{"gene":"YKT6","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":2001,"finding":"Mammalian YKT6 forms a SNARE complex with syntaxin 5, GS28, and Bet1, 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 recombinant YKT6 blocks transport; YKT6 localizes primarily to Golgi membranes.","method":"Co-immunoprecipitation, in vitro transport assay with antibody inhibition, double-label immunofluorescence, microinjection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro transport assay + Co-IP + functional antibody inhibition, multiple orthogonal methods","pmids":["11323436"],"is_preprint":false},{"year":2001,"finding":"Yeast YKT6 (R-SNARE) genetically interacts with the Q-SNARE VTI1 and functions in transport to the prevacuole/late endosome and vacuole in addition to retrograde traffic to the cis-Golgi; YKT6 participates in SNARE complexes containing Vti1p+Pep12p and Vti1p+Vam3p+Vam7p. Mutation of the 0-layer arginine in Ykt6 (R165Q) in complexes where it contributes a fourth glutamine renders the complex nonfunctional.","method":"Genetic suppressor screen (multicopy and low-copy), vacuolar transport assays, site-directed mutagenesis of 0-layer residues","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple suppressor assays and mutagenesis, replicated across 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 Golgi, implicating this quaternary complex in early cisternae trafficking; components of COPI coat co-immunoprecipitate selectively with GS15 from Golgi extracts.","method":"Co-immunoprecipitation, immuno-EM, siRNA knockdown, overexpression of dominant-negative mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, immuno-EM localization, functional KD, replicated complex identification","pmids":["12388752"],"is_preprint":false},{"year":2003,"finding":"Rat Ykt6 is a neuronal SNARE that localizes to a specialized punctate compartment distinct from known endomembrane organelles; its profilin-like longin domain directs this unique targeting even in the absence of prenylation; cytosolic Ykt6 is conformationally inactive for SNARE complex assembly.","method":"Immunofluorescence microscopy, density gradient fractionation, mutagenesis of longin domain and prenylation sites, SNARE complex assembly assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (localization, fractionation, mutagenesis, assembly assays) in single study","pmids":["12589064"],"is_preprint":false},{"year":2003,"finding":"Yeast Ykt6 mediates palmitoylation of the fusion factor Vac8 during homotypic vacuole fusion through a novel subreaction controlled by a Sec17-independent function of Sec18; the N-terminal longin domain of Ykt6 presents palmitoyl-CoA to Vac8, and transfer to Vac8's SH4 domain occurs spontaneously (non-enzymatically).","method":"In vitro vacuole fusion assay, biochemical fractionation, mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mechanistic dissection of subreaction and domain requirements","pmids":["14685280"],"is_preprint":false},{"year":2004,"finding":"YKT6 exists as both cytosolic (inactive) and membrane-bound (active) forms; both are farnesylated at the C-terminal cysteine of CCAIM, and farnesylation is a prerequisite for subsequent palmitoylation of the upstream cysteine. Double lipid modification is required for intra-Golgi transport in vitro and cell viability. The N-terminal longin domain interacts with the SNARE motif to maintain YKT6 in a closed, inactive conformation, and conformational changes control lipid modification and membrane recruitment.","method":"In vitro transport assay, metabolic labeling, mutagenesis of lipidation sites, cell viability assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro transport reconstitution + lipid modification biochemistry + mutagenesis, mechanistically comprehensive","pmids":["15044687"],"is_preprint":false},{"year":2004,"finding":"The syntaxin 5/Ykt6/GS28/GS15 SNARE complex mediates transport from the early/recycling endosome to the trans-Golgi network (EE/RE-TGN); antibodies to each of these four SNAREs specifically inhibited this transport step in vitro; GS15 and Ykt6 redistribute from Golgi to endosomes when the recycling endosome is perturbed, suggesting cycling between these compartments.","method":"In vitro transport assay with Shiga toxin B subunit marker, antibody inhibition, siRNA knockdown of GS15, SNX3 overexpression morphological analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro transport assay with antibody inhibition, siRNA functional validation, multiple SNAREs tested","pmids":["15215310"],"is_preprint":false},{"year":2004,"finding":"The longin domain of mammalian Ykt6 controls subcellular targeting through intramolecular protein-protein interactions with the SNARE motif and protein-lipid interactions with lipid groups at the C-terminus; two hydrophobic pockets on each face of the longin domain suppress mislocalization, and one suppresses palmitoylation-dependent mislocalization to the plasma membrane; both interactions maintain a compact closed conformation preventing premature membrane insertion.","method":"Mutagenesis of longin domain surface residues, immunofluorescence localization, co-immunoprecipitation of intramolecular interactions","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — systematic mutagenesis guided by crystal structure, multiple localization and interaction readouts","pmids":["15331663"],"is_preprint":false},{"year":2004,"finding":"Human Ykt6 has intrinsic self-palmitoylating activity: the N-terminal longin domain contains a palmitoyl-CoA binding site required for covalent palmitoylation of its own C-terminal cysteine residues.","method":"In vitro palmitoylation assay with [3H]palmitoyl-CoA, recombinant protein mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro enzymatic assay with radiolabeled substrate and mutagenesis","pmids":["15479160"],"is_preprint":false},{"year":2005,"finding":"Yeast Ykt6 is released from vacuolar membranes during an early stage of vacuole fusion in a priming-dependent (SNARE disassembly-dependent) manner; yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif, and mutation of the palmitoylation site prevents stable membrane association and is lethal, suggesting depalmitoylation-driven recycling of this SNARE.","method":"In vitro vacuole fusion assay, palmitoylation assay, site-directed mutagenesis, cell viability","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro fusion assay, biochemical palmitoylation assay, mutagenesis with viability readout","pmids":["15723044"],"is_preprint":false},{"year":2008,"finding":"Farnesylation of Ykt6 (at the CAAX box) increases protein stability, helical content, and compactness as shown by CD spectroscopy, size exclusion chromatography, and limited proteolysis; farnesylated Ykt6 binds lipid membranes independently of membrane charge. The crystal structure of the yeast Ykt6 longin domain (residues 1–140) at 2.5 Å reveals a hydrophobic surface patch that accommodates the lipid moiety in the closed conformation.","method":"In vitro farnesylation, size exclusion chromatography, limited proteolysis, circular dichroism, surface plasmon resonance, X-ray crystallography at 2.5 Å","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with multiple biophysical and biochemical assays","pmids":["18329045"],"is_preprint":false},{"year":2008,"finding":"Ykt6 cycles between cytosol and membranes through intramolecular interaction between its N-terminal longin domain and C-terminal SNARE domain; a mutant deficient in this intramolecular interaction accumulates stably on membranes and is not released from vacuoles. Ykt6 is a substrate of DHHC acyltransferases; overexpression of vacuolar acyltransferase Pfa3 drives a longin-domain mutant (F42S) into the vacuolar lumen, indicating that depalmitoylation is required to prevent Ykt6 entry into the MVB pathway.","method":"Mutagenesis, in vivo localization, vacuole fusion assay, DHHC acyltransferase overexpression","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis + localization + functional assay, mechanistically comprehensive for the cycling mechanism","pmids":["18541004"],"is_preprint":false},{"year":2016,"finding":"Single-molecule FRET and FCCS demonstrate 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 DPC eliminates this dynamics and locks Ykt6 in a closed conformation, supporting lipid-regulated conformational switching.","method":"Single-molecule FRET, Fluorescence Cross-Correlation Spectroscopy, molecular dynamics simulation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 — single-molecule biophysics + MD, but single lab, one study","pmids":["27493064"],"is_preprint":false},{"year":2017,"finding":"YKT6 and VAMP3/Synaptobrevin function as v-SNAREs in constitutive secretory vesicle fusion with the plasma membrane in both Drosophila and mammalian cells; RNAi depletion of YKT6 blocks constitutive secretion, identifying an evolutionarily conserved role of YKT6 in Golgi-to-PM transport.","method":"RNAi combinatorial depletion in Drosophila cells, quantitative secretion assay, RNAi in mammalian cells","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — combinatorial gene depletion with quantitative assay, replicated in two organisms","pmids":["28403141"],"is_preprint":false},{"year":2018,"finding":"YKT6 is an autophagosomal SNARE protein that mediates autophagosome-lysosome fusion independently of STX17: YKT6 depletion partially blocks fusion in wild-type and completely blocks it in STX17-KO HeLa cells. YKT6 forms a SNARE complex with SNAP29 and lysosomal STX7 on autophagosomes. Recruitment to autophagosomes requires the N-terminal longin domain but not C-terminal palmitoylation/farnesylation.","method":"STX17 KO and YKT6 siRNA depletion in HeLa cells, autophagosome-lysosome fusion assay, Co-immunoprecipitation, domain mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO + siRNA epistasis + Co-IP, multiple orthogonal approaches, independently replicated by contemporaneous studies","pmids":["29789439"],"is_preprint":false},{"year":2018,"finding":"In Drosophila, Ykt6 is required for autophagosome-lysosome fusion and localizes to lysosomes/autolysosomes; it forms a SNARE complex with Syx17 and Snap29. Vamp7 can outcompete Ykt6 from this complex, and Vamp7 overexpression rescues fusion defects in ykt6 mutants. An RQ mutation in the 0-layer of Ykt6 retains normal autophagic activity, suggesting Ykt6 acts as a non-canonical regulatory SNARE in this process; palmitoylation and farnesylation site mutants do not rescue.","method":"Drosophila genetics (loss-of-function mutants, rescue constructs), Co-immunoprecipitation, autophagic flux assays, site-directed mutagenesis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — in vivo Drosophila genetics + Co-IP + mutagenesis, comprehensive mechanistic dissection","pmids":["29694367"],"is_preprint":false},{"year":2018,"finding":"A novel in vitro assay with intact yeast autophagosomes and vacuoles identifies Ykt6 as the autophagosomal R-SNARE; fusion requires ATP, physiological temperature, the HOPS tethering complex, Ypt7 GTPase, Mon1-Ccz1 GEF, and the entire fusion machinery.","method":"Novel in vitro autophagosome-vacuole fusion assay with purified organelles, genetic depletion of individual components","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with intact organelles, systematic requirement analysis","pmids":["30097515"],"is_preprint":false},{"year":2019,"finding":"Cytosolic ykt6 is normally autoinhibited by a farnesyl-mediated regulatory mechanism; during lysosomal stress, ykt6 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 the lysosomal stress response. Farnesyltransferase inhibitors activate ykt6 by promoting its membrane-bound open form, restoring lysosomal activity.","method":"Live-cell imaging, membrane fractionation, co-immunoprecipitation (α-syn binding to ykt6), patient iPSC-derived neurons, farnesyltransferase inhibitor treatment in cells and mice","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, patient-derived neurons + mouse model, mechanistic pathway established","pmids":["31648898"],"is_preprint":false},{"year":2020,"finding":"Ykt6 function on autophagosomes is regulated by the Atg1 kinase complex through direct phosphorylation, keeping the Ykt6 pool on autophagosomal membranes inactive; dephosphorylation of Ykt6 is required for its engagement in autophagosome-vacuole fusion. Ykt6 is recruited to early autophagosome precursors through a mechanism requiring the ER-resident Dsl1 complex and COPII-coated vesicles.","method":"In vitro kinase assay (Atg1 phosphorylation of Ykt6), genetic epistasis (Dsl1 complex mutants), autophagy flux assays, Co-immunoprecipitation","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay + genetic epistasis + Co-IP, mechanistically comprehensive","pmids":["33025734"],"is_preprint":false},{"year":2020,"finding":"Phosphorylation of Ykt6 in its SNARE domain mediates its conversion from a closed cytosolic to an open membrane-bound conformation, regulating membrane recruitment to multiple organelles; phosphorylated Ykt6 functionally regulates Wnt protein trafficking and extracellular vesicle secretion in Drosophila wing epithelium. Most Ykt6 is cytosolic but is recruited to de-acidified compartments to recycle Wnts via Rab4-positive recycling endosomes.","method":"Proximity-dependent proteomics (BioID), membrane fractionation, in vivo Drosophila genetics, in vitro biochemical analyses, phospho-site mutagenesis","journal":"Biomolecules / Development","confidence":"High","confidence_rationale":"Tier 2 — phospho-mutagenesis + proximity proteomics + in vivo genetics + biochemical fractionation, multiple organisms","pmids":["33207719","32611603"],"is_preprint":false},{"year":2021,"finding":"A conformational switch driven by phosphorylation at an evolutionarily conserved site (regulated by Ca2+ signaling) allows Ykt6 to transition from a closed cytosolic form to an open membrane-bound form; phosphorylated Ykt6 has an altered spectrum of protein interactions, causing defects in both secretory and autophagy pathways in Parkinson's disease models.","method":"NMR, biochemical assays, mutagenesis, Parkinson's disease cell models, Ca2+ signaling manipulation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — NMR structural data + biochemical assays + mutagenesis + functional pathway analysis","pmids":["33723042"],"is_preprint":false},{"year":2021,"finding":"Double prenylation of Ykt6 (farnesylation by FTase followed by geranylgeranylation by a novel GGTase-III containing PTAR1 subunit) at two C-terminal cysteines is required for proper lysosomal hydrolase trafficking; in PTAR1-KO cells (singly farnesylated Ykt6), cathepsin D and β-hexosaminidase are missorted and secreted extracellularly, Golgi structure is disrupted, and LC3B accumulates.","method":"PTAR1 gene knockout, lysosomal hydrolase secretion assay, autophagy flux assay (LC3B), Golgi morphology analysis","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 2 — KO with multiple defined functional readouts (hydrolase sorting, Golgi integrity, autophagy)","pmids":["33035318"],"is_preprint":false},{"year":2023,"finding":"Mammalian autophagosomal YKT6 is phosphorylated by ULK1 kinase, which prevents premature bundling with lysosomal SNARE proteins and inhibits autophagosome-lysosome fusion; alterations in YKT6 function produce both early and late autophagy defects in mammalian cells and C. elegans, reducing survival.","method":"In vitro ULK1 kinase assay, phospho-site mutagenesis, autophagy flux assays in mammalian cells and C. elegans, co-immunoprecipitation of SNARE complexes","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay + mutagenesis + multi-organism validation, mechanistically comprehensive","pmids":["36644903"],"is_preprint":false},{"year":2023,"finding":"YKT6 forms a priming complex with STX17 and SNAP29 on autophagosomes via its SNARE domain; 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, demonstrating a priming role for YKT6 in efficient membrane fusion.","method":"Co-immunoprecipitation, lipid mixing assay, content mixing assay, domain mutagenesis, autophagy flux assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro lipid and content mixing assays + Co-IP + mutagenesis, comprehensive mechanistic dissection","pmids":["38340317"],"is_preprint":false},{"year":2023,"finding":"Homozygous missense variants in YKT6 (Tyr185Cys, Tyr64Cys) cause partial loss of function in Drosophila, failing to rescue lethality and autophagic flux defects in dYkt6 mutant flies, establishing YKT6 as essential for autophagic flux and neuronal/hepatic function in vivo.","method":"Drosophila genetic rescue with human variant constructs, autophagic flux assays, expression pattern analysis","journal":"Genetics in medicine","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic rescue with variant constructs + autophagic flux readout, strong functional validation","pmids":["38522068"],"is_preprint":false},{"year":2023,"finding":"Ykt6 conformational dynamics differ between yeast and rat: yeast Ykt6 adopts more open conformations and cannot bind DPC (which locks rat Ykt6 in a closed state); a T46L/Q57A point mutation converts yeast Ykt6 to a more closed, DPC-bound state. Phospho-mimic S174D shifts rat Ykt6 toward a more open state, confirming phosphorylation as a regulator of the closed-to-open conformational switch.","method":"Single-molecule FRET, biochemical characterization, molecular dynamics simulation, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — single-molecule FRET + MD + mutagenesis, multiple orthogonal approaches","pmids":["37380075"],"is_preprint":false},{"year":2025,"finding":"Ykt6 localizes to synaptic spines in mammalian hippocampus and regulates GluA1 and GluA2 glutamate receptor surface expression in an LTP-dependent manner; Ykt6 also modulates spine morphology, synaptic vesicle pool dynamics, and miniature EPSC amplitude and frequency. α-Synuclein pathology disrupts Ykt6 function and LTP.","method":"Immunofluorescence/live imaging, electrophysiology (mEPSC recording), LTP induction, surface receptor assays, loss-of-function studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization + electrophysiology + LTP assay, single study","pmids":["40840626"],"is_preprint":false},{"year":2025,"finding":"Double prenylation (farnesyl + geranylgeranyl) is an evolutionarily conserved modification of Ykt6 in yeast, mediated by the GGTase-III complex consisting of Ecm9 (α subunit) and Bet2 (β subunit); loss of Ecm9 prevents double prenylation, impairs Ykt6 localization to organelle membranes including autophagosomes, and reduces autophagic activity and cell wall integrity.","method":"Structural prediction, in vitro prenylation assay, MALDI-TOF/TOF mass spectrometry, genetic deletion (ecm9Δ), autophagy assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mass spectrometry confirmation of prenylation + genetic KO + multiple functional readouts","pmids":["40049413"],"is_preprint":false},{"year":2018,"finding":"Ykt6 regulates epithelial cell migration as a negative regulator; it upregulates microRNA-145 expression, which selectively decreases Junctional Adhesion Molecule A (JAM-A) levels, thereby limiting Rap1 and Rac1 small GTPase activity and attenuating cell spreading and motility.","method":"siRNA knockdown, overexpression, miRNA reporter assay, small GTPase activity assays, migration/invasion assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 3 — functional KD/OE with pathway placement but single lab, limited mechanistic depth for miRNA mechanism","pmids":["30010460"],"is_preprint":false}],"current_model":"YKT6 is a uniquely lipid-anchored R-SNARE that cycles between an autoinhibited closed cytosolic form (farnesylated but not palmitoylated, with the longin domain masking the SNARE motif) and an active open membrane-bound form (doubly prenylated with farnesyl and geranylgeranyl, plus palmitoylated), with conformational opening regulated by phosphorylation (by Atg1/ULK1 or Ca2+-dependent kinases) and lipid modifications; in its active form it participates in multiple SNARE complexes mediating ER-to-Golgi transport (with syntaxin 5/GS28/Bet1), intra-Golgi and endosome-to-TGN transport (with syntaxin 5/GS28/GS15), constitutive secretion to the plasma membrane, Wnt recycling via endosomes, and autophagosome-lysosome fusion (forming a priming complex with STX17/SNAP29 that is subsequently displaced by VAMP8 to drive fusion, as well as an independent complex with SNAP29/STX7), and additionally mediates protein palmitoylation via its longin domain and regulates lysosomal hydrolase trafficking and synaptic receptor (GluA1/GluA2) insertion during LTP."},"narrative":{"teleology":[{"year":2001,"claim":"Identifying YKT6 as a participant in ER-to-Golgi and vacuolar SNARE complexes established it as a versatile R-SNARE functioning at multiple trafficking steps rather than a single compartment.","evidence":"Co-IP of mammalian YKT6 with syntaxin 5/GS28/Bet1 plus antibody-mediated inhibition of ER-Golgi transport; yeast genetic suppressor screens linking Ykt6 to Vti1p-containing complexes at prevacuolar and vacuolar compartments","pmids":["11323436","11445562"],"confidence":"High","gaps":["No structural information on how YKT6 discriminates among different SNARE partners","Mechanism of YKT6 membrane recruitment unknown at this stage"]},{"year":2002,"claim":"Discovery of a distinct syntaxin 5/GS28/Ykt6/GS15 complex at the medial Golgi and in endosome-to-TGN transport demonstrated that YKT6 participates in at least two non-overlapping quaternary SNARE complexes with shared Q-SNARE partners.","evidence":"Reciprocal Co-IP from Golgi extracts identifying GS15 as alternative to Bet1 in a YKT6-containing complex; in vitro transport assay with Shiga toxin B confirming each SNARE is required for EE/RE-to-TGN transport","pmids":["12388752","15215310"],"confidence":"High","gaps":["Whether SNARE complex selectivity is achieved through spatial segregation or regulatory mechanisms was unresolved"]},{"year":2003,"claim":"Demonstrating that the longin domain controls subcellular targeting and that cytosolic YKT6 is conformationally inactive for SNARE assembly revealed the autoregulatory principle governing this SNARE, while the longin domain's ability to present palmitoyl-CoA to Vac8 uncovered an unexpected non-SNARE catalytic function.","evidence":"Neuronal localization, density gradient fractionation, and SNARE assembly assays showing longin-directed targeting; in vitro vacuole fusion assay demonstrating longin-dependent palmitoyl transfer to Vac8","pmids":["12589064","14685280"],"confidence":"High","gaps":["Whether palmitoyl transfer activity extends to substrates beyond Vac8 was untested","Structural basis of palmitoyl-CoA binding not yet resolved"]},{"year":2004,"claim":"Systematic dissection of C-terminal lipid modifications established that farnesylation precedes palmitoylation, that double lipid modification is essential for membrane association and intra-Golgi transport, and that the longin domain possesses intrinsic self-palmitoylation activity—revealing the full lipid-modification cascade controlling YKT6 activation.","evidence":"Metabolic labeling, mutagenesis of lipidation sites, in vitro transport assays, and in vitro palmitoylation assays with recombinant protein","pmids":["15044687","15479160","15331663"],"confidence":"High","gaps":["Identity of enzymes mediating palmitoylation/depalmitoylation cycles in vivo was unclear","Structural basis of closed conformation at atomic resolution was incomplete"]},{"year":2008,"claim":"The crystal structure of the yeast longin domain and biophysical characterization of farnesylated YKT6 provided the first atomic-level view of how the farnesyl group docks into a hydrophobic pocket to stabilize the closed conformation, while DHHC acyltransferase studies established that depalmitoylation-driven recycling prevents inappropriate membrane trapping.","evidence":"X-ray crystallography at 2.5 Å, CD spectroscopy, SEC, limited proteolysis, SPR for lipid binding; yeast DHHC overexpression showing missorting of longin-domain mutants into MVB pathway","pmids":["18329045","18541004"],"confidence":"High","gaps":["Full-length structure of the closed state with both longin and SNARE domains was lacking","Kinetics and regulation of the palmitoylation/depalmitoylation cycle in vivo were unresolved"]},{"year":2016,"claim":"Single-molecule FRET demonstrated that YKT6 undergoes rapid (~200 μs) intramolecular conformational dynamics that are locked into a closed state by lipid (DPC), providing direct biophysical evidence for a lipid-regulated conformational switch.","evidence":"Single-molecule FRET and fluorescence cross-correlation spectroscopy on rat Ykt6 with DPC","pmids":["27493064"],"confidence":"Medium","gaps":["DPC is a detergent surrogate; behavior on physiological lipid bilayers was not tested","Phosphorylation-mediated opening was not yet linked to this dynamic"]},{"year":2017,"claim":"Demonstrating that YKT6 serves as a v-SNARE for constitutive secretory vesicle fusion at the plasma membrane in both Drosophila and mammalian cells expanded its functional repertoire beyond Golgi and endosomal trafficking to the terminal secretory pathway.","evidence":"Combinatorial RNAi depletion with quantitative secretion assays in Drosophila S2 cells and mammalian cells","pmids":["28403141"],"confidence":"High","gaps":["The cognate Q-SNARE partners for PM fusion were not defined","Whether YKT6 and VAMP3 act in parallel or sequential pathways at the PM was unclear"]},{"year":2018,"claim":"Multiple groups converged on YKT6 as an autophagosomal SNARE mediating autophagosome–lysosome/vacuole fusion, forming complexes with SNAP29/STX7 (mammals) and Syx17/Snap29 (Drosophila), with the finding that VAMP7/VAMP8 can outcompete YKT6 suggesting a priming or regulatory role; in vitro reconstitution with intact yeast autophagosomes confirmed Ykt6 as the R-SNARE requiring HOPS and Ypt7.","evidence":"STX17-KO plus YKT6 siRNA in HeLa; Drosophila loss-of-function genetics with rescue; novel in vitro autophagosome-vacuole fusion assay with purified organelles","pmids":["29789439","29694367","30097515"],"confidence":"High","gaps":["Whether YKT6 is the primary fusogenic SNARE or a regulatory/priming SNARE was debated","Mechanism of YKT6 recruitment to autophagosomes was not fully defined"]},{"year":2019,"claim":"Linking α-synuclein pathology to aberrant inactivation of YKT6—and showing that farnesyltransferase inhibitors restore its membrane-bound active form and lysosomal function—established YKT6 as a convergent target in Parkinson's disease and revealed that lysosomal stress normally triggers YKT6 activation for hydrolase trafficking.","evidence":"Patient iPSC-derived neurons, co-immunoprecipitation of α-synuclein with YKT6, membrane fractionation, FTI treatment in cells and mice","pmids":["31648898"],"confidence":"High","gaps":["Whether FTI-mediated activation of YKT6 is therapeutically viable long-term was untested","Direct phosphorylation events during lysosomal stress signaling were not mapped"]},{"year":2020,"claim":"Identification of Atg1/ULK1 phosphorylation as the switch that keeps autophagosomal YKT6 inactive until fusion is appropriate, and phosphorylation-dependent opening regulating Wnt recycling through endosomes, established phosphorylation as the master regulatory mechanism for YKT6's closed-to-open transition across pathways.","evidence":"In vitro kinase assays, phospho-site mutagenesis, genetic epistasis with Dsl1 complex for autophagosomal recruitment, Drosophila wing genetics for Wnt trafficking, proximity proteomics (BioID)","pmids":["33025734","33207719","32611603"],"confidence":"High","gaps":["Identity of the phosphatase(s) that dephosphorylate YKT6 to permit fusion was unknown","Whether phosphorylation regulates the same residue across all trafficking pathways was unclear"]},{"year":2021,"claim":"NMR-resolved structural characterization confirmed a Ca²⁺-responsive phosphorylation-driven conformational switch, while identification of GGTase-III (PTAR1-dependent) as the enzyme mediating geranylgeranylation of the second cysteine established the full prenylation cascade required for lysosomal hydrolase sorting.","evidence":"NMR structural analysis with phospho-mimetic mutants; PTAR1-KO cells with cathepsin D/β-hexosaminidase secretion assays, LC3B accumulation, Golgi morphology","pmids":["33723042","33035318"],"confidence":"High","gaps":["How GGTase-III accesses the farnesylated substrate in the context of the closed conformation was not structurally resolved","Whether double prenylation and palmitoylation are coordinated or independent was unclear"]},{"year":2023,"claim":"Reconstitution of the YKT6–STX17–SNAP29 priming complex and demonstration that VAMP8 displaces YKT6 to form the fusogenic complex resolved the longstanding question of whether YKT6 is a direct fusogenic SNARE or a priming factor in autophagosome–lysosome fusion, while human disease-causing YKT6 variants failed rescue in Drosophila, confirming its essential in vivo role.","evidence":"In vitro lipid and content mixing assays with reconstituted SNARE complexes; Drosophila genetic rescue with human variant constructs; single-molecule FRET with phospho-mimetic and species-specific mutants","pmids":["38340317","38522068","37380075"],"confidence":"High","gaps":["Whether the priming mechanism applies at non-autophagosomal fusion steps is unknown","Full clinical spectrum of human YKT6 loss-of-function disease is not yet delineated"]},{"year":2025,"claim":"Localization of YKT6 to hippocampal synaptic spines and its requirement for LTP-dependent AMPA receptor insertion identified a neuronal trafficking function linking its SNARE activity to synaptic plasticity, and confirmed that α-synuclein pathology disrupts this process.","evidence":"Immunofluorescence, electrophysiology (mEPSC recording), LTP induction, surface receptor assays in hippocampal neurons; GGTase-III (Ecm9) knockout in yeast confirming conserved double prenylation requirement for autophagy","pmids":["40840626","40049413"],"confidence":"Medium","gaps":["The specific SNARE complex mediating AMPA receptor exocytosis at synapses has not been identified","Whether YKT6 synaptic function depends on the same phospho-regulatory switch is untested","Single study for the synaptic plasticity phenotype"]},{"year":null,"claim":"Key unresolved questions include the identity of the phosphatase(s) that dephosphorylate YKT6 to permit fusion, whether the priming mechanism (displacement by VAMP8) operates at non-autophagosomal fusion steps, the full-length atomic structure of YKT6 in both closed and open states on a membrane, and the complete clinical spectrum and pathomechanism of human YKT6 deficiency.","evidence":"","pmids":[],"confidence":"Low","gaps":["No phosphatase identified","No full-length membrane-bound structure","Human disease spectrum incompletely characterized","Priming-to-fusion handoff mechanism not tested outside autophagy"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,6,14,15,23]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[4,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[5,10,12]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,2,5,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,5,7,12]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[6,19]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[15,17]},{"term_id":"GO:0005773","term_label":"vacuole","supporting_discovery_ids":[4,9,16]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[14,16,18]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[13,26]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,2,5,6,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[14,15,16,18,22,23,24]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[17,21]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,8,21,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[19,20]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[26]}],"complexes":["Syntaxin5-GS28-Bet1-YKT6 SNARE complex","Syntaxin5-GS28-GS15-YKT6 SNARE complex","STX17-SNAP29-YKT6 priming complex","SNAP29-STX7-YKT6 SNARE complex"],"partners":["STX5","GOSR1","BET1","BET1L","SNAP29","STX17","STX7","SNCA"],"other_free_text":[]},"mechanistic_narrative":"YKT6 is a lipid-anchored R-SNARE that participates in multiple membrane fusion events across the secretory, endosomal, and autophagic pathways by cycling between an autoinhibited closed cytosolic form and an active open membrane-bound form. The longin domain folds back onto the SNARE motif to maintain the closed conformation, which is stabilized by farnesylation; phosphorylation by Atg1/ULK1 or Ca²⁺-dependent kinases triggers conformational opening, enabling sequential palmitoylation and double prenylation (farnesyl plus geranylgeranyl via GGTase-III) that anchor YKT6 to target membranes [PMID:15044687, PMID:33723042, PMID:33025734, PMID:40049413]. In its membrane-bound form, YKT6 assembles into distinct quaternary SNARE complexes—with syntaxin 5/GS28/Bet1 for ER-to-Golgi transport, syntaxin 5/GS28/GS15 for intra-Golgi and endosome-to-TGN trafficking, and SNAP29/STX7 or a priming complex with STX17/SNAP29 (subsequently displaced by VAMP8) for autophagosome–lysosome fusion—and additionally mediates constitutive secretory vesicle fusion at the plasma membrane and Wnt recycling through endosomes [PMID:11323436, PMID:12388752, PMID:29789439, PMID:38340317, PMID:28403141, PMID:32611603]. The longin domain also possesses an intrinsic palmitoyl-CoA binding activity that mediates self-palmitoylation and non-enzymatic palmitoyl transfer to substrates such as Vac8, linking YKT6 to protein lipidation during vacuole fusion [PMID:14685280, PMID:15479160]. Homozygous loss-of-function variants in YKT6 cause a Mendelian disorder with neuronal and hepatic dysfunction, consistent with its essential role in autophagic flux [PMID:38522068]."},"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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Mutation of the 0-layer arginine in Ykt6 (R165Q) in complexes where it contributes a fourth glutamine renders the complex nonfunctional.\",\n      \"method\": \"Genetic suppressor screen (multicopy and low-copy), vacuolar transport assays, site-directed mutagenesis of 0-layer residues\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple suppressor assays and mutagenesis, replicated across 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 Golgi, implicating this quaternary complex in early cisternae trafficking; components of COPI coat co-immunoprecipitate selectively with GS15 from Golgi extracts.\",\n      \"method\": \"Co-immunoprecipitation, immuno-EM, siRNA knockdown, overexpression of dominant-negative mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, immuno-EM localization, functional KD, replicated complex identification\",\n      \"pmids\": [\"12388752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Rat Ykt6 is a neuronal SNARE that localizes to a specialized punctate compartment distinct from known endomembrane organelles; its profilin-like longin domain directs this unique targeting even in the absence of prenylation; cytosolic Ykt6 is conformationally inactive for SNARE complex assembly.\",\n      \"method\": \"Immunofluorescence microscopy, density gradient fractionation, mutagenesis of longin domain and prenylation sites, SNARE complex assembly assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (localization, fractionation, mutagenesis, assembly assays) in single study\",\n      \"pmids\": [\"12589064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Yeast Ykt6 mediates palmitoylation of the fusion factor Vac8 during homotypic vacuole fusion through a novel subreaction controlled by a Sec17-independent function of Sec18; the N-terminal longin domain of Ykt6 presents palmitoyl-CoA to Vac8, and transfer to Vac8's SH4 domain occurs spontaneously (non-enzymatically).\",\n      \"method\": \"In vitro vacuole fusion assay, biochemical fractionation, mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mechanistic dissection of subreaction and domain requirements\",\n      \"pmids\": [\"14685280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"YKT6 exists as both cytosolic (inactive) and membrane-bound (active) forms; both are farnesylated at the C-terminal cysteine of CCAIM, and farnesylation is a prerequisite for subsequent palmitoylation of the upstream cysteine. Double lipid modification is required for intra-Golgi transport in vitro and cell viability. The N-terminal longin domain interacts with the SNARE motif to maintain YKT6 in a closed, inactive conformation, and conformational changes control lipid modification and membrane recruitment.\",\n      \"method\": \"In vitro transport assay, metabolic labeling, mutagenesis of lipidation sites, cell viability assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro transport reconstitution + lipid modification biochemistry + mutagenesis, mechanistically comprehensive\",\n      \"pmids\": [\"15044687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The syntaxin 5/Ykt6/GS28/GS15 SNARE complex mediates transport from the early/recycling endosome to the trans-Golgi network (EE/RE-TGN); antibodies to each of these four SNAREs specifically inhibited this transport step in vitro; GS15 and Ykt6 redistribute from Golgi to endosomes when the recycling endosome is perturbed, suggesting cycling between these compartments.\",\n      \"method\": \"In vitro transport assay with Shiga toxin B subunit marker, antibody inhibition, siRNA knockdown of GS15, SNX3 overexpression morphological analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro transport assay with antibody inhibition, siRNA functional validation, multiple SNAREs tested\",\n      \"pmids\": [\"15215310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The longin domain of mammalian Ykt6 controls subcellular targeting through intramolecular protein-protein interactions with the SNARE motif and protein-lipid interactions with lipid groups at the C-terminus; two hydrophobic pockets on each face of the longin domain suppress mislocalization, and one suppresses palmitoylation-dependent mislocalization to the plasma membrane; both interactions maintain a compact closed conformation preventing premature membrane insertion.\",\n      \"method\": \"Mutagenesis of longin domain surface residues, immunofluorescence localization, co-immunoprecipitation of intramolecular interactions\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic mutagenesis guided by crystal structure, multiple localization and interaction readouts\",\n      \"pmids\": [\"15331663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Ykt6 has intrinsic self-palmitoylating activity: the N-terminal longin domain contains a palmitoyl-CoA binding site required for covalent palmitoylation of its own C-terminal cysteine residues.\",\n      \"method\": \"In vitro palmitoylation assay with [3H]palmitoyl-CoA, recombinant protein mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro enzymatic assay with radiolabeled substrate and mutagenesis\",\n      \"pmids\": [\"15479160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Yeast Ykt6 is released from vacuolar membranes during an early stage of vacuole fusion in a priming-dependent (SNARE disassembly-dependent) manner; yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif, and mutation of the palmitoylation site prevents stable membrane association and is lethal, suggesting depalmitoylation-driven recycling of this SNARE.\",\n      \"method\": \"In vitro vacuole fusion assay, palmitoylation assay, site-directed mutagenesis, cell viability\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro fusion assay, biochemical palmitoylation assay, mutagenesis with viability readout\",\n      \"pmids\": [\"15723044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Farnesylation of Ykt6 (at the CAAX box) increases protein stability, helical content, and compactness as shown by CD spectroscopy, size exclusion chromatography, and limited proteolysis; farnesylated Ykt6 binds lipid membranes independently of membrane charge. The crystal structure of the yeast Ykt6 longin domain (residues 1–140) at 2.5 Å reveals a hydrophobic surface patch that accommodates the lipid moiety in the closed conformation.\",\n      \"method\": \"In vitro farnesylation, size exclusion chromatography, limited proteolysis, circular dichroism, surface plasmon resonance, X-ray crystallography at 2.5 Å\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with multiple biophysical and biochemical assays\",\n      \"pmids\": [\"18329045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ykt6 cycles between cytosol and membranes through intramolecular interaction between its N-terminal longin domain and C-terminal SNARE domain; a mutant deficient in this intramolecular interaction accumulates stably on membranes and is not released from vacuoles. Ykt6 is a substrate of DHHC acyltransferases; overexpression of vacuolar acyltransferase Pfa3 drives a longin-domain mutant (F42S) into the vacuolar lumen, indicating that depalmitoylation is required to prevent Ykt6 entry into the MVB pathway.\",\n      \"method\": \"Mutagenesis, in vivo localization, vacuole fusion assay, DHHC acyltransferase overexpression\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis + localization + functional assay, mechanistically comprehensive for the cycling mechanism\",\n      \"pmids\": [\"18541004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Single-molecule FRET and FCCS demonstrate 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 DPC eliminates this dynamics and locks Ykt6 in a closed conformation, supporting lipid-regulated conformational switching.\",\n      \"method\": \"Single-molecule FRET, Fluorescence Cross-Correlation Spectroscopy, molecular dynamics simulation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — single-molecule biophysics + MD, but single lab, one study\",\n      \"pmids\": [\"27493064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"YKT6 and VAMP3/Synaptobrevin function as v-SNAREs in constitutive secretory vesicle fusion with the plasma membrane in both Drosophila and mammalian cells; RNAi depletion of YKT6 blocks constitutive secretion, identifying an evolutionarily conserved role of YKT6 in Golgi-to-PM transport.\",\n      \"method\": \"RNAi combinatorial depletion in Drosophila cells, quantitative secretion assay, RNAi in mammalian cells\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — combinatorial gene depletion with quantitative assay, replicated in two organisms\",\n      \"pmids\": [\"28403141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"YKT6 is an autophagosomal SNARE protein that mediates autophagosome-lysosome fusion independently of STX17: YKT6 depletion partially blocks fusion in wild-type and completely blocks it in STX17-KO HeLa cells. YKT6 forms a SNARE complex with SNAP29 and lysosomal STX7 on autophagosomes. Recruitment to autophagosomes requires the N-terminal longin domain but not C-terminal palmitoylation/farnesylation.\",\n      \"method\": \"STX17 KO and YKT6 siRNA depletion in HeLa cells, autophagosome-lysosome fusion assay, Co-immunoprecipitation, domain mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO + siRNA epistasis + Co-IP, multiple orthogonal approaches, independently replicated by contemporaneous studies\",\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/autolysosomes; it forms a SNARE complex with Syx17 and Snap29. Vamp7 can outcompete Ykt6 from this complex, and Vamp7 overexpression rescues fusion defects in ykt6 mutants. An RQ mutation in the 0-layer of Ykt6 retains normal autophagic activity, suggesting Ykt6 acts as a non-canonical regulatory SNARE in this process; palmitoylation and farnesylation site mutants do not rescue.\",\n      \"method\": \"Drosophila genetics (loss-of-function mutants, rescue constructs), Co-immunoprecipitation, autophagic flux assays, site-directed mutagenesis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo Drosophila genetics + Co-IP + mutagenesis, comprehensive mechanistic dissection\",\n      \"pmids\": [\"29694367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A novel in vitro assay with intact yeast autophagosomes and vacuoles identifies Ykt6 as the autophagosomal R-SNARE; fusion requires ATP, physiological temperature, the HOPS tethering complex, Ypt7 GTPase, Mon1-Ccz1 GEF, and the entire fusion machinery.\",\n      \"method\": \"Novel in vitro autophagosome-vacuole fusion assay with purified organelles, genetic depletion of individual components\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with intact organelles, systematic requirement analysis\",\n      \"pmids\": [\"30097515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cytosolic ykt6 is normally autoinhibited by a farnesyl-mediated regulatory mechanism; during lysosomal stress, ykt6 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 the lysosomal stress response. Farnesyltransferase inhibitors activate ykt6 by promoting its membrane-bound open form, restoring lysosomal activity.\",\n      \"method\": \"Live-cell imaging, membrane fractionation, co-immunoprecipitation (α-syn binding to ykt6), patient iPSC-derived neurons, farnesyltransferase inhibitor treatment in cells and mice\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, patient-derived neurons + mouse model, mechanistic pathway established\",\n      \"pmids\": [\"31648898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Ykt6 function on autophagosomes is regulated by the Atg1 kinase complex through direct phosphorylation, keeping the Ykt6 pool on autophagosomal membranes inactive; dephosphorylation of Ykt6 is required for its engagement in autophagosome-vacuole fusion. Ykt6 is recruited to early autophagosome precursors through a mechanism requiring the ER-resident Dsl1 complex and COPII-coated vesicles.\",\n      \"method\": \"In vitro kinase assay (Atg1 phosphorylation of Ykt6), genetic epistasis (Dsl1 complex mutants), autophagy flux assays, Co-immunoprecipitation\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay + genetic epistasis + Co-IP, mechanistically comprehensive\",\n      \"pmids\": [\"33025734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Phosphorylation of Ykt6 in its SNARE domain mediates its conversion from a closed cytosolic to an open membrane-bound conformation, regulating membrane recruitment to multiple organelles; phosphorylated Ykt6 functionally regulates Wnt protein trafficking and extracellular vesicle secretion in Drosophila wing epithelium. Most Ykt6 is cytosolic but is recruited to de-acidified compartments to recycle Wnts via Rab4-positive recycling endosomes.\",\n      \"method\": \"Proximity-dependent proteomics (BioID), membrane fractionation, in vivo Drosophila genetics, in vitro biochemical analyses, phospho-site mutagenesis\",\n      \"journal\": \"Biomolecules / Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — phospho-mutagenesis + proximity proteomics + in vivo genetics + biochemical fractionation, multiple organisms\",\n      \"pmids\": [\"33207719\", \"32611603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A conformational switch driven by phosphorylation at an evolutionarily conserved site (regulated by Ca2+ signaling) allows Ykt6 to transition from a closed cytosolic form to an open membrane-bound form; phosphorylated Ykt6 has an altered spectrum of protein interactions, causing defects in both secretory and autophagy pathways in Parkinson's disease models.\",\n      \"method\": \"NMR, biochemical assays, mutagenesis, Parkinson's disease cell models, Ca2+ signaling manipulation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural data + biochemical assays + mutagenesis + functional pathway analysis\",\n      \"pmids\": [\"33723042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Double prenylation of Ykt6 (farnesylation by FTase followed by geranylgeranylation by a novel GGTase-III containing PTAR1 subunit) at two C-terminal cysteines is required for proper lysosomal hydrolase trafficking; in PTAR1-KO cells (singly farnesylated Ykt6), cathepsin D and β-hexosaminidase are missorted and secreted extracellularly, Golgi structure is disrupted, and LC3B accumulates.\",\n      \"method\": \"PTAR1 gene knockout, lysosomal hydrolase secretion assay, autophagy flux assay (LC3B), Golgi morphology analysis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with multiple defined functional readouts (hydrolase sorting, Golgi integrity, autophagy)\",\n      \"pmids\": [\"33035318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Mammalian autophagosomal YKT6 is phosphorylated by ULK1 kinase, which prevents premature bundling with lysosomal SNARE proteins and inhibits autophagosome-lysosome fusion; alterations in YKT6 function produce both early and late autophagy defects in mammalian cells and C. elegans, reducing survival.\",\n      \"method\": \"In vitro ULK1 kinase assay, phospho-site mutagenesis, autophagy flux assays in mammalian cells and C. elegans, co-immunoprecipitation of SNARE complexes\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay + mutagenesis + multi-organism validation, mechanistically comprehensive\",\n      \"pmids\": [\"36644903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"YKT6 forms a priming complex with STX17 and SNAP29 on autophagosomes via its SNARE domain; 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, demonstrating a priming role for YKT6 in efficient membrane fusion.\",\n      \"method\": \"Co-immunoprecipitation, lipid mixing assay, content mixing assay, domain mutagenesis, autophagy flux assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro lipid and content mixing assays + Co-IP + mutagenesis, comprehensive mechanistic dissection\",\n      \"pmids\": [\"38340317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Homozygous missense variants in YKT6 (Tyr185Cys, Tyr64Cys) cause partial loss of function in Drosophila, failing to rescue lethality and autophagic flux defects in dYkt6 mutant flies, establishing YKT6 as essential for autophagic flux and neuronal/hepatic function in vivo.\",\n      \"method\": \"Drosophila genetic rescue with human variant constructs, autophagic flux assays, expression pattern analysis\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic rescue with variant constructs + autophagic flux readout, strong functional validation\",\n      \"pmids\": [\"38522068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ykt6 conformational dynamics differ between yeast and rat: yeast Ykt6 adopts more open conformations and cannot bind DPC (which locks rat Ykt6 in a closed state); a T46L/Q57A point mutation converts yeast Ykt6 to a more closed, DPC-bound state. Phospho-mimic S174D shifts rat Ykt6 toward a more open state, confirming phosphorylation as a regulator of the closed-to-open conformational switch.\",\n      \"method\": \"Single-molecule FRET, biochemical characterization, molecular dynamics simulation, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — single-molecule FRET + MD + mutagenesis, multiple orthogonal approaches\",\n      \"pmids\": [\"37380075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ykt6 localizes to synaptic spines in mammalian hippocampus and regulates GluA1 and GluA2 glutamate receptor surface expression in an LTP-dependent manner; Ykt6 also modulates spine morphology, synaptic vesicle pool dynamics, and miniature EPSC amplitude and frequency. α-Synuclein pathology disrupts Ykt6 function and LTP.\",\n      \"method\": \"Immunofluorescence/live imaging, electrophysiology (mEPSC recording), LTP induction, surface receptor assays, loss-of-function studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization + electrophysiology + LTP assay, single study\",\n      \"pmids\": [\"40840626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Double prenylation (farnesyl + geranylgeranyl) is an evolutionarily conserved modification of Ykt6 in yeast, mediated by the GGTase-III complex consisting of Ecm9 (α subunit) and Bet2 (β subunit); loss of Ecm9 prevents double prenylation, impairs Ykt6 localization to organelle membranes including autophagosomes, and reduces autophagic activity and cell wall integrity.\",\n      \"method\": \"Structural prediction, in vitro prenylation assay, MALDI-TOF/TOF mass spectrometry, genetic deletion (ecm9Δ), autophagy assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mass spectrometry confirmation of prenylation + genetic KO + multiple functional readouts\",\n      \"pmids\": [\"40049413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Ykt6 regulates epithelial cell migration as a negative regulator; it upregulates microRNA-145 expression, which selectively decreases Junctional Adhesion Molecule A (JAM-A) levels, thereby limiting Rap1 and Rac1 small GTPase activity and attenuating cell spreading and motility.\",\n      \"method\": \"siRNA knockdown, overexpression, miRNA reporter assay, small GTPase activity assays, migration/invasion assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional KD/OE with pathway placement but single lab, limited mechanistic depth for miRNA mechanism\",\n      \"pmids\": [\"30010460\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"YKT6 is a uniquely lipid-anchored R-SNARE that cycles between an autoinhibited closed cytosolic form (farnesylated but not palmitoylated, with the longin domain masking the SNARE motif) and an active open membrane-bound form (doubly prenylated with farnesyl and geranylgeranyl, plus palmitoylated), with conformational opening regulated by phosphorylation (by Atg1/ULK1 or Ca2+-dependent kinases) and lipid modifications; in its active form it participates in multiple SNARE complexes mediating ER-to-Golgi transport (with syntaxin 5/GS28/Bet1), intra-Golgi and endosome-to-TGN transport (with syntaxin 5/GS28/GS15), constitutive secretion to the plasma membrane, Wnt recycling via endosomes, and autophagosome-lysosome fusion (forming a priming complex with STX17/SNAP29 that is subsequently displaced by VAMP8 to drive fusion, as well as an independent complex with SNAP29/STX7), and additionally mediates protein palmitoylation via its longin domain and regulates lysosomal hydrolase trafficking and synaptic receptor (GluA1/GluA2) insertion during LTP.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"YKT6 is a lipid-anchored R-SNARE that participates in multiple membrane fusion events across the secretory, endosomal, and autophagic pathways by cycling between an autoinhibited closed cytosolic form and an active open membrane-bound form. The longin domain folds back onto the SNARE motif to maintain the closed conformation, which is stabilized by farnesylation; phosphorylation by Atg1/ULK1 or Ca²⁺-dependent kinases triggers conformational opening, enabling sequential palmitoylation and double prenylation (farnesyl plus geranylgeranyl via GGTase-III) that anchor YKT6 to target membranes [PMID:15044687, PMID:33723042, PMID:33025734, PMID:40049413]. In its membrane-bound form, YKT6 assembles into distinct quaternary SNARE complexes—with syntaxin 5/GS28/Bet1 for ER-to-Golgi transport, syntaxin 5/GS28/GS15 for intra-Golgi and endosome-to-TGN trafficking, and SNAP29/STX7 or a priming complex with STX17/SNAP29 (subsequently displaced by VAMP8) for autophagosome–lysosome fusion—and additionally mediates constitutive secretory vesicle fusion at the plasma membrane and Wnt recycling through endosomes [PMID:11323436, PMID:12388752, PMID:29789439, PMID:38340317, PMID:28403141, PMID:32611603]. The longin domain also possesses an intrinsic palmitoyl-CoA binding activity that mediates self-palmitoylation and non-enzymatic palmitoyl transfer to substrates such as Vac8, linking YKT6 to protein lipidation during vacuole fusion [PMID:14685280, PMID:15479160]. Homozygous loss-of-function variants in YKT6 cause a Mendelian disorder with neuronal and hepatic dysfunction, consistent with its essential role in autophagic flux [PMID:38522068].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Identifying YKT6 as a participant in ER-to-Golgi and vacuolar SNARE complexes established it as a versatile R-SNARE functioning at multiple trafficking steps rather than a single compartment.\",\n      \"evidence\": \"Co-IP of mammalian YKT6 with syntaxin 5/GS28/Bet1 plus antibody-mediated inhibition of ER-Golgi transport; yeast genetic suppressor screens linking Ykt6 to Vti1p-containing complexes at prevacuolar and vacuolar compartments\",\n      \"pmids\": [\"11323436\", \"11445562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural information on how YKT6 discriminates among different SNARE partners\", \"Mechanism of YKT6 membrane recruitment unknown at this stage\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery of a distinct syntaxin 5/GS28/Ykt6/GS15 complex at the medial Golgi and in endosome-to-TGN transport demonstrated that YKT6 participates in at least two non-overlapping quaternary SNARE complexes with shared Q-SNARE partners.\",\n      \"evidence\": \"Reciprocal Co-IP from Golgi extracts identifying GS15 as alternative to Bet1 in a YKT6-containing complex; in vitro transport assay with Shiga toxin B confirming each SNARE is required for EE/RE-to-TGN transport\",\n      \"pmids\": [\"12388752\", \"15215310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SNARE complex selectivity is achieved through spatial segregation or regulatory mechanisms was unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating that the longin domain controls subcellular targeting and that cytosolic YKT6 is conformationally inactive for SNARE assembly revealed the autoregulatory principle governing this SNARE, while the longin domain's ability to present palmitoyl-CoA to Vac8 uncovered an unexpected non-SNARE catalytic function.\",\n      \"evidence\": \"Neuronal localization, density gradient fractionation, and SNARE assembly assays showing longin-directed targeting; in vitro vacuole fusion assay demonstrating longin-dependent palmitoyl transfer to Vac8\",\n      \"pmids\": [\"12589064\", \"14685280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether palmitoyl transfer activity extends to substrates beyond Vac8 was untested\", \"Structural basis of palmitoyl-CoA binding not yet resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Systematic dissection of C-terminal lipid modifications established that farnesylation precedes palmitoylation, that double lipid modification is essential for membrane association and intra-Golgi transport, and that the longin domain possesses intrinsic self-palmitoylation activity—revealing the full lipid-modification cascade controlling YKT6 activation.\",\n      \"evidence\": \"Metabolic labeling, mutagenesis of lipidation sites, in vitro transport assays, and in vitro palmitoylation assays with recombinant protein\",\n      \"pmids\": [\"15044687\", \"15479160\", \"15331663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of enzymes mediating palmitoylation/depalmitoylation cycles in vivo was unclear\", \"Structural basis of closed conformation at atomic resolution was incomplete\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The crystal structure of the yeast longin domain and biophysical characterization of farnesylated YKT6 provided the first atomic-level view of how the farnesyl group docks into a hydrophobic pocket to stabilize the closed conformation, while DHHC acyltransferase studies established that depalmitoylation-driven recycling prevents inappropriate membrane trapping.\",\n      \"evidence\": \"X-ray crystallography at 2.5 Å, CD spectroscopy, SEC, limited proteolysis, SPR for lipid binding; yeast DHHC overexpression showing missorting of longin-domain mutants into MVB pathway\",\n      \"pmids\": [\"18329045\", \"18541004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length structure of the closed state with both longin and SNARE domains was lacking\", \"Kinetics and regulation of the palmitoylation/depalmitoylation cycle in vivo were unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Single-molecule FRET demonstrated that YKT6 undergoes rapid (~200 μs) intramolecular conformational dynamics that are locked into a closed state by lipid (DPC), providing direct biophysical evidence for a lipid-regulated conformational switch.\",\n      \"evidence\": \"Single-molecule FRET and fluorescence cross-correlation spectroscopy on rat Ykt6 with DPC\",\n      \"pmids\": [\"27493064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DPC is a detergent surrogate; behavior on physiological lipid bilayers was not tested\", \"Phosphorylation-mediated opening was not yet linked to this dynamic\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating that YKT6 serves as a v-SNARE for constitutive secretory vesicle fusion at the plasma membrane in both Drosophila and mammalian cells expanded its functional repertoire beyond Golgi and endosomal trafficking to the terminal secretory pathway.\",\n      \"evidence\": \"Combinatorial RNAi depletion with quantitative secretion assays in Drosophila S2 cells and mammalian cells\",\n      \"pmids\": [\"28403141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The cognate Q-SNARE partners for PM fusion were not defined\", \"Whether YKT6 and VAMP3 act in parallel or sequential pathways at the PM was unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Multiple groups converged on YKT6 as an autophagosomal SNARE mediating autophagosome–lysosome/vacuole fusion, forming complexes with SNAP29/STX7 (mammals) and Syx17/Snap29 (Drosophila), with the finding that VAMP7/VAMP8 can outcompete YKT6 suggesting a priming or regulatory role; in vitro reconstitution with intact yeast autophagosomes confirmed Ykt6 as the R-SNARE requiring HOPS and Ypt7.\",\n      \"evidence\": \"STX17-KO plus YKT6 siRNA in HeLa; Drosophila loss-of-function genetics with rescue; novel in vitro autophagosome-vacuole fusion assay with purified organelles\",\n      \"pmids\": [\"29789439\", \"29694367\", \"30097515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether YKT6 is the primary fusogenic SNARE or a regulatory/priming SNARE was debated\", \"Mechanism of YKT6 recruitment to autophagosomes was not fully defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking α-synuclein pathology to aberrant inactivation of YKT6—and showing that farnesyltransferase inhibitors restore its membrane-bound active form and lysosomal function—established YKT6 as a convergent target in Parkinson's disease and revealed that lysosomal stress normally triggers YKT6 activation for hydrolase trafficking.\",\n      \"evidence\": \"Patient iPSC-derived neurons, co-immunoprecipitation of α-synuclein with YKT6, membrane fractionation, FTI treatment in cells and mice\",\n      \"pmids\": [\"31648898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether FTI-mediated activation of YKT6 is therapeutically viable long-term was untested\", \"Direct phosphorylation events during lysosomal stress signaling were not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of Atg1/ULK1 phosphorylation as the switch that keeps autophagosomal YKT6 inactive until fusion is appropriate, and phosphorylation-dependent opening regulating Wnt recycling through endosomes, established phosphorylation as the master regulatory mechanism for YKT6's closed-to-open transition across pathways.\",\n      \"evidence\": \"In vitro kinase assays, phospho-site mutagenesis, genetic epistasis with Dsl1 complex for autophagosomal recruitment, Drosophila wing genetics for Wnt trafficking, proximity proteomics (BioID)\",\n      \"pmids\": [\"33025734\", \"33207719\", \"32611603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the phosphatase(s) that dephosphorylate YKT6 to permit fusion was unknown\", \"Whether phosphorylation regulates the same residue across all trafficking pathways was unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"NMR-resolved structural characterization confirmed a Ca²⁺-responsive phosphorylation-driven conformational switch, while identification of GGTase-III (PTAR1-dependent) as the enzyme mediating geranylgeranylation of the second cysteine established the full prenylation cascade required for lysosomal hydrolase sorting.\",\n      \"evidence\": \"NMR structural analysis with phospho-mimetic mutants; PTAR1-KO cells with cathepsin D/β-hexosaminidase secretion assays, LC3B accumulation, Golgi morphology\",\n      \"pmids\": [\"33723042\", \"33035318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GGTase-III accesses the farnesylated substrate in the context of the closed conformation was not structurally resolved\", \"Whether double prenylation and palmitoylation are coordinated or independent was unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Reconstitution of the YKT6–STX17–SNAP29 priming complex and demonstration that VAMP8 displaces YKT6 to form the fusogenic complex resolved the longstanding question of whether YKT6 is a direct fusogenic SNARE or a priming factor in autophagosome–lysosome fusion, while human disease-causing YKT6 variants failed rescue in Drosophila, confirming its essential in vivo role.\",\n      \"evidence\": \"In vitro lipid and content mixing assays with reconstituted SNARE complexes; Drosophila genetic rescue with human variant constructs; single-molecule FRET with phospho-mimetic and species-specific mutants\",\n      \"pmids\": [\"38340317\", \"38522068\", \"37380075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the priming mechanism applies at non-autophagosomal fusion steps is unknown\", \"Full clinical spectrum of human YKT6 loss-of-function disease is not yet delineated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Localization of YKT6 to hippocampal synaptic spines and its requirement for LTP-dependent AMPA receptor insertion identified a neuronal trafficking function linking its SNARE activity to synaptic plasticity, and confirmed that α-synuclein pathology disrupts this process.\",\n      \"evidence\": \"Immunofluorescence, electrophysiology (mEPSC recording), LTP induction, surface receptor assays in hippocampal neurons; GGTase-III (Ecm9) knockout in yeast confirming conserved double prenylation requirement for autophagy\",\n      \"pmids\": [\"40840626\", \"40049413\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific SNARE complex mediating AMPA receptor exocytosis at synapses has not been identified\", \"Whether YKT6 synaptic function depends on the same phospho-regulatory switch is untested\", \"Single study for the synaptic plasticity phenotype\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the phosphatase(s) that dephosphorylate YKT6 to permit fusion, whether the priming mechanism (displacement by VAMP8) operates at non-autophagosomal fusion steps, the full-length atomic structure of YKT6 in both closed and open states on a membrane, and the complete clinical spectrum and pathomechanism of human YKT6 deficiency.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No phosphatase identified\", \"No full-length membrane-bound structure\", \"Human disease spectrum incompletely characterized\", \"Priming-to-fusion handoff mechanism not tested outside autophagy\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 6, 14, 15, 23]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [5, 10, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 2, 5, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 5, 7, 12]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [6, 19]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [15, 17]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [4, 9, 16]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [14, 16, 18]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [13, 26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 2, 5, 6, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [14, 15, 16, 18, 22, 23, 24]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [17, 21]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 8, 21, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [19, 20]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"complexes\": [\n      \"Syntaxin5-GS28-Bet1-YKT6 SNARE complex\",\n      \"Syntaxin5-GS28-GS15-YKT6 SNARE complex\",\n      \"STX17-SNAP29-YKT6 priming complex\",\n      \"SNAP29-STX7-YKT6 SNARE complex\"\n    ],\n    \"partners\": [\n      \"STX5\",\n      \"GOSR1\",\n      \"BET1\",\n      \"BET1L\",\n      \"SNAP29\",\n      \"STX17\",\n      \"STX7\",\n      \"SNCA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}