High","gaps":["Structural basis of α-synuclein–YKT6 interaction not defined","How α-synuclein binding alters lipidation or conformation unresolved"]},{"year":2020,"claim":"Established phosphorylation as a conformational switch and identified the kinases and recruitment machinery controlling YKT6 activation.","evidence":"Phosphomimetic mutagenesis, BioID, in vitro Atg1 kinase assays, and genetic epistasis with Dsl1/COPII in yeast and Drosophila","pmids":["33207719","33025734","32611603"],"confidence":"Medium","gaps":["Phosphatases reversing YKT6 phosphorylation not identified","Cross-talk between phosphorylation and lipidation states incompletely defined"]},{"year":2021,"claim":"Resolved the prenylation requirement and a Ca2+-regulated phosphosite, connecting double prenylation and signaling to YKT6's interactome and pathway choice.","evidence":"PTAR1 KO with prenylation assays, NMR structural analysis, mass spectrometry, and Ca2+ modulation in mammalian cells and PD models","pmids":["33035318","33723042"],"confidence":"High","gaps":["Upstream signals controlling the Ca2+-dependent kinase unknown","How prenylation and phosphorylation are coordinated temporally unresolved"]},{"year":2023,"claim":"Defined ULK1-mediated phosphorylation as a conserved brake preventing premature autophagic SNARE assembly, and reinforced the α-synuclein/FTase axis in patient neurons.","evidence":"In vitro ULK1 kinase assays and phospho-mutant phenotypes across yeast, mammals, and C. elegans; Co-IP of YKT6-SNAP29 and FTase intervention in PD iPSC neurons and mice","pmids":["36644903","36788031"],"confidence":"High","gaps":["Timing of dephosphorylation relative to fusion not precisely mapped","Whether ULK1 directly senses autophagosome maturation state unknown"]},{"year":2024,"claim":"Assigned YKT6 a priming rather than fusogenic role in autophagy and demonstrated functional redundancy with other R-SNAREs.","evidence":"Co-IP, VAMP8 displacement assays, in vitro lipid/content mixing reconstitution in mammalian cells; double-mutant genetics in yeast","pmids":["38340317","38588809"],"confidence":"High","gaps":["Molecular trigger for VAMP8 displacement of YKT6 not defined","Physiological conditions selecting priming vs fusogenic complexes unclear"]},{"year":2025,"claim":"Expanded YKT6's roles to exosome secretion and synaptic plasticity, and identified Ecm9 as the yeast GGTase-III α subunit completing the prenylation pathway.","evidence":"In vitro Ecm9/Bet2 prenylation with MALDI MS and ecm9Δ phenotyping in yeast; EV nanoparticle tracking with FTase inhibition in neurons; AMPA receptor and mEPSC electrophysiology in hippocampal neurons","pmids":["40049413","39794126","40840626"],"confidence":"Medium","gaps":["Mechanistic link between YKT6 and AMPA receptor insertion not defined","Which YKT6 SNARE complex mediates exosome release unresolved"]},{"year":null,"claim":"The structural basis of the open, membrane-engaged YKT6 conformation and how lipidation, phosphorylation, and partner selection are integrated to direct YKT6 into one of its many distinct SNARE complexes remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of full-length open/membrane-bound YKT6","Rules governing which SNARE complex YKT6 enters in a given compartment unknown","Phosphatases and depalmitoylases acting on YKT6 unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,8,14,25]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[25,14]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[5,11,22]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,11,17,19]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[14,22]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[8,19]},{"term_id":"GO:0005773","term_label":"vacuole","supporting_discovery_ids":[3,9,16,26]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[13,28]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[14,16,23,25]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,2,8,13]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[22,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,22,27]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[17,24,29]}],"complexes":["STX5-GS28-Bet1-YKT6 (ER-Golgi SNARE complex)","STX5-GS28-GS15-YKT6 (Golgi/endosome-TGN SNARE complex)","YKT6-SNAP29-STX7 (autophagosomal SNARE complex)","YKT6-STX17-SNAP29 (autophagosome priming complex)"],"partners":["STX5","GS28","BET1","GS15","STX17","SNAP29","STX7","VAMP8"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O15498","full_name":"Synaptobrevin homolog YKT6","aliases":[],"length_aa":198,"mass_kda":22.4,"function":"Vesicular soluble NSF attachment protein receptor (v-SNARE) mediating vesicle docking and fusion to a specific acceptor cellular compartment (PubMed:9211930, PubMed:15215310). Functions in endoplasmic reticulum to Golgi transport; as part of a SNARE complex composed of GOSR1, GOSR2 and STX5 (By similarity). Functions in early/recycling endosome to TGN transport; as part of a SNARE complex composed of BET1L, GOSR1 and STX5 (PubMed:15215310). Has a S-palmitoyl transferase activity (PubMed:15479160, PubMed:15044687). Essential for the structural and functional organization of the Golgi apparatus (PubMed:32128853)","subcellular_location":"Cytoplasm, cytosol; Cytoplasmic vesicle membrane; Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/O15498/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/YKT6","classification":"Common Essential","n_dependent_lines":1202,"n_total_lines":1208,"dependency_fraction":0.9950331125827815},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000106636","cell_line_id":"CID000744","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"NAPA","stoichiometry":0.2},{"gene":"SCFD1","stoichiometry":0.2},{"gene":"STX5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000744","total_profiled":1310},"omim":[{"mim_id":"621024","title":"PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT REPEAT-CONTAINING PROTEIN 1; PTAR1","url":"https://www.omim.org/entry/621024"},{"mim_id":"618483","title":"LLGL SCRIBBLE CELL POLARITY COMPLEX COMPONENT 2; LLGL2","url":"https://www.omim.org/entry/618483"},{"mim_id":"615417","title":"BET1-LIKE PROTEIN; BET1L","url":"https://www.omim.org/entry/615417"},{"mim_id":"606209","title":"YKT6 v-SNARE HOMOLOG; YKT6","url":"https://www.omim.org/entry/606209"},{"mim_id":"600182","title":"SOLUTE CARRIER FAMILY 7 (CATIONIC AMINO ACID TRANSPORTER, y+ SYSTEM), MEMBER 5; SLC7A5","url":"https://www.omim.org/entry/600182"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/YKT6"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O15498","domains":[{"cath_id":"3.30.450.50","chopping":"2-161","consensus_level":"high","plddt":93.2929,"start":2,"end":161}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15498","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15498-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15498-F1-predicted_aligned_error_v6.png","plddt_mean":90.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=YKT6","jax_strain_url":"https://www.jax.org/strain/search?query=YKT6"},"sequence":{"accession":"O15498","fasta_url":"https://rest.uniprot.org/uniprotkb/O15498.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15498/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15498"}},"corpus_meta":[{"pmid":"29789439","id":"PMC_29789439","title":"Autophagosomal YKT6 is required for fusion with 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YKT6-dependent exosome biogenesis and c-Met cargo selection.","date":"2025","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/40834974","citation_count":2,"is_preprint":false},{"pmid":"39806148","id":"PMC_39806148","title":"Use of Biotin-Labeled Geranyl Pyrophosphate for Analysis of Ykt6 Geranylgeranylation.","date":"2025","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/39806148","citation_count":2,"is_preprint":false},{"pmid":"41298248","id":"PMC_41298248","title":"YKT6 Promotes Bladder Cancer Progression by Stabilizing β-catenin Through USP7-Mediated Deubiquitination.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41298248","citation_count":1,"is_preprint":false},{"pmid":"40840626","id":"PMC_40840626","title":"The SNARE protein Ykt6 drives insertion of the GluA1 and GluA2 glutamate receptors at synaptic spines during long-term 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Study.","date":"2025","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/41477365","citation_count":0,"is_preprint":false},{"pmid":"42177960","id":"PMC_42177960","title":"Artematrolide F suppresses communication between hepatocellular carcinoma cells and hepatic stellate cells to attenuate liver cancer progression through targeting YKT6.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/42177960","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.18.665422","title":"Engineered Extracellular Vesicles Enriched with the miR-214/199a Cluster Enhance the Efficacy of Chemotherapy for Ovarian Cancer","date":"2025-07-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.18.665422","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":29368,"output_tokens":9158,"usd":0.112737,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18968,"output_tokens":5371,"usd":0.114558,"stage2_stop_reason":"end_turn"},"total_usd":0.227295,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"Mammalian YKT6 forms a SNARE complex with syntaxin 5, GS28, and Bet1, localizes primarily to Golgi membranes, and functions at a late stage in ER-to-Golgi transport; antibodies against YKT6 inhibit in vitro ER-Golgi transport of VSVG before the EGTA-sensitive stage, and microinjection of YKT6 antibodies fragments the Golgi apparatus.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ER-Golgi transport assay, antibody inhibition, microinjection, immunofluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus in vitro transport assay with antibody inhibition, two orthogonal methods in a single study\",\n      \"pmids\": [\"11323436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"In yeast, YKT6 (R-SNARE) acts as a multicopy and low-copy suppressor of vti1-2 defects, functionally interacting with VTI1 in transport to the prevacuole and vacuole; YKT6 participates in SNARE complexes containing Pep12p and Vam3p/Vam7p. Mutation of the zero ionic layer arginine (ykt6-R165Q) renders these complexes nonfunctional, establishing that arginine in the 0-layer is essential.\",\n      \"method\": \"Genetic suppressor screen, double-mutant analysis, in vivo transport assays, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis combined with mutagenesis of the catalytic zero-layer residue, replicated across multiple transport steps\",\n      \"pmids\": [\"11445562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GS15 forms a distinct SNARE complex with syntaxin 5, GS28, and Ykt6 in the medial-cisternae of the Golgi; co-immunoprecipitation of COPI coat components with GS15 from Golgi extracts links this complex to early Golgi trafficking.\",\n      \"method\": \"Co-immunoprecipitation, immuno-electron microscopy, siRNA knockdown, dominant-negative overexpression\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, immuno-EM localization, and functional siRNA knockdown, multiple orthogonal methods in one study\",\n      \"pmids\": [\"12388752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The yeast SNARE Ykt6 mediates palmitoylation of the vacuolar fusion factor Vac8 via its N-terminal longin domain, which presents palmitoyl-CoA (Pal-CoA) to Vac8; transfer to Vac8's SH4 domain occurs spontaneously rather than enzymatically. This acyltransferase activity operates during a Sec17-independent subreaction of vacuole fusion controlled by Sec18.\",\n      \"method\": \"In vitro vacuole fusion assay, palmitoylation assay, domain mutagenesis, biochemical reconstitution\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of palmitoylation activity with domain mutagenesis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"14685280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Rat neuronal Ykt6 localizes to a specialized punctate compartment distinct from conventional endomembrane markers; targeting to this compartment is directed by the profilin-like longin domain even in the absence of prenylation. Cytosolic Ykt6 is conformationally inactive for SNARE complex assembly, suggesting autoinhibition.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, domain deletion/mutagenesis, SNARE complex assembly assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with domain mutagenesis and SNARE assembly assays, single lab\",\n      \"pmids\": [\"12589064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Both cytosolic and membrane-bound mammalian Ykt6 are farnesylated at the C-terminal CCAIM cysteine; farnesylation is a prerequisite for subsequent palmitoylation of the upstream cysteine, enabling stable membrane association. The double lipid modification (farnesyl + palmitoyl) is essential for intra-Golgi transport in vitro and cell survival in vivo. The N-terminal longin domain interacts with the SNARE motif, maintaining Ykt6 in an inactive closed conformation that controls membrane recruitment and palmitoylation.\",\n      \"method\": \"Metabolic labeling, in vitro intra-Golgi transport assay, site-directed mutagenesis of CAAX cysteines, cell viability assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of Golgi transport combined with lipid modification biochemistry and mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"15044687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Ykt6 has intrinsic self-palmitoylating activity: incubation of recombinant hYkt6 with [3H]Pal-CoA leads to covalent attachment of palmitate to C-terminal cysteine residues. The N-terminal longin domain contains a Pal-CoA binding site and is required for the reaction.\",\n      \"method\": \"In vitro palmitoylation assay with [3H]palmitoyl-CoA and recombinant protein, domain deletion\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic assay with radiolabeled substrate and domain mutagenesis, single lab\",\n      \"pmids\": [\"15479160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In mammalian neuronal Ykt6, the longin domain controls conformation and subcellular targeting through intramolecular protein-protein interactions with the SNARE motif and protein-lipid interactions with C-terminal lipid groups. Two hydrophobic pockets on opposite faces of the longin domain participate; one suppresses palmitoylation-dependent mislocalization to the plasma membrane. Both protein-protein and protein-lipid intramolecular interactions are required for a tightly closed, autoinhibited conformation.\",\n      \"method\": \"Site-directed mutagenesis of longin domain surface residues, immunofluorescence localization, cell fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-guided mutagenesis combined with direct localization experiments, single lab\",\n      \"pmids\": [\"15331663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Syntaxin 5, GS28, Ykt6, and GS15 function as a SNARE complex mediating transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN); antibodies to each of these four SNAREs specifically inhibit STxB transport in vitro. GS15 and Ykt6 redistribute from the Golgi to endosomes when the recycling endosome is perturbed, indicating they cycle between these compartments.\",\n      \"method\": \"In vitro EE/RE-to-TGN transport assay with STxB, antibody inhibition, siRNA knockdown of GS15, overexpression of SNX3 to perturb recycling endosomes, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro transport assay with antibody inhibition validated by siRNA knockdown and morphological redistribution, multiple orthogonal methods\",\n      \"pmids\": [\"15215310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Ykt6 is released from yeast vacuolar membranes during an early stage of homotypic vacuole fusion in a manner dependent on SNARE disassembly (priming by Sec18). Yeast Ykt6 undergoes palmitoylation at its C-terminal CAAX motif in vitro; mutagenesis of the palmitoylation site prevents stable membrane association and is lethal, indicating depalmitoylation drives Ykt6 cycling between membranes and cytosol.\",\n      \"method\": \"In vitro vacuole fusion assay, [3H]palmitate labeling, site-directed mutagenesis of palmitoylation site, cell viability assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of vacuole fusion combined with palmitoylation biochemistry and lethality of mutagenesis, multiple methods\",\n      \"pmids\": [\"15723044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The Ykt6 longin domain–SNARE domain intramolecular interaction controls cycling between cytosol and membranes; a mutant deficient in this interaction accumulates on membranes and is not released from vacuoles. Ykt6 is a substrate of the DHHC acyltransferase network; overexpression of the vacuolar acyltransferase Pfa3 drives a constitutively membrane-associated Ykt6 mutant into the vacuolar lumen via the MVB pathway, showing that depalmitoylation and release are required to prevent entry into the MVB pathway.\",\n      \"method\": \"Site-directed mutagenesis of longin-SNARE interface, overexpression of Pfa3 acyltransferase, vacuole isolation and fractionation, fluorescence microscopy\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis and acyltransferase overexpression with organelle fractionation, single lab, two orthogonal approaches\",\n      \"pmids\": [\"18541004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In vitro farnesylation of the C-terminal CAAX box of recombinant Ykt6 stabilizes the native protein, increases helical content, and promotes a more compact structure. The farnesyl moiety folds onto a hydrophobic groove in the longin domain, consistent with a closed autoinhibited conformation; the crystal structure of the yeast Ykt6 longin domain (residues 1–140) was determined at 2.5 Å resolution.\",\n      \"method\": \"In vitro farnesylation assay, size exclusion chromatography, limited proteolysis, circular dichroism spectroscopy, surface plasmon resonance, X-ray crystallography\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with multiple biophysical assays (CD, SPR, proteolysis) and in vitro enzymatic modification in one study\",\n      \"pmids\": [\"18329045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Single-molecule FRET and fluorescence cross-correlation spectroscopy reveal that rat Ykt6 undergoes intramolecular conformational dynamics between its longin domain and SNARE core at a timescale of ~200 μs. The presence of the lipid dodecylphosphocholine (DPC) regulates and can eliminate these dynamics, locking Ykt6 in a closed conformation; molecular dynamics simulations show that the SNARE core is flexible while the longin domain is relatively stable in the apo state.\",\n      \"method\": \"Single-molecule FRET, fluorescence cross-correlation spectroscopy (FCCS), molecular dynamics simulation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule biophysical measurements combined with MD simulation, single lab, two orthogonal methods\",\n      \"pmids\": [\"27493064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNAi depletion of YKT6 in Drosophila cells blocks constitutive secretory carrier fusion with the plasma membrane; YKT6 participates in at least two SNARE complexes mediating Golgi-to-PM transport (STX1/SNAP24-29/YKT6 and STX4/SNAP24/Syb). RNAi depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion, establishing an evolutionarily conserved role.\",\n      \"method\": \"Quantitative secretion assay, combinatorial RNAi in Drosophila cells, RNAi in mammalian cells\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative loss-of-function assay replicated across Drosophila and mammalian cells, single lab\",\n      \"pmids\": [\"28403141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In mammalian cells, YKT6 is an autophagosomal SNARE that mediates autophagosome-lysosome fusion independently of STX17. YKT6 forms a SNARE complex with SNAP29 and lysosomal STX7. Recruitment of YKT6 to autophagosomes requires its N-terminal longin domain but not C-terminal palmitoylation/farnesylation (which are required for Golgi localization). YKT6 depletion completely blocks autophagosome-lysosome fusion in STX17 KO cells, indicating two independent SNARE complexes mediate this fusion.\",\n      \"method\": \"SNARE screen by siRNA, STX17 CRISPR KO, YKT6 depletion, co-immunoprecipitation of SNARE complex (YKT6/SNAP29/STX7), domain mutant analysis, autophagy flux assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, CRISPR KO epistasis, domain mutagenesis, and functional autophagy flux measurements, multiple orthogonal methods\",\n      \"pmids\": [\"29789439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Drosophila, Ykt6 is required for autophagosome-lysosome fusion and localizes to lysosomes and autolysosomes. Ykt6 forms a SNARE complex with Syx17 and Snap29 but can be outcompeted by Vamp7; Vamp7 overexpression rescues the fusion defect of ykt6 loss-of-function cells. A zero-ionic-layer mutation (R→Q) in Ykt6 does not impair autophagic activity, whereas palmitoylation/farnesylation site mutations do, supporting a non-canonical regulatory (non-fusogenic) role for Ykt6 in this complex.\",\n      \"method\": \"Drosophila genetics (loss-of-function mutants, transgenic rescue), Co-immunoprecipitation, immunofluorescence localization, Vamp7 overexpression rescue, site-directed mutagenesis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with rescue experiments, Co-IP, localization, and mutagenesis across multiple orthogonal methods in vivo\",\n      \"pmids\": [\"29694367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In yeast, Ykt6 is the autophagosomal SNARE required for autophagosome-vacuole fusion. A novel in vitro fusion assay using intact autophagosomes and vacuoles demonstrated that Ykt6 localizes to the autophagosome side of the fusion machinery, and that this process requires ATP, physiological temperature, HOPS tethering complex, Ypt7 GTPase, and Mon1-Ccz1 GEF.\",\n      \"method\": \"Novel in vitro autophagosome-vacuole fusion assay, SNARE localization by biochemical fractionation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of fusion with intact organelles, direct assignment of Ykt6 as autophagosomal SNARE, single lab rigorous study\",\n      \"pmids\": [\"30097515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"During lysosomal stress, cytosolic ykt6 (normally autoinhibited by a farnesyl-mediated regulatory mechanism) activates and redistributes to membranes to promote lysosomal hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, disabling this lysosomal stress response. Farnesyltransferase inhibitors restore ykt6 activity and reduce α-synuclein in patient-derived neurons and mice.\",\n      \"method\": \"Membrane fractionation, co-immunoprecipitation of α-synuclein with ykt6, patient-derived iPSC neurons, farnesyltransferase inhibitor treatment, lysosomal activity assays, mouse in vivo experiments\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of α-syn/ykt6, farnesylation-dependent mechanism validated in patient neurons and in vivo mouse models, multiple orthogonal methods\",\n      \"pmids\": [\"31648898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In yeast, Ykt6 is recruited to autophagosomes at an early stage of their formation via a mechanism dependent on the ER-resident Dsl1 complex and COPII-coated vesicles. The Atg1 kinase complex directly phosphorylates Ykt6 on autophagosomes to keep it inactive; dephosphorylation of Ykt6 allows its engagement in autophagosome-vacuole fusion.\",\n      \"method\": \"In vitro kinase assay (Atg1 phosphorylation of Ykt6), genetic epistasis with Dsl1 complex and COPII mutants, fluorescence microscopy, autophagy flux assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay combined with genetic epistasis and imaging, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33025734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In Drosophila wing epithelium, most Ykt6 is cytosolic but is recruited to de-acidified endosomal compartments where it recycles Wnt/Wingless to the plasma membrane via Rab4-positive recycling endosomes; this recycling is required for proper Wnt secretion. Proximity-dependent proteomics and biochemical analyses confirmed Ykt6 interactions within endosomal compartments.\",\n      \"method\": \"In vivo Drosophila genetics, proximity-dependent proteomics (BioID), immunofluorescence co-localization with Rab4, membrane fractionation, Wnt trafficking assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetics and proximity proteomics with Rab4 co-localization and functional Wnt trafficking readout, single lab\",\n      \"pmids\": [\"32611603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Phosphorylation of Ykt6 SNARE domain serine residues drives the conformational switch from a closed cytosolic form to an open membrane-bound form; phosphorylation mediates Ykt6 recruitment to several organelle membranes and functionally regulates Wnt protein trafficking and extracellular vesicle secretion.\",\n      \"method\": \"Phosphomimetic and phospho-dead mutagenesis, proximity-dependent labeling (BioID), membrane fractionation, Wnt trafficking assay, extracellular vesicle quantification\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphomimetic mutagenesis combined with proximity labeling and functional trafficking assays, single lab\",\n      \"pmids\": [\"33207719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In mammalian cells, Ykt6 is phosphorylated at an evolutionarily conserved site regulated by Ca2+ signaling; this phosphorylation triggers a conformational change from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways and enhanced toxicity in Parkinson's disease models.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation site, NMR structural analysis, phosphomimetic/phospho-dead mutagenesis, Ca2+ signaling modulation, Co-IP of interaction partners, autophagy and secretory pathway assays, PD model toxicity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure combined with phosphorylation site identification, mutagenesis, and functional pathway assays, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"33723042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Double prenylation of Ykt6 (farnesylation of Cys195 by farnesyltransferase followed by geranylgeranylation of Cys194 by a novel GGTase-III consisting of PTAR1/Bet2) is required for efficient trafficking of lysosomal hydrolases (cathepsin D and β-hexosaminidase) from the trans-Golgi network to lysosomes. In PTAR1 KO cells (singly farnesylated Ykt6), hydrolases are missorted and secreted extracellularly, their maturation is impaired, and LC3B accumulates indicating autophagic defects.\",\n      \"method\": \"PTAR1 knockout cells, lysosomal hydrolase secretion assay, hydrolase maturation analysis, LC3B accumulation by immunoblot, in vitro prenylation assays\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO combined with biochemical prenylation analysis and functional hydrolase trafficking assays, replicated across multiple readouts\",\n      \"pmids\": [\"33035318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 (mammalian Atg1 ortholog) phosphorylates YKT6 on autophagosomes, preventing premature SNARE complex assembly with lysosomal SNAREs and inhibiting autophagosome-lysosome fusion; this regulation is conserved in yeast, mammalian cells, and C. elegans. Alterations in YKT6 phosphorylation status produce both early and late autophagy defects and reduce cellular survival.\",\n      \"method\": \"In vitro ULK1 kinase assay, phosphomimetic/phospho-dead YKT6 mutants, autophagy flux assays in mammalian cells and C. elegans, survival assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay confirmed with phospho-mutant phenotypes replicated across three organisms (yeast, mammals, nematodes)\",\n      \"pmids\": [\"36644903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Parkinson's disease patient iPSC-derived midbrain neurons, chronic endogenous α-synuclein accumulation directly impairs autophagosome-lysosome fusion by blocking ykt6-SNAP-29 complex formation. ykt6 depletion causes near-complete block of autophagic flux in human neurons. Increased farnesyltransferase activity in PD suppresses active (membrane-associated) ykt6; farnesyltransferase inhibitors restore autophagic flux by promoting active ykt6.\",\n      \"method\": \"iPSC-derived midbrain neuron culture from PD patients, Co-immunoprecipitation of ykt6-SNAP29 complex, autophagic flux assays, farnesyltransferase activity measurement, FTase inhibitor treatment in neurons and mice\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of endogenous complex in patient-derived neurons, loss-of-function with defined phenotype, pharmacological intervention replicated in mice\",\n      \"pmids\": [\"36788031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"YKT6 forms a priming SNARE complex with STX17 and SNAP29 on autophagosomes via its SNARE domain, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex to form the fusogenic STX17-SNAP29-VAMP8 complex. The YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, indicating YKT6 plays a priming (not direct fusogenic) role for efficient autophagosome-lysosome fusion.\",\n      \"method\": \"Co-immunoprecipitation of YKT6/STX17/SNAP29 complex, VAMP8 displacement assay, in vitro lipid mixing and content mixing fusion assays, domain mutagenesis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro lipid and content mixing reconstitution assays combined with Co-IP and competitive displacement experiments, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"38340317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In yeast, Ykt6 and Nyv1 are functionally redundant R-SNAREs in homotypic vacuole fusion; a ykt6-104 nyv1Δ double mutant exhibits highly fragmented vacuoles while neither single mutant does. Ykt6 can also substitute for exocytic R-SNAREs Snc1/Snc2 when those lose the ability to assemble into exocytic SNARE complexes, indicating Ykt6 can function as a backup R-SNARE maintaining robustness of the vesicular transport network.\",\n      \"method\": \"Yeast genetic double-mutant analysis, vacuole morphology by fluorescence microscopy, SNARE complex assembly assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with double mutants and SNARE complex assembly assays, single lab\",\n      \"pmids\": [\"38588809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In budding yeast, the uncharacterized protein Ecm9 is the functional α subunit of yeast GGTase-III; Ecm9 forms a complex with Bet2 and transfers a geranylgeranyl group to mono-farnesylated Ykt6. MALDI-TOF/TOF mass spectrometry confirmed double prenylation (farnesyl + geranylgeranyl) of Ykt6 in wild-type but not ecm9Δ cells. Loss of Ecm9 impairs Ykt6 localization to organelle membranes including autophagosomes, reduces autophagic activity, and causes Golgi mannosyltransferase mislocalization and cell wall fragility.\",\n      \"method\": \"Structural prediction, in vitro prenylation assay with recombinant Ecm9/Bet2 complex, MALDI-TOF/TOF mass spectrometry of Ykt6 prenylation state, ecm9Δ yeast phenotyping, immunofluorescence of Ykt6 localization and mannosyltransferases, autophagy flux assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — MS confirmation of double prenylation, in vitro enzymatic reconstitution, and genetic KO phenotypes across multiple readouts in one study\",\n      \"pmids\": [\"40049413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ykt6 is highly expressed in the mammalian hippocampus, localizes to synaptic spines, and is required for LTP-dependent insertion of GluA1 and GluA2 AMPA receptor subunits at the postsynaptic membrane. Loss of Ykt6 function alters synaptic vesicle pool dynamics and the amplitude and frequency of miniature excitatory postsynaptic currents, modulates spine morphology, and impairs LTP.\",\n      \"method\": \"Immunofluorescence localization in hippocampal neurons, loss-of-function with defined synaptic phenotype, surface GluA1/GluA2 expression assay, mEPSC electrophysiology, spine morphology analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct synaptic localization and loss-of-function with multiple electrophysiological and biochemical readouts, single lab\",\n      \"pmids\": [\"40840626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Elevated α-synuclein reduces membrane-associated YKT6, impairing YKT6-mediated extracellular vesicle (exosome) secretion in H4 cells and iPSC-derived dopaminergic neurons. Pharmacological farnesyltransferase inhibition (FTI) decreases both membrane-associated YKT6 and EV secretion, establishing that farnesylation-dependent membrane association of YKT6 is required for EV secretion and that α-synuclein disrupts this by reducing membrane-associated YKT6.\",\n      \"method\": \"α-Synuclein-inducible H4 cells and iPSC-derived dopaminergic neurons, nanoparticle tracking analysis of EVs, membrane fractionation of YKT6, farnesyltransferase inhibitor treatment\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic link of α-syn to YKT6 membrane localization and EV secretion confirmed in two cell models with pharmacological intervention, single lab\",\n      \"pmids\": [\"39794126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In mammalian prostate epithelial cells, Ykt6 acts as a negative regulator of cell migration and invasion by upregulating microRNA-145, which decreases Junctional Adhesion Molecule A (JAM-A) expression, thereby reducing Rap1 and Rac1 GTPase activity and attenuating cell spreading and motility.\",\n      \"method\": \"Ykt6 overexpression/knockdown in prostate epithelial cells, migration and invasion assays, miR-145 quantification, JAM-A protein level measurement, Rap1/Rac1 activity assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect pathway (via miRNA upregulation) with functional readouts but no direct biochemical link between Ykt6 and miR-145 induction mechanism\",\n      \"pmids\": [\"30010460\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"YKT6 is an evolutionarily conserved, lipid-anchored R-SNARE that lacks a transmembrane domain and instead cycles between an autoinhibited closed cytosolic form (farnesylated only) and an active open membrane-bound form (doubly prenylated by farnesyltransferase and GGTase-III, plus palmitoylated); this conformational switch is regulated by intramolecular longin domain–SNARE motif interactions, double prenylation state, and phosphorylation by Atg1/ULK1 kinases. YKT6 participates in multiple distinct SNARE complexes mediating: (1) late ER-to-Golgi and intra-Golgi transport (with STX5/GS28/Bet1 or GS15); (2) endosome-to-TGN recycling (with STX5/GS28/GS15); (3) constitutive secretory carrier fusion with the plasma membrane; (4) autophagosome-lysosome fusion, where it first forms a priming complex with STX17/SNAP29 on autophagosomes before being displaced by VAMP8 for the fusogenic step, and also independently forms a fusogenic complex with SNAP29/STX7; YKT6 additionally serves as a palmitoyl-CoA-presenting acyltransferase for Vac8/palmitoylation events at the vacuole. Farnesylation-dependent membrane association of YKT6 is also essential for lysosomal hydrolase trafficking, exosome secretion, and hippocampal AMPA receptor insertion during LTP, while α-synuclein pathologically inhibits YKT6 activity to impair autophagosomal–lysosomal fusion and cellular clearance in Parkinson's disease models.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"YKT6 is an evolutionarily conserved R-SNARE that, uniquely among SNAREs, lacks a transmembrane domain and instead cycles between an autoinhibited cytosolic pool and an active membrane-bound pool to drive fusion at multiple steps of the secretory and degradative pathways [#0, #4, #5]. Membrane recruitment is governed by C-terminal lipidation: farnesylation of the CAAX cysteine is a prerequisite for palmitoylation of the upstream cysteine, and this double lipid modification, together with a longin domain–SNARE motif intramolecular interaction that maintains a closed conformation, controls stable membrane association and activity [#5, #11]. Efficient targeting further requires double prenylation, in which the singly farnesylated protein is geranylgeranylated by a GGTase-III (PTAR1/Bet2 in mammals; Ecm9/Bet2 in yeast), a step needed for lysosomal hydrolase trafficking from the TGN and for organelle membrane localization including autophagosomes [#22, #27]. The closed–open conformational switch is additionally driven by phosphorylation of SNARE-domain serines, including a Ca2+-regulated site, which redistributes YKT6 to membranes and reshapes its interactome [#20, #21]. In its open form YKT6 assembles into distinct SNARE complexes mediating late ER-to-Golgi and intra-Golgi transport (with syntaxin 5, GS28, Bet1 or GS15), endosome-to-TGN recycling, and constitutive secretory carrier fusion at the plasma membrane [#0, #2, #8, #13]. At the autophagosome, YKT6 forms a priming complex with STX17 and SNAP29 that is subsequently displaced by VAMP8 for the fusogenic step, and it can also act in a STX17-independent complex with SNAP29 and STX7, with its activity gated by ULK1/Atg1 phosphorylation that prevents premature complex assembly [#14, #23, #25]. Beyond fusion, the yeast longin domain presents palmitoyl-CoA to the vacuolar factor Vac8, conferring a non-enzymatic acyltransferase-like activity [#3, #6]. YKT6 also supports exosome secretion and hippocampal AMPA receptor insertion during LTP [#28, #29], and pathological α-synuclein binds and deactivates YKT6 to block YKT6–SNAP29 complex formation and impair autophagosome-lysosome fusion in Parkinson's disease neuron models, a defect reversible by farnesyltransferase inhibitors [#17, #24].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established YKT6 as a functional SNARE in the early secretory pathway, answering where this R-SNARE acts and with which partners.\",\n      \"evidence\": \"Co-IP and in vitro ER-Golgi transport assay with antibody inhibition in mammalian cells, plus genetic suppressor analysis in yeast\",\n      \"pmids\": [\"11323436\", \"11445562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how YKT6 associates with membranes without a transmembrane domain\", \"Mechanism of cycling between compartments unaddressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined a distinct GS15-containing SNARE complex, showing YKT6 participates in more than one Golgi fusion complex.\",\n      \"evidence\": \"Reciprocal Co-IP, immuno-EM, and siRNA knockdown in Golgi extracts\",\n      \"pmids\": [\"12388752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional discrimination between GS15 and Bet1 complexes not fully resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed a non-fusion biochemical activity, with the longin domain presenting palmitoyl-CoA to Vac8, broadening YKT6's molecular repertoire.\",\n      \"evidence\": \"In vitro vacuole fusion and palmitoylation assays with domain mutagenesis in yeast\",\n      \"pmids\": [\"14685280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this acyltransferase-like activity operates on substrates beyond Vac8 unknown\", \"Relevance in mammalian cells not established\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established the lipidation hierarchy and autoinhibition model, explaining how a TM-less SNARE achieves regulated membrane association.\",\n      \"evidence\": \"Metabolic labeling, in vitro intra-Golgi transport, recombinant self-palmitoylation assays, and longin-domain surface mutagenesis in mammalian/neuronal cells\",\n      \"pmids\": [\"15044687\", \"15479160\", \"15331663\", \"12589064\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for opening the closed conformation in vivo not identified\", \"Identity of palmitoyltransferase in vivo unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated that depalmitoylation drives YKT6 cycling off membranes, linking lipid turnover to its trafficking life cycle.\",\n      \"evidence\": \"In vitro vacuole fusion, [3H]palmitate labeling, and lethal palmitoylation-site mutagenesis in yeast\",\n      \"pmids\": [\"15723044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Depalmitoylating enzyme not identified\", \"Coupling of disassembly to depalmitoylation mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided structural and biophysical proof of the closed autoinhibited conformation, with the farnesyl group folding into the longin domain.\",\n      \"evidence\": \"X-ray crystallography of the yeast longin domain plus CD, SPR, limited proteolysis, and acyltransferase overexpression in yeast\",\n      \"pmids\": [\"18329045\", \"18541004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the full-length open membrane-bound form\", \"How conformational opening is triggered physiologically unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantified the conformational dynamics of the longin–SNARE switch and showed lipid environment locks the closed state.\",\n      \"evidence\": \"Single-molecule FRET, FCCS, and MD simulation on rat YKT6\",\n      \"pmids\": [\"27493064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dynamics measured in detergent/lipid mimics, not native membranes\", \"Link between observed microsecond dynamics and SNARE engagement not directly demonstrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended YKT6 function to constitutive secretory fusion at the plasma membrane, showing a conserved exocytic role.\",\n      \"evidence\": \"Quantitative secretion assays with combinatorial RNAi in Drosophila and mammalian cells\",\n      \"pmids\": [\"28403141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration of the proposed PM SNARE complexes biochemically incomplete\", \"Regulation of YKT6 recruitment to secretory carriers unaddressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified YKT6 as an autophagosomal SNARE acting independently of STX17, redefining the autophagosome-lysosome fusion machinery.\",\n      \"evidence\": \"siRNA SNARE screen, STX17 CRISPR KO epistasis, Co-IP, and autophagy flux assays in mammalian cells; genetics and in vitro fusion in Drosophila and yeast\",\n      \"pmids\": [\"29789439\", \"29694367\", \"30097515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether YKT6 acts fusogenically or regulatorily in autophagy not yet settled at this stage\", \"Relationship between STX7- and STX17-containing complexes unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked YKT6 to a lysosomal stress response and to Parkinson's disease, showing α-synuclein deactivates YKT6 and FTase inhibitors rescue it.\",\n      \"evidence\": \"Membrane fractionation, Co-IP of α-synuclein, patient iPSC neurons, and FTase inhibitor treatment in neurons and mice\",\n      \"pmids\": [\"31648898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of α-synuclein–YKT6 interaction not defined\", \"How α-synuclein binding alters lipidation or conformation unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established phosphorylation as a conformational switch and identified the kinases and recruitment machinery controlling YKT6 activation.\",\n      \"evidence\": \"Phosphomimetic mutagenesis, BioID, in vitro Atg1 kinase assays, and genetic epistasis with Dsl1/COPII in yeast and Drosophila\",\n      \"pmids\": [\"33207719\", \"33025734\", \"32611603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphatases reversing YKT6 phosphorylation not identified\", \"Cross-talk between phosphorylation and lipidation states incompletely defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the prenylation requirement and a Ca2+-regulated phosphosite, connecting double prenylation and signaling to YKT6's interactome and pathway choice.\",\n      \"evidence\": \"PTAR1 KO with prenylation assays, NMR structural analysis, mass spectrometry, and Ca2+ modulation in mammalian cells and PD models\",\n      \"pmids\": [\"33035318\", \"33723042\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling the Ca2+-dependent kinase unknown\", \"How prenylation and phosphorylation are coordinated temporally unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined ULK1-mediated phosphorylation as a conserved brake preventing premature autophagic SNARE assembly, and reinforced the α-synuclein/FTase axis in patient neurons.\",\n      \"evidence\": \"In vitro ULK1 kinase assays and phospho-mutant phenotypes across yeast, mammals, and C. elegans; Co-IP of YKT6-SNAP29 and FTase intervention in PD iPSC neurons and mice\",\n      \"pmids\": [\"36644903\", \"36788031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Timing of dephosphorylation relative to fusion not precisely mapped\", \"Whether ULK1 directly senses autophagosome maturation state unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Assigned YKT6 a priming rather than fusogenic role in autophagy and demonstrated functional redundancy with other R-SNAREs.\",\n      \"evidence\": \"Co-IP, VAMP8 displacement assays, in vitro lipid/content mixing reconstitution in mammalian cells; double-mutant genetics in yeast\",\n      \"pmids\": [\"38340317\", \"38588809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger for VAMP8 displacement of YKT6 not defined\", \"Physiological conditions selecting priming vs fusogenic complexes unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded YKT6's roles to exosome secretion and synaptic plasticity, and identified Ecm9 as the yeast GGTase-III α subunit completing the prenylation pathway.\",\n      \"evidence\": \"In vitro Ecm9/Bet2 prenylation with MALDI MS and ecm9Δ phenotyping in yeast; EV nanoparticle tracking with FTase inhibition in neurons; AMPA receptor and mEPSC electrophysiology in hippocampal neurons\",\n      \"pmids\": [\"40049413\", \"39794126\", \"40840626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between YKT6 and AMPA receptor insertion not defined\", \"Which YKT6 SNARE complex mediates exosome release unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of the open, membrane-engaged YKT6 conformation and how lipidation, phosphorylation, and partner selection are integrated to direct YKT6 into one of its many distinct SNARE complexes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of full-length open/membrane-bound YKT6\", \"Rules governing which SNARE complex YKT6 enters in a given compartment unknown\", \"Phosphatases and depalmitoylases acting on YKT6 unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 8, 14, 25]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [25, 14]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [5, 11, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 11, 17, 19]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [14, 22]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [8, 19]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [3, 9, 16, 26]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [13, 28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [14, 16, 23, 25]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 2, 8, 13]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [22, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 22, 27]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [17, 24, 29]}\n    ],\n    \"complexes\": [\n      \"STX5-GS28-Bet1-YKT6 (ER-Golgi SNARE complex)\",\n      \"STX5-GS28-GS15-YKT6 (Golgi/endosome-TGN SNARE complex)\",\n      \"YKT6-SNAP29-STX7 (autophagosomal SNARE complex)\",\n      \"YKT6-STX17-SNAP29 (autophagosome priming complex)\"\n    ],\n    \"partners\": [\n      \"STX5\",\n      \"GS28\",\n      \"Bet1\",\n      \"GS15\",\n      \"STX17\",\n      \"SNAP29\",\n      \"STX7\",\n      \"VAMP8\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}