{"gene":"STX5","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":1992,"finding":"Sed5p (yeast ortholog of STX5) is an integral membrane protein required for ER-to-Golgi vesicular transport; depletion blocks carboxypeptidase Y transport to the Golgi and causes dramatic accumulation of ER membranes and vesicles; overexpression reduces ER-to-Golgi transport efficiency and causes vesicle accumulation. Immunofluorescence shows Sed5p localizes to punctate cytoplasmic structures, not ER membranes.","method":"Gene depletion/overexpression with cargo transport assay (carboxypeptidase Y), immunofluorescence, electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (depletion, overexpression, transport assay, immunofluorescence, EM) in a foundational study, foundational to the field","pmids":["1400588"],"is_preprint":false},{"year":1994,"finding":"Sed5p is a member of the syntaxin family and localizes to the cis-Golgi network (tubulo-vesicular compartment on the cis side of the Golgi). Both its transmembrane domain and cytoplasmic domain contribute independently to its Golgi localization; the transmembrane domain contains targeting information, but cytoplasmic domain provides an additional targeting mechanism sufficient when the transmembrane domain is altered.","method":"Expression of epitope-tagged Sed5 and chimeric molecules in COS cells, immuno-EM, domain-swap mutagenesis, in vivo yeast functional assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (immuno-EM, chimeric mutagenesis, in vivo complementation), replicated across yeast/Drosophila/rat homologs","pmids":["7929581"],"is_preprint":false},{"year":1998,"finding":"The N-terminal helix of Sed5 is responsible for binding Sly1 protein, whereas Sly1 binding to syntaxin 1 requires almost the entire molecule. The N-terminal region of Sed5 also binds to its own C-terminal helix (intramolecular interaction), and Sly1 binding interferes with this intramolecular interaction.","method":"Protein-protein interaction assays dissecting individual helices of Sed5; pull-down/binding assays with truncation/domain fragments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — in vitro binding assays with domain mapping, single lab, multiple domain fragments tested","pmids":["9753609"],"is_preprint":false},{"year":2002,"finding":"Sly1 (Sec1/Munc18 family protein) binds to the t-SNARE Sed5 and enhances formation of the early Golgi SNARE complex (Sed5–Bet1 trans-SNARE complex). A temperature-sensitive sly1 mutant with reduced Sed5 binding fails to promote SNARE complex formation; addition of recombinant Sly1 to yeast lysate enhances in vitro SNARE complex formation.","method":"Co-immunoprecipitation, in vitro SNARE complex formation assay with recombinant Sly1 added to yeast lysate, temperature-sensitive mutant analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — reconstitution (in vitro complex formation) combined with co-IP and mutant analysis, single lab but multiple orthogonal methods","pmids":["12186954"],"is_preprint":false},{"year":2005,"finding":"Phosphorylation of Sed5 at a conserved PKA consensus site (serine-317) regulates ER-Golgi transport and Golgi morphology. A phosphomimetic (S317D) substitution causes ER elaboration, defects in retrograde Golgi-to-ER transport, accumulation of small transport vesicles, and growth inhibition. A non-phosphorylatable (S317A) substitution has no transport defects but causes ordering of the Golgi into a mammalian-like stacked structure dependent on Sed5 retrograde recycling (absent in sec21-2 retrograde transport mutants).","method":"Site-directed mutagenesis (S317A and S317D), phosphoprotein analysis, intracellular transport assays, fluorescence microscopy, genetic epistasis with sec21-2","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis at defined phosphosite, multiple transport and morphology readouts, epistasis with retrograde transport mutant, single lab","pmids":["16093353"],"is_preprint":false},{"year":2007,"finding":"The COG (conserved oligomeric Golgi) tethering complex colocalizes and co-immunoprecipitates with intra-Golgi SNARE molecules. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a (the mammalian ortholog of Sed5p), as confirmed by FRET. COG complex knockdown decreases Golgi SNARE mobility, causes accumulation of free Syntaxin5, and decreases steady-state levels of intra-Golgi SNARE complexes. Overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes.","method":"Co-immunoprecipitation, FRET (in vivo interaction), RNAi knockdown, FRAP, dominant-negative overexpression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, FRET, RNAi, FRAP, dominant-negative) in both yeast and mammalian cells, replicated across systems","pmids":["18086915"],"is_preprint":false},{"year":2013,"finding":"STX5 physically interacts with the C-terminal domain of the VLDL-receptor (VLDL-R). Overexpression of Stx5 prevents advanced Golgi-maturation of VLDL-R without causing ER accumulation, instead translocating ER-/N-glycosylated VLDL-R to the plasma membrane via a BFA-insensitive and low-temperature-insensitive pathway, and interferes with VLDL-R reaching the trans-Golgi network.","method":"Co-immunoprecipitation, in vitro binding assays, glycosylation maturation assays, overexpression, BFA treatment, subcellular fractionation/trafficking assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple in vitro and cell-based approaches (co-IP, glycosylation assays, BFA treatment), single lab, mechanism partially characterized","pmids":["23701949"],"is_preprint":false},{"year":2017,"finding":"Yeast Sed5 (cis-Golgi t-SNARE) plays a role in autophagy by regulating anterograde trafficking of Atg9-containing vesicles to the phagophore assembly site (PAS). In sed5-1 mutant cells, Atg8 is not properly transported to the PAS (multiple Atg8 dots dispersed in cytoplasm or trapped in Golgi), and Atg23 and Atg27 fail to localize properly to the Golgi. Overexpression of SFT1 or SFT2 (suppressors of sed5-ts) rescues these autophagy defects, indicating genetic interaction between Sft1/2 and Sed5 is required for autophagy.","method":"Temperature-sensitive sed5-1 mutant analysis, fluorescence microscopy of Atg8/Atg9/Atg23/Atg27 localization, genetic suppression by SFT1/SFT2 overexpression","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — defined mutant with multiple autophagy readouts and genetic suppression, single lab","pmids":["28927260"],"is_preprint":false}],"current_model":"STX5/Sed5p is a cis-Golgi-localized t-SNARE that mediates anterograde ER-to-Golgi and intra-Golgi vesicular transport by forming SNARE complexes with cognate v-SNAREs; its activity is positively regulated by the Sec1/Munc18 protein Sly1/Scfd1, which binds the N-terminal domain of Sed5/STX5 to enhance SNARE complex assembly, and by the COG tethering complex, which binds Syntaxin5a/Sed5 (via Cog4/Cog6) to stabilize intra-Golgi SNARE complexes; PKA-mediated phosphorylation of Sed5 at serine-317 cycles the protein between active and inactive states to control Golgi morphology and retrograde transport; STX5 also interacts with VLDL-R to influence its trafficking and glycosylation, and Sed5 participates in autophagy by regulating Atg9-containing vesicle trafficking to the phagophore assembly site."},"narrative":{"mechanistic_narrative":"STX5 (yeast Sed5p) is a cis-Golgi-localized t-SNARE of the syntaxin family that mediates anterograde ER-to-Golgi and intra-Golgi vesicular transport, with depletion blocking cargo delivery to the Golgi and driving accumulation of ER membranes and transport vesicles [PMID:1400588, PMID:7929581]. Both its transmembrane and cytoplasmic domains independently encode cis-Golgi targeting information [PMID:7929581]. SNARE complex assembly by Sed5/STX5 is positively regulated by the Sec1/Munc18 protein Sly1, which binds the N-terminal helix of Sed5—relieving an autoinhibitory intramolecular interaction between the N-terminal region and its own C-terminal helix—and enhances formation of the early Golgi Sed5–Bet1 trans-SNARE complex [PMID:9753609, PMID:12186954]. Intra-Golgi SNARE complexes containing the mammalian ortholog Syntaxin5a are further stabilized by the COG tethering complex through direct Cog4 and Cog6 interactions, loss of which leaves Syntaxin5 free and reduces steady-state SNARE complex levels and mobility [PMID:18086915]. PKA-site phosphorylation of Sed5 at serine-317 toggles the protein between states controlling ER-Golgi transport, retrograde recycling, and Golgi stacking morphology [PMID:16093353]. Beyond core trafficking, STX5 binds the VLDL-receptor C-terminal domain to influence its Golgi maturation, glycosylation, and surface delivery [PMID:23701949], and Sed5 supports autophagy by directing Atg9-containing vesicle trafficking to the phagophore assembly site in concert with Sft1/Sft2 [PMID:28927260].","teleology":[{"year":1992,"claim":"Established that Sed5p is an essential integral membrane component of the early secretory pathway, defining the gene's core role before any molecular activity was known.","evidence":"Gene depletion/overexpression with carboxypeptidase Y transport assay, immunofluorescence, and EM in yeast","pmids":["1400588"],"confidence":"High","gaps":["Did not define the protein family or SNARE activity","Precise sub-Golgi localization not yet resolved"]},{"year":1994,"claim":"Placed Sed5 in the syntaxin family and mapped its cis-Golgi targeting determinants, showing both transmembrane and cytoplasmic domains independently direct localization.","evidence":"Epitope-tagged and chimeric Sed5 expression in COS cells, immuno-EM, domain-swap mutagenesis, in vivo yeast assays","pmids":["7929581"],"confidence":"High","gaps":["Did not identify cognate v-SNAREs or regulators","Targeting receptor/machinery for the cytoplasmic-domain signal unknown"]},{"year":1998,"claim":"Resolved how the regulator Sly1 engages Sed5, localizing binding to the N-terminal helix and linking it to an autoinhibitory intramolecular interaction.","evidence":"In vitro binding/pull-down assays with individual Sed5 helices and truncation fragments","pmids":["9753609"],"confidence":"Medium","gaps":["In vitro domain-mapping not validated in cells","Functional consequence of relieving autoinhibition not demonstrated here"]},{"year":2002,"claim":"Demonstrated that Sly1 binding to Sed5 is functionally productive, enhancing assembly of the early Golgi Sed5-Bet1 trans-SNARE complex.","evidence":"Co-IP, in vitro SNARE complex formation with recombinant Sly1 added to yeast lysate, temperature-sensitive sly1 mutant","pmids":["12186954"],"confidence":"High","gaps":["Full reconstituted membrane fusion not shown","Quantitative kinetics of Sly1-stimulated assembly not defined"]},{"year":2005,"claim":"Identified phosphoregulation of Sed5 at PKA-site S317 as a switch controlling transport directionality and Golgi morphology.","evidence":"S317A/S317D site-directed mutagenesis, transport and morphology readouts, genetic epistasis with sec21-2 in yeast","pmids":["16093353"],"confidence":"High","gaps":["Direct demonstration of PKA as the in vivo kinase incomplete","Mechanism by which phosphostate alters SNARE assembly not resolved"]},{"year":2007,"claim":"Connected Syntaxin5/Sed5 to the COG tethering complex, showing COG stabilizes intra-Golgi SNARE complexes via Cog4/Cog6 binding.","evidence":"Co-IP, FRET, RNAi knockdown, FRAP, dominant-negative overexpression in yeast and mammalian cells","pmids":["18086915"],"confidence":"High","gaps":["Structural basis of Cog4/Cog6-Syntaxin5 interface unknown","Whether COG acts on assembly versus disassembly not distinguished"]},{"year":2013,"claim":"Extended STX5 function to cargo-specific trafficking by showing direct interaction with the VLDL-receptor influencing its glycosylation and surface delivery.","evidence":"Co-IP, in vitro binding, glycosylation maturation assays, overexpression, BFA treatment, trafficking assays","pmids":["23701949"],"confidence":"Medium","gaps":["Single-lab characterization without reciprocal in vivo validation","Physiological role of the STX5-VLDLR interaction not established"]},{"year":2017,"claim":"Implicated Sed5 in autophagy by linking it to anterograde trafficking of Atg9 vesicles to the phagophore assembly site.","evidence":"Temperature-sensitive sed5-1 mutant, fluorescence microscopy of Atg8/Atg9/Atg23/Atg27, genetic suppression by SFT1/SFT2 overexpression in yeast","pmids":["28927260"],"confidence":"Medium","gaps":["Direct physical role of Sed5 in Atg9 vesicle fusion not shown","Conservation of this autophagy role in mammalian STX5 untested"]},{"year":null,"claim":"How phosphoregulation, Sly1 binding, and COG tethering are integrated to coordinate the directionality and fidelity of STX5-mediated fusion remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of STX5 in an assembled trans-SNARE complex","Crosstalk between S317 phosphostate and Sly1/COG regulation undefined","Mammalian autophagy and cargo-selective roles not mechanistically unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,3,5]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,1,5]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,3,5]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]}],"complexes":["cis-Golgi SNARE complex"],"partners":["SLY1/SCFD1","BET1","COG4","COG6","VLDLR","SFT1","SFT2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13190","full_name":"Syntaxin-5","aliases":[],"length_aa":355,"mass_kda":39.7,"function":"Mediates endoplasmic reticulum to Golgi transport. Together with p115/USO1 and GM130/GOLGA2, involved in vesicle tethering and fusion at the cis-Golgi membrane to maintain the stacked and inter-connected structure of the Golgi apparatus Required for Golgi to endoplasmic reticulum retrogade transport, and for intra-Golgi transport (Microbial infection) Required for the efficient production of infectious virion during human cytomegalovirus infection. Mechanistically, participates in the formation of the cytoplasmic viral assembly compartment where tegument acquisition and envelopment occur","subcellular_location":"Endoplasmic reticulum-Golgi intermediate compartment membrane; Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q13190/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/STX5","classification":"Common Essential","n_dependent_lines":1178,"n_total_lines":1208,"dependency_fraction":0.9751655629139073},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000162236","cell_line_id":"CID000756","localizations":[{"compartment":"golgi","grade":3},{"compartment":"er","grade":2}],"interactors":[{"gene":"GOSR2","stoichiometry":10.0},{"gene":"SCFD1","stoichiometry":10.0},{"gene":"SEC22B","stoichiometry":10.0},{"gene":"NSF","stoichiometry":10.0},{"gene":"BET1L","stoichiometry":10.0},{"gene":"SFT2D3","stoichiometry":4.0},{"gene":"GORASP2","stoichiometry":0.2},{"gene":"NAPA","stoichiometry":0.2},{"gene":"PGRMC1","stoichiometry":0.2},{"gene":"VAMP4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000756","total_profiled":1310},"omim":[{"mim_id":"620633","title":"GOLGI TRANSPORT 1A; GOLT1A","url":"https://www.omim.org/entry/620633"},{"mim_id":"620454","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE IIaa; CDG2AA","url":"https://www.omim.org/entry/620454"},{"mim_id":"620201","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE IIz; CDG2Z","url":"https://www.omim.org/entry/620201"},{"mim_id":"620200","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE IIy; CDG2Y","url":"https://www.omim.org/entry/620200"},{"mim_id":"619659","title":"SYNAPTOSOME-ASSOCIATED PROTEIN 47; SNAP47","url":"https://www.omim.org/entry/619659"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/STX5"},"hgnc":{"alias_symbol":["SED5"],"prev_symbol":["STX5A"]},"alphafold":{"accession":"Q13190","domains":[{"cath_id":"1.20.58.70","chopping":"89-214_258-300","consensus_level":"medium","plddt":83.6609,"start":89,"end":300},{"cath_id":"1.20.5","chopping":"314-355","consensus_level":"medium","plddt":80.729,"start":314,"end":355}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13190","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13190-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13190-F1-predicted_aligned_error_v6.png","plddt_mean":70.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=STX5","jax_strain_url":"https://www.jax.org/strain/search?query=STX5"},"sequence":{"accession":"Q13190","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13190.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13190/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13190"}},"corpus_meta":[{"pmid":"1400588","id":"PMC_1400588","title":"SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex.","date":"1992","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/1400588","citation_count":269,"is_preprint":false},{"pmid":"7929581","id":"PMC_7929581","title":"Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains.","date":"1994","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/7929581","citation_count":151,"is_preprint":false},{"pmid":"18086915","id":"PMC_18086915","title":"Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability.","date":"2007","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18086915","citation_count":93,"is_preprint":false},{"pmid":"31357511","id":"PMC_31357511","title":"Stx5-Mediated ER-Golgi Transport in Mammals and Yeast.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31357511","citation_count":43,"is_preprint":false},{"pmid":"16107716","id":"PMC_16107716","title":"Immunoisolaton of the yeast Golgi subcompartments and characterization of a novel membrane protein, Svp26, discovered in the Sed5-containing compartments.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16107716","citation_count":43,"is_preprint":false},{"pmid":"16093353","id":"PMC_16093353","title":"Control of Golgi morphology and function by Sed5 t-SNARE phosphorylation.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16093353","citation_count":31,"is_preprint":false},{"pmid":"12186954","id":"PMC_12186954","title":"Binding of Sly1 to Sed5 enhances formation of the yeast early Golgi SNARE complex.","date":"2002","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/12186954","citation_count":30,"is_preprint":false},{"pmid":"9753609","id":"PMC_9753609","title":"Protein-protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct from its neural plasma membrane cognate syntaxin 1.","date":"1998","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/9753609","citation_count":21,"is_preprint":false},{"pmid":"16699179","id":"PMC_16699179","title":"Identification of a Sed5-like SNARE gene LjSYP32-1 that contributes to nodule tissue formation of Lotus japonicus.","date":"2006","source":"Plant & cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16699179","citation_count":13,"is_preprint":false},{"pmid":"23701949","id":"PMC_23701949","title":"Stx5 is a novel interactor of VLDL-R to affect its intracellular trafficking and processing.","date":"2013","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/23701949","citation_count":12,"is_preprint":false},{"pmid":"28927260","id":"PMC_28927260","title":"The Roles of the SNARE Protein Sed5 in Autophagy in Saccharomyces cerevisiae.","date":"2017","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/28927260","citation_count":7,"is_preprint":false},{"pmid":"36969886","id":"PMC_36969886","title":"STX5 Inhibits Hepatocellular Carcinoma Adhesion and Promotes Metastasis by Regulating the PI3K/mTOR Pathway.","date":"2023","source":"Journal of clinical and translational hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/36969886","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7600,"output_tokens":2594,"usd":0.030855,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9662,"output_tokens":2853,"usd":0.059818,"stage2_stop_reason":"end_turn"},"total_usd":0.090673,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"Sed5p (yeast ortholog of STX5) is an integral membrane protein required for ER-to-Golgi vesicular transport; depletion blocks carboxypeptidase Y transport to the Golgi and causes dramatic accumulation of ER membranes and vesicles; overexpression reduces ER-to-Golgi transport efficiency and causes vesicle accumulation. Immunofluorescence shows Sed5p localizes to punctate cytoplasmic structures, not ER membranes.\",\n      \"method\": \"Gene depletion/overexpression with cargo transport assay (carboxypeptidase Y), immunofluorescence, electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (depletion, overexpression, transport assay, immunofluorescence, EM) in a foundational study, foundational to the field\",\n      \"pmids\": [\"1400588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Sed5p is a member of the syntaxin family and localizes to the cis-Golgi network (tubulo-vesicular compartment on the cis side of the Golgi). Both its transmembrane domain and cytoplasmic domain contribute independently to its Golgi localization; the transmembrane domain contains targeting information, but cytoplasmic domain provides an additional targeting mechanism sufficient when the transmembrane domain is altered.\",\n      \"method\": \"Expression of epitope-tagged Sed5 and chimeric molecules in COS cells, immuno-EM, domain-swap mutagenesis, in vivo yeast functional assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (immuno-EM, chimeric mutagenesis, in vivo complementation), replicated across yeast/Drosophila/rat homologs\",\n      \"pmids\": [\"7929581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The N-terminal helix of Sed5 is responsible for binding Sly1 protein, whereas Sly1 binding to syntaxin 1 requires almost the entire molecule. The N-terminal region of Sed5 also binds to its own C-terminal helix (intramolecular interaction), and Sly1 binding interferes with this intramolecular interaction.\",\n      \"method\": \"Protein-protein interaction assays dissecting individual helices of Sed5; pull-down/binding assays with truncation/domain fragments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — in vitro binding assays with domain mapping, single lab, multiple domain fragments tested\",\n      \"pmids\": [\"9753609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Sly1 (Sec1/Munc18 family protein) binds to the t-SNARE Sed5 and enhances formation of the early Golgi SNARE complex (Sed5–Bet1 trans-SNARE complex). A temperature-sensitive sly1 mutant with reduced Sed5 binding fails to promote SNARE complex formation; addition of recombinant Sly1 to yeast lysate enhances in vitro SNARE complex formation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro SNARE complex formation assay with recombinant Sly1 added to yeast lysate, temperature-sensitive mutant analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — reconstitution (in vitro complex formation) combined with co-IP and mutant analysis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12186954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Phosphorylation of Sed5 at a conserved PKA consensus site (serine-317) regulates ER-Golgi transport and Golgi morphology. A phosphomimetic (S317D) substitution causes ER elaboration, defects in retrograde Golgi-to-ER transport, accumulation of small transport vesicles, and growth inhibition. A non-phosphorylatable (S317A) substitution has no transport defects but causes ordering of the Golgi into a mammalian-like stacked structure dependent on Sed5 retrograde recycling (absent in sec21-2 retrograde transport mutants).\",\n      \"method\": \"Site-directed mutagenesis (S317A and S317D), phosphoprotein analysis, intracellular transport assays, fluorescence microscopy, genetic epistasis with sec21-2\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis at defined phosphosite, multiple transport and morphology readouts, epistasis with retrograde transport mutant, single lab\",\n      \"pmids\": [\"16093353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The COG (conserved oligomeric Golgi) tethering complex colocalizes and co-immunoprecipitates with intra-Golgi SNARE molecules. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a (the mammalian ortholog of Sed5p), as confirmed by FRET. COG complex knockdown decreases Golgi SNARE mobility, causes accumulation of free Syntaxin5, and decreases steady-state levels of intra-Golgi SNARE complexes. Overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes.\",\n      \"method\": \"Co-immunoprecipitation, FRET (in vivo interaction), RNAi knockdown, FRAP, dominant-negative overexpression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, FRET, RNAi, FRAP, dominant-negative) in both yeast and mammalian cells, replicated across systems\",\n      \"pmids\": [\"18086915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"STX5 physically interacts with the C-terminal domain of the VLDL-receptor (VLDL-R). Overexpression of Stx5 prevents advanced Golgi-maturation of VLDL-R without causing ER accumulation, instead translocating ER-/N-glycosylated VLDL-R to the plasma membrane via a BFA-insensitive and low-temperature-insensitive pathway, and interferes with VLDL-R reaching the trans-Golgi network.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, glycosylation maturation assays, overexpression, BFA treatment, subcellular fractionation/trafficking assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple in vitro and cell-based approaches (co-IP, glycosylation assays, BFA treatment), single lab, mechanism partially characterized\",\n      \"pmids\": [\"23701949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Yeast Sed5 (cis-Golgi t-SNARE) plays a role in autophagy by regulating anterograde trafficking of Atg9-containing vesicles to the phagophore assembly site (PAS). In sed5-1 mutant cells, Atg8 is not properly transported to the PAS (multiple Atg8 dots dispersed in cytoplasm or trapped in Golgi), and Atg23 and Atg27 fail to localize properly to the Golgi. Overexpression of SFT1 or SFT2 (suppressors of sed5-ts) rescues these autophagy defects, indicating genetic interaction between Sft1/2 and Sed5 is required for autophagy.\",\n      \"method\": \"Temperature-sensitive sed5-1 mutant analysis, fluorescence microscopy of Atg8/Atg9/Atg23/Atg27 localization, genetic suppression by SFT1/SFT2 overexpression\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — defined mutant with multiple autophagy readouts and genetic suppression, single lab\",\n      \"pmids\": [\"28927260\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"STX5/Sed5p is a cis-Golgi-localized t-SNARE that mediates anterograde ER-to-Golgi and intra-Golgi vesicular transport by forming SNARE complexes with cognate v-SNAREs; its activity is positively regulated by the Sec1/Munc18 protein Sly1/Scfd1, which binds the N-terminal domain of Sed5/STX5 to enhance SNARE complex assembly, and by the COG tethering complex, which binds Syntaxin5a/Sed5 (via Cog4/Cog6) to stabilize intra-Golgi SNARE complexes; PKA-mediated phosphorylation of Sed5 at serine-317 cycles the protein between active and inactive states to control Golgi morphology and retrograde transport; STX5 also interacts with VLDL-R to influence its trafficking and glycosylation, and Sed5 participates in autophagy by regulating Atg9-containing vesicle trafficking to the phagophore assembly site.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"STX5 (yeast Sed5p) is a cis-Golgi-localized t-SNARE of the syntaxin family that mediates anterograde ER-to-Golgi and intra-Golgi vesicular transport, with depletion blocking cargo delivery to the Golgi and driving accumulation of ER membranes and transport vesicles [#0, #1]. Both its transmembrane and cytoplasmic domains independently encode cis-Golgi targeting information [#1]. SNARE complex assembly by Sed5/STX5 is positively regulated by the Sec1/Munc18 protein Sly1, which binds the N-terminal helix of Sed5—relieving an autoinhibitory intramolecular interaction between the N-terminal region and its own C-terminal helix—and enhances formation of the early Golgi Sed5–Bet1 trans-SNARE complex [#2, #3]. Intra-Golgi SNARE complexes containing the mammalian ortholog Syntaxin5a are further stabilized by the COG tethering complex through direct Cog4 and Cog6 interactions, loss of which leaves Syntaxin5 free and reduces steady-state SNARE complex levels and mobility [#5]. PKA-site phosphorylation of Sed5 at serine-317 toggles the protein between states controlling ER-Golgi transport, retrograde recycling, and Golgi stacking morphology [#4]. Beyond core trafficking, STX5 binds the VLDL-receptor C-terminal domain to influence its Golgi maturation, glycosylation, and surface delivery [#6], and Sed5 supports autophagy by directing Atg9-containing vesicle trafficking to the phagophore assembly site in concert with Sft1/Sft2 [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established that Sed5p is an essential integral membrane component of the early secretory pathway, defining the gene's core role before any molecular activity was known.\",\n      \"evidence\": \"Gene depletion/overexpression with carboxypeptidase Y transport assay, immunofluorescence, and EM in yeast\",\n      \"pmids\": [\"1400588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the protein family or SNARE activity\", \"Precise sub-Golgi localization not yet resolved\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Placed Sed5 in the syntaxin family and mapped its cis-Golgi targeting determinants, showing both transmembrane and cytoplasmic domains independently direct localization.\",\n      \"evidence\": \"Epitope-tagged and chimeric Sed5 expression in COS cells, immuno-EM, domain-swap mutagenesis, in vivo yeast assays\",\n      \"pmids\": [\"7929581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify cognate v-SNAREs or regulators\", \"Targeting receptor/machinery for the cytoplasmic-domain signal unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Resolved how the regulator Sly1 engages Sed5, localizing binding to the N-terminal helix and linking it to an autoinhibitory intramolecular interaction.\",\n      \"evidence\": \"In vitro binding/pull-down assays with individual Sed5 helices and truncation fragments\",\n      \"pmids\": [\"9753609\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro domain-mapping not validated in cells\", \"Functional consequence of relieving autoinhibition not demonstrated here\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated that Sly1 binding to Sed5 is functionally productive, enhancing assembly of the early Golgi Sed5-Bet1 trans-SNARE complex.\",\n      \"evidence\": \"Co-IP, in vitro SNARE complex formation with recombinant Sly1 added to yeast lysate, temperature-sensitive sly1 mutant\",\n      \"pmids\": [\"12186954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full reconstituted membrane fusion not shown\", \"Quantitative kinetics of Sly1-stimulated assembly not defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified phosphoregulation of Sed5 at PKA-site S317 as a switch controlling transport directionality and Golgi morphology.\",\n      \"evidence\": \"S317A/S317D site-directed mutagenesis, transport and morphology readouts, genetic epistasis with sec21-2 in yeast\",\n      \"pmids\": [\"16093353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration of PKA as the in vivo kinase incomplete\", \"Mechanism by which phosphostate alters SNARE assembly not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected Syntaxin5/Sed5 to the COG tethering complex, showing COG stabilizes intra-Golgi SNARE complexes via Cog4/Cog6 binding.\",\n      \"evidence\": \"Co-IP, FRET, RNAi knockdown, FRAP, dominant-negative overexpression in yeast and mammalian cells\",\n      \"pmids\": [\"18086915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Cog4/Cog6-Syntaxin5 interface unknown\", \"Whether COG acts on assembly versus disassembly not distinguished\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended STX5 function to cargo-specific trafficking by showing direct interaction with the VLDL-receptor influencing its glycosylation and surface delivery.\",\n      \"evidence\": \"Co-IP, in vitro binding, glycosylation maturation assays, overexpression, BFA treatment, trafficking assays\",\n      \"pmids\": [\"23701949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab characterization without reciprocal in vivo validation\", \"Physiological role of the STX5-VLDLR interaction not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Implicated Sed5 in autophagy by linking it to anterograde trafficking of Atg9 vesicles to the phagophore assembly site.\",\n      \"evidence\": \"Temperature-sensitive sed5-1 mutant, fluorescence microscopy of Atg8/Atg9/Atg23/Atg27, genetic suppression by SFT1/SFT2 overexpression in yeast\",\n      \"pmids\": [\"28927260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical role of Sed5 in Atg9 vesicle fusion not shown\", \"Conservation of this autophagy role in mammalian STX5 untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How phosphoregulation, Sly1 binding, and COG tethering are integrated to coordinate the directionality and fidelity of STX5-mediated fusion remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of STX5 in an assembled trans-SNARE complex\", \"Crosstalk between S317 phosphostate and Sly1/COG regulation undefined\", \"Mammalian autophagy and cargo-selective roles not mechanistically unified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"cis-Golgi SNARE complex\"],\n    \"partners\": [\"SLY1/SCFD1\", \"BET1\", \"COG4\", \"COG6\", \"VLDLR\", \"SFT1\", \"SFT2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}