{"gene":"OST4","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1996,"finding":"OST4 encodes an unusually small 36-amino-acid (~3.6 kDa) protein that is required for normal oligosaccharyltransferase (OTase) activity in yeast; deletion of OST4 greatly diminishes but does not abolish OTase activity in vivo and in vitro, and the null mutant is inviable at 37°C, demonstrating OST4 is an essential OTase subunit or accessory component at high temperature.","method":"Genetic disruption/null mutant analysis, in vivo and in vitro OTase activity assays, biochemical characterization of the OST4 gene product","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined biochemical phenotype (OTase activity assay), replicated in multiple subsequent studies","pmids":["8621712"],"is_preprint":false},{"year":1997,"finding":"Ost4p (3.6 kDa) is a bona fide subunit of the yeast oligosaccharyltransferase complex present in equimolar stoichiometry with seven other subunits; mild denaturation of the immunoprecipitated complex releases a stable subcomplex comprising Stt3p, Ost3p, and Ost4p, indicating Ost4p bridges these two subunits within the OST.","method":"Epitope-tagging of Ost3p, co-immunoprecipitation of all OST subunits, quantification of radiolabeled subunits, partial denaturation to isolate subcomplexes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with quantitative stoichiometry, subcomplex isolation, replicated by subsequent studies","pmids":["9405463"],"is_preprint":false},{"year":1997,"finding":"Overexpression of OST4 suppresses conditional stt3 alleles in yeast, providing genetic epistasis evidence that Ost4p functionally interacts with the catalytic Stt3p subunit of the OST complex.","method":"Genetic suppressor analysis — overexpression of OST4 in stt3 conditional mutants","journal":"Molecular & general genetics : MGG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (suppressor assay), single lab, one method","pmids":["9435788"],"is_preprint":false},{"year":2003,"finding":"Ost4p adopts an N(lumen)-C(cyto) type I membrane topology in the ER membrane, with its transmembrane segment spanning residues ~10–25; mutation of residues 18–25 to ionizable amino acids disrupts interactions with both Stt3p and Ost3p, and ost4 temperature-sensitive mutants cause loss of direct Ost3p–Stt3p interaction, demonstrating that the cytoplasmic-leaflet region of Ost4p physically bridges Ost3p and Stt3p in the OST subcomplex.","method":"In vivo membrane topology determination, site-directed mutagenesis of transmembrane residues, CD analysis of synthetic Ost4p in liposomes, co-immunoprecipitation in ost4 mutant backgrounds","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — topology mapping combined with mutagenesis and Co-IP showing functional consequence, multiple orthogonal methods in one study","pmids":["12810948"],"is_preprint":false},{"year":2005,"finding":"Ost4p is required for the assembly of two functionally distinct OST complexes in yeast: one containing Ost3p and one containing Ost6p. In the absence of Ost4p, neither Ost3p nor Ost6p is incorporated into the OST complex, showing that Ost4p is essential for the assembly of both OST isoforms.","method":"Blue native gel electrophoresis of OST complexes from wild-type and ost4 deletion strains, immunodetection of individual subunits","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — blue native PAGE with defined genetic background (deletion), clear biochemical readout, single lab but multiple subunits tracked","pmids":["16096346"],"is_preprint":false},{"year":2005,"finding":"The eukaryotic OST complex in yeast contains Ost4p as one of eight equimolar subunits, and the STT3 subunit is critical for catalytic activity; Ost4p participates in the Stt3p-Ost3p/Ost6p subcomplex.","method":"Review synthesizing co-immunoprecipitation, blue native gel, and genetic evidence from multiple laboratories","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Strong — review synthesizing replicated biochemical and genetic findings from multiple labs, no new primary data","pmids":["16317064"],"is_preprint":false},{"year":2011,"finding":"Solution NMR of human OST4 in detergent solvent reveals that residues 5–30 adopt an α-helical structure with a kink in the transmembrane domain, providing the first structural characterization of the human OST4 protein.","method":"NMR spectroscopy (solution structure determination in detergent)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure determined, single lab, no mutagenesis or functional validation in same study","pmids":["21609714"],"is_preprint":false},{"year":2013,"finding":"Human OST4 is assembled into native OST complexes containing either the catalytic STT3A or STT3B isoforms; OST4 co-immunoprecipitates with both STT3 isoforms and with ribophorin I; a single amino acid change in the OST4 transmembrane region perturbs these interactions; siRNA depletion of OST4 destabilizes native OST complexes (producing a novel ribophorin I subcomplex) and causes a defect in N-glycosylation of endogenous prosaposin similar to STT3A depletion, consistent with OST4 stabilizing STT3A-containing isoforms for co-translational N-glycosylation.","method":"Co-immunoprecipitation, site-directed mutagenesis of transmembrane domain, siRNA knockdown, blue native gel electrophoresis, N-glycosylation assay of endogenous substrate (prosaposin)","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, siRNA KD with defined glycosylation phenotype and native gel analysis; multiple orthogonal methods in single study","pmids":["23606741"],"is_preprint":false},{"year":2021,"finding":"NMR solution structures and MD simulations of yeast Ost4 and the V23D mutant show that while the V23D point mutation does not alter the overall helical structure of Ost4, it changes its position and solvent exposure in the membrane-mimetic environment; MD simulations of the membrane-embedded OST complex show that V23D disrupts hydrophobic helix–helix interactions between Ost4 and TM12/TM13 of Stt3, causing disengagement of Ost4V23D and exposure of residue D23 in the hydrophobic pocket, providing a structural mechanism for OST complex destabilization.","method":"Solution NMR structure determination (micelles), molecular dynamics simulations of membrane-bound OST complex with WT and V23D Ost4","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure plus MD simulation with mutagenesis, single lab, no reconstitution activity assay","pmids":["33442744"],"is_preprint":false},{"year":2024,"finding":"Solid-state NMR of yeast Ost4 and Ost4V23D reconstituted in POPC/POPE lipid bilayers shows significant chemical shift changes upon V23D mutation, indicating a dramatic change in the chemical environment of the transmembrane helix in a native-like lipid bilayer context.","method":"Solid-state NMR with magic-angle spinning, reconstitution in lipid bilayers","journal":"Journal of biomolecular NMR","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — solid-state NMR in lipid bilayers, single lab, no functional activity assay","pmids":["38421550"],"is_preprint":false},{"year":2025,"finding":"Ost4 is a substrate of the AAA+ ATPase Msp1 when mislocalized to mitochondria; an Msp1-protease chimera captures Ost4 as a novel Msp1 substrate by mass spectrometry; topology experiments show that mislocalized Ost4 adopts mixed membrane orientations at mitochondria, and Msp1 extracts mislocalized Ost4 regardless of its orientation.","method":"Msp1-protease chimera (substrate trapping), mass spectrometry identification, topology assay of mislocalized Ost4","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — novel chimeric trapping tool with MS identification and topology experiment, preprint, single lab","pmids":["40236206"],"is_preprint":true}],"current_model":"OST4 encodes an extremely small (36-residue, ~3.6 kDa) single-pass ER membrane protein that functions as a structural subunit of the oligosaccharyltransferase (OST) complex in both yeast and mammals: its cytoplasmic-leaflet transmembrane residues (Met18–Ile24 in yeast) mediate hydrophobic helix–helix contacts with the catalytic Stt3/STT3 subunit, physically bridging Stt3 to the accessory subunits Ost3/Ost6 (yeast) or ribophorin I (mammals), thereby stabilizing the assembled OST complex and supporting efficient co-translational N-glycosylation; loss or mutation of Ost4 disassembles these subcomplexes and reduces N-glycosylation activity, while mislocalized Ost4 is extracted from the outer mitochondrial membrane by the AAA+ ATPase Msp1."},"narrative":{"mechanistic_narrative":"OST4 encodes an unusually small (~3.6 kDa, 36-residue) single-pass ER membrane protein that functions as a structural subunit of the oligosaccharyltransferase (OST) complex required for efficient co-translational N-glycosylation in both yeast and mammals [PMID:8621712, PMID:9405463]. In yeast, Ost4p is an equimolar subunit of the eight-subunit OST and adopts an N(lumen)-C(cytosol) type I topology, with its cytoplasmic-leaflet transmembrane residues (~18-25) physically bridging the catalytic Stt3 subunit to the accessory subunit Ost3p; mutation of these residues to ionizable amino acids severs both the Ost4-Stt3 and the Ost3-Stt3 contacts [PMID:9405463, PMID:12810948]. Ost4p is required for assembly of two functionally distinct OST isoforms, since loss of Ost4p prevents incorporation of either Ost3p or Ost6p into the complex [PMID:16096346]. The human ortholog assembles into both STT3A- and STT3B-containing OST complexes and contacts ribophorin I; a single transmembrane substitution or siRNA depletion of OST4 destabilizes native complexes and reduces N-glycosylation of endogenous substrate, paralleling the yeast role in stabilizing the assembled catalytic core [PMID:23606741]. Structurally, the destabilizing V23D substitution exposes a charged residue in the membrane and disrupts hydrophobic helix-helix packing against Stt3 transmembrane segments TM12/TM13, explaining how a point mutation disassembles the complex [PMID:33442744]. When mislocalized to the outer mitochondrial membrane, Ost4 is recognized and extracted by the AAA+ ATPase Msp1 irrespective of its membrane orientation [PMID:40236206].","teleology":[{"year":1996,"claim":"Established that the OST4 gene product, despite its tiny size, is functionally required for normal oligosaccharyltransferase activity, defining it as a genuine OST component rather than an incidental polypeptide.","evidence":"Null-mutant genetics with in vivo and in vitro OTase activity assays in yeast","pmids":["8621712"],"confidence":"High","gaps":["Did not resolve whether Ost4 acts catalytically or structurally","No subunit-contact map defined"]},{"year":1997,"claim":"Defined Ost4p as a stoichiometric OST subunit and localized its position within the complex, showing it bridges the catalytic Stt3p to the accessory Ost3p subunit.","evidence":"Co-immunoprecipitation of all subunits with quantitative stoichiometry and partial-denaturation subcomplex isolation in yeast","pmids":["9405463"],"confidence":"High","gaps":["The molecular interface mediating bridging was not mapped","Topology of Ost4p unknown"]},{"year":1997,"claim":"Provided genetic confirmation that Ost4p functionally interacts with the catalytic Stt3p subunit, independent of biochemical co-purification.","evidence":"Suppression of conditional stt3 alleles by OST4 overexpression in yeast","pmids":["9435788"],"confidence":"Medium","gaps":["Genetic epistasis does not establish a direct physical contact","Single method, single lab"]},{"year":2003,"claim":"Mapped Ost4p topology and identified the specific cytoplasmic-leaflet transmembrane residues whose integrity is needed to hold Stt3p and Ost3p together, converting the bridging model into residue-level mechanism.","evidence":"In vivo topology mapping, transmembrane mutagenesis, CD of synthetic peptide in liposomes, and Co-IP in ost4 mutant backgrounds","pmids":["12810948"],"confidence":"High","gaps":["No high-resolution structure of the contact","Did not address mammalian conservation"]},{"year":2005,"claim":"Demonstrated that Ost4p is required for biogenesis of both the Ost3p- and Ost6p-containing OST isoforms, broadening its role from a single complex to assembly of functionally distinct OST forms.","evidence":"Blue native PAGE of OST complexes from wild-type and ost4 deletion strains with subunit immunodetection","pmids":["16096346"],"confidence":"High","gaps":["Functional differences between the two isoforms not attributed to Ost4 directly","Order of assembly events unresolved"]},{"year":2011,"claim":"Provided the first structural view of human OST4, showing an α-helical transmembrane segment with a kink.","evidence":"Solution NMR of human OST4 in detergent","pmids":["21609714"],"confidence":"Medium","gaps":["Structure in detergent rather than native bilayer","No functional or interaction data in the same study"]},{"year":2013,"claim":"Extended the yeast subunit-stabilization model to mammals, showing human OST4 incorporates into both STT3A- and STT3B-containing complexes and stabilizes them to support co-translational N-glycosylation.","evidence":"Co-IP with both STT3 isoforms and ribophorin I, transmembrane mutagenesis, siRNA knockdown with native gel and prosaposin glycosylation assay","pmids":["23606741"],"confidence":"High","gaps":["Precise mammalian subunit interface not resolved at atomic level","Substrate specificity consequences beyond prosaposin not characterized"]},{"year":2021,"claim":"Provided a structural mechanism for OST destabilization by the V23D mutation, showing it disrupts hydrophobic packing against Stt3 TM12/TM13 and exposes a buried charge.","evidence":"Solution NMR of WT and V23D Ost4 plus MD simulation of the membrane-embedded OST complex","pmids":["33442744"],"confidence":"Medium","gaps":["No reconstituted activity assay confirming functional disengagement","Simulation-derived, not experimentally resolved complex"]},{"year":2024,"claim":"Confirmed in a native-like lipid bilayer that the V23D mutation dramatically alters the transmembrane helix environment, validating membrane-context effects beyond micelles.","evidence":"Magic-angle-spinning solid-state NMR of Ost4 and Ost4V23D in POPC/POPE bilayers","pmids":["38421550"],"confidence":"Medium","gaps":["No functional activity assay","Effect on intact OST complex not directly observed in bilayer"]},{"year":2025,"claim":"Identified Ost4 as a quality-control substrate, showing that mislocalized Ost4 at mitochondria is extracted by the AAA+ ATPase Msp1 regardless of orientation, linking OST4 biogenesis to membrane-protein surveillance.","evidence":"Msp1-protease chimera substrate trapping, mass spectrometry identification, and topology assay of mislocalized Ost4 (preprint)","pmids":["40236206"],"confidence":"Medium","gaps":["Preprint, single lab","Physiological frequency of Ost4 mislocalization unknown","Fate of extracted Ost4 not defined"]},{"year":null,"claim":"How Ost4 acts within the catalytic cycle of an intact, reconstituted OST complex and whether its bridging function modulates glycosylation efficiency at the level of enzyme turnover remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No reconstituted activity assay isolating Ost4 contribution","No atomic structure of the full Ost4-Stt3-Ost3 interface","Physiological consequences of OST4 loss in mammals beyond cell-based glycosylation assays uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,3,4,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,3]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,7]}],"complexes":["oligosaccharyltransferase (OST) complex"],"partners":["STT3","STT3A","STT3B","OST3","OST6","RPN1","MSP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P0C6T2","full_name":"Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4","aliases":[],"length_aa":37,"mass_kda":4.2,"function":"Subunit of the oligosaccharyl transferase (OST) complex that catalyzes the initial transfer of a defined glycan (Glc(3)Man(9)GlcNAc(2) in eukaryotes) from the lipid carrier dolichol-pyrophosphate to an asparagine residue within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains, the first step in protein N-glycosylation (PubMed:31831667). N-glycosylation occurs cotranslationally and the complex associates with the Sec61 complex at the channel-forming translocon complex that mediates protein translocation across the endoplasmic reticulum (ER). All subunits are required for a maximal enzyme activity. Specifically involved in maintaining stability of STT3A-containing OST complexes","subcellular_location":"Endoplasmic reticulum; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/P0C6T2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OST4","classification":"Not Classified","n_dependent_lines":378,"n_total_lines":1090,"dependency_fraction":0.3467889908256881},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000228474","cell_line_id":"CID000185","localizations":[{"compartment":"er","grade":3}],"interactors":[{"gene":"LIN54","stoichiometry":0.2},{"gene":"SUPT6H","stoichiometry":0.2},{"gene":"GIGYF2","stoichiometry":0.2},{"gene":"NFXL1","stoichiometry":0.2},{"gene":"RIF1","stoichiometry":0.2},{"gene":"ZC3H13","stoichiometry":0.2},{"gene":"RBM26","stoichiometry":0.2},{"gene":"RNF10","stoichiometry":0.2},{"gene":"KRTCAP2","stoichiometry":0.2},{"gene":"PACRGL","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000185","total_profiled":1310},"omim":[{"mim_id":"619029","title":"KERATINOCYTE-ASSOCIATED PROTEIN 2; KRTCAP2","url":"https://www.omim.org/entry/619029"},{"mim_id":"618932","title":"OLIGOSACCHARYLTRANSFERASE COMPLEX, SUBUNIT 4, NONCATALYTIC; OST4","url":"https://www.omim.org/entry/618932"},{"mim_id":"608605","title":"OLIGOSACCHARYLTRANSFERASE COMPLEX, CATALYTIC SUBUNIT STT3B; STT3B","url":"https://www.omim.org/entry/608605"},{"mim_id":"601134","title":"OLIGOSACCHARYLTRANSFERASE COMPLEX, CATALYTIC SUBUNIT STT3A; STT3A","url":"https://www.omim.org/entry/601134"},{"mim_id":"180470","title":"RIBOPHORIN I; RPN1","url":"https://www.omim.org/entry/180470"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/OST4"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P0C6T2","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C6T2","model_url":"https://alphafold.ebi.ac.uk/files/AF-P0C6T2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P0C6T2-F1-predicted_aligned_error_v6.png","plddt_mean":90.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OST4","jax_strain_url":"https://www.jax.org/strain/search?query=OST4"},"sequence":{"accession":"P0C6T2","fasta_url":"https://rest.uniprot.org/uniprotkb/P0C6T2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P0C6T2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C6T2"}},"corpus_meta":[{"pmid":"16317064","id":"PMC_16317064","title":"An evolving view of the eukaryotic 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Cancer & chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/6575722","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16172,"output_tokens":2972,"usd":0.046548,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10219,"output_tokens":3214,"usd":0.065723,"stage2_stop_reason":"end_turn"},"total_usd":0.112271,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"OST4 encodes an unusually small 36-amino-acid (~3.6 kDa) protein that is required for normal oligosaccharyltransferase (OTase) activity in yeast; deletion of OST4 greatly diminishes but does not abolish OTase activity in vivo and in vitro, and the null mutant is inviable at 37°C, demonstrating OST4 is an essential OTase subunit or accessory component at high temperature.\",\n      \"method\": \"Genetic disruption/null mutant analysis, in vivo and in vitro OTase activity assays, biochemical characterization of the OST4 gene product\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined biochemical phenotype (OTase activity assay), replicated in multiple subsequent studies\",\n      \"pmids\": [\"8621712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Ost4p (3.6 kDa) is a bona fide subunit of the yeast oligosaccharyltransferase complex present in equimolar stoichiometry with seven other subunits; mild denaturation of the immunoprecipitated complex releases a stable subcomplex comprising Stt3p, Ost3p, and Ost4p, indicating Ost4p bridges these two subunits within the OST.\",\n      \"method\": \"Epitope-tagging of Ost3p, co-immunoprecipitation of all OST subunits, quantification of radiolabeled subunits, partial denaturation to isolate subcomplexes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with quantitative stoichiometry, subcomplex isolation, replicated by subsequent studies\",\n      \"pmids\": [\"9405463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Overexpression of OST4 suppresses conditional stt3 alleles in yeast, providing genetic epistasis evidence that Ost4p functionally interacts with the catalytic Stt3p subunit of the OST complex.\",\n      \"method\": \"Genetic suppressor analysis — overexpression of OST4 in stt3 conditional mutants\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (suppressor assay), single lab, one method\",\n      \"pmids\": [\"9435788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Ost4p adopts an N(lumen)-C(cyto) type I membrane topology in the ER membrane, with its transmembrane segment spanning residues ~10–25; mutation of residues 18–25 to ionizable amino acids disrupts interactions with both Stt3p and Ost3p, and ost4 temperature-sensitive mutants cause loss of direct Ost3p–Stt3p interaction, demonstrating that the cytoplasmic-leaflet region of Ost4p physically bridges Ost3p and Stt3p in the OST subcomplex.\",\n      \"method\": \"In vivo membrane topology determination, site-directed mutagenesis of transmembrane residues, CD analysis of synthetic Ost4p in liposomes, co-immunoprecipitation in ost4 mutant backgrounds\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — topology mapping combined with mutagenesis and Co-IP showing functional consequence, multiple orthogonal methods in one study\",\n      \"pmids\": [\"12810948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Ost4p is required for the assembly of two functionally distinct OST complexes in yeast: one containing Ost3p and one containing Ost6p. In the absence of Ost4p, neither Ost3p nor Ost6p is incorporated into the OST complex, showing that Ost4p is essential for the assembly of both OST isoforms.\",\n      \"method\": \"Blue native gel electrophoresis of OST complexes from wild-type and ost4 deletion strains, immunodetection of individual subunits\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — blue native PAGE with defined genetic background (deletion), clear biochemical readout, single lab but multiple subunits tracked\",\n      \"pmids\": [\"16096346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The eukaryotic OST complex in yeast contains Ost4p as one of eight equimolar subunits, and the STT3 subunit is critical for catalytic activity; Ost4p participates in the Stt3p-Ost3p/Ost6p subcomplex.\",\n      \"method\": \"Review synthesizing co-immunoprecipitation, blue native gel, and genetic evidence from multiple laboratories\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Strong — review synthesizing replicated biochemical and genetic findings from multiple labs, no new primary data\",\n      \"pmids\": [\"16317064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Solution NMR of human OST4 in detergent solvent reveals that residues 5–30 adopt an α-helical structure with a kink in the transmembrane domain, providing the first structural characterization of the human OST4 protein.\",\n      \"method\": \"NMR spectroscopy (solution structure determination in detergent)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure determined, single lab, no mutagenesis or functional validation in same study\",\n      \"pmids\": [\"21609714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human OST4 is assembled into native OST complexes containing either the catalytic STT3A or STT3B isoforms; OST4 co-immunoprecipitates with both STT3 isoforms and with ribophorin I; a single amino acid change in the OST4 transmembrane region perturbs these interactions; siRNA depletion of OST4 destabilizes native OST complexes (producing a novel ribophorin I subcomplex) and causes a defect in N-glycosylation of endogenous prosaposin similar to STT3A depletion, consistent with OST4 stabilizing STT3A-containing isoforms for co-translational N-glycosylation.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of transmembrane domain, siRNA knockdown, blue native gel electrophoresis, N-glycosylation assay of endogenous substrate (prosaposin)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, siRNA KD with defined glycosylation phenotype and native gel analysis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"23606741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NMR solution structures and MD simulations of yeast Ost4 and the V23D mutant show that while the V23D point mutation does not alter the overall helical structure of Ost4, it changes its position and solvent exposure in the membrane-mimetic environment; MD simulations of the membrane-embedded OST complex show that V23D disrupts hydrophobic helix–helix interactions between Ost4 and TM12/TM13 of Stt3, causing disengagement of Ost4V23D and exposure of residue D23 in the hydrophobic pocket, providing a structural mechanism for OST complex destabilization.\",\n      \"method\": \"Solution NMR structure determination (micelles), molecular dynamics simulations of membrane-bound OST complex with WT and V23D Ost4\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus MD simulation with mutagenesis, single lab, no reconstitution activity assay\",\n      \"pmids\": [\"33442744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Solid-state NMR of yeast Ost4 and Ost4V23D reconstituted in POPC/POPE lipid bilayers shows significant chemical shift changes upon V23D mutation, indicating a dramatic change in the chemical environment of the transmembrane helix in a native-like lipid bilayer context.\",\n      \"method\": \"Solid-state NMR with magic-angle spinning, reconstitution in lipid bilayers\",\n      \"journal\": \"Journal of biomolecular NMR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — solid-state NMR in lipid bilayers, single lab, no functional activity assay\",\n      \"pmids\": [\"38421550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ost4 is a substrate of the AAA+ ATPase Msp1 when mislocalized to mitochondria; an Msp1-protease chimera captures Ost4 as a novel Msp1 substrate by mass spectrometry; topology experiments show that mislocalized Ost4 adopts mixed membrane orientations at mitochondria, and Msp1 extracts mislocalized Ost4 regardless of its orientation.\",\n      \"method\": \"Msp1-protease chimera (substrate trapping), mass spectrometry identification, topology assay of mislocalized Ost4\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — novel chimeric trapping tool with MS identification and topology experiment, preprint, single lab\",\n      \"pmids\": [\"40236206\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"OST4 encodes an extremely small (36-residue, ~3.6 kDa) single-pass ER membrane protein that functions as a structural subunit of the oligosaccharyltransferase (OST) complex in both yeast and mammals: its cytoplasmic-leaflet transmembrane residues (Met18–Ile24 in yeast) mediate hydrophobic helix–helix contacts with the catalytic Stt3/STT3 subunit, physically bridging Stt3 to the accessory subunits Ost3/Ost6 (yeast) or ribophorin I (mammals), thereby stabilizing the assembled OST complex and supporting efficient co-translational N-glycosylation; loss or mutation of Ost4 disassembles these subcomplexes and reduces N-glycosylation activity, while mislocalized Ost4 is extracted from the outer mitochondrial membrane by the AAA+ ATPase Msp1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"OST4 encodes an unusually small (~3.6 kDa, 36-residue) single-pass ER membrane protein that functions as a structural subunit of the oligosaccharyltransferase (OST) complex required for efficient co-translational N-glycosylation in both yeast and mammals [#0, #1]. In yeast, Ost4p is an equimolar subunit of the eight-subunit OST and adopts an N(lumen)-C(cytosol) type I topology, with its cytoplasmic-leaflet transmembrane residues (~18-25) physically bridging the catalytic Stt3 subunit to the accessory subunit Ost3p; mutation of these residues to ionizable amino acids severs both the Ost4-Stt3 and the Ost3-Stt3 contacts [#1, #3]. Ost4p is required for assembly of two functionally distinct OST isoforms, since loss of Ost4p prevents incorporation of either Ost3p or Ost6p into the complex [#4]. The human ortholog assembles into both STT3A- and STT3B-containing OST complexes and contacts ribophorin I; a single transmembrane substitution or siRNA depletion of OST4 destabilizes native complexes and reduces N-glycosylation of endogenous substrate, paralleling the yeast role in stabilizing the assembled catalytic core [#7]. Structurally, the destabilizing V23D substitution exposes a charged residue in the membrane and disrupts hydrophobic helix-helix packing against Stt3 transmembrane segments TM12/TM13, explaining how a point mutation disassembles the complex [#8]. When mislocalized to the outer mitochondrial membrane, Ost4 is recognized and extracted by the AAA+ ATPase Msp1 irrespective of its membrane orientation [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that the OST4 gene product, despite its tiny size, is functionally required for normal oligosaccharyltransferase activity, defining it as a genuine OST component rather than an incidental polypeptide.\",\n      \"evidence\": \"Null-mutant genetics with in vivo and in vitro OTase activity assays in yeast\",\n      \"pmids\": [\"8621712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether Ost4 acts catalytically or structurally\", \"No subunit-contact map defined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Defined Ost4p as a stoichiometric OST subunit and localized its position within the complex, showing it bridges the catalytic Stt3p to the accessory Ost3p subunit.\",\n      \"evidence\": \"Co-immunoprecipitation of all subunits with quantitative stoichiometry and partial-denaturation subcomplex isolation in yeast\",\n      \"pmids\": [\"9405463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The molecular interface mediating bridging was not mapped\", \"Topology of Ost4p unknown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Provided genetic confirmation that Ost4p functionally interacts with the catalytic Stt3p subunit, independent of biochemical co-purification.\",\n      \"evidence\": \"Suppression of conditional stt3 alleles by OST4 overexpression in yeast\",\n      \"pmids\": [\"9435788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genetic epistasis does not establish a direct physical contact\", \"Single method, single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped Ost4p topology and identified the specific cytoplasmic-leaflet transmembrane residues whose integrity is needed to hold Stt3p and Ost3p together, converting the bridging model into residue-level mechanism.\",\n      \"evidence\": \"In vivo topology mapping, transmembrane mutagenesis, CD of synthetic peptide in liposomes, and Co-IP in ost4 mutant backgrounds\",\n      \"pmids\": [\"12810948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of the contact\", \"Did not address mammalian conservation\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated that Ost4p is required for biogenesis of both the Ost3p- and Ost6p-containing OST isoforms, broadening its role from a single complex to assembly of functionally distinct OST forms.\",\n      \"evidence\": \"Blue native PAGE of OST complexes from wild-type and ost4 deletion strains with subunit immunodetection\",\n      \"pmids\": [\"16096346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional differences between the two isoforms not attributed to Ost4 directly\", \"Order of assembly events unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided the first structural view of human OST4, showing an α-helical transmembrane segment with a kink.\",\n      \"evidence\": \"Solution NMR of human OST4 in detergent\",\n      \"pmids\": [\"21609714\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure in detergent rather than native bilayer\", \"No functional or interaction data in the same study\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the yeast subunit-stabilization model to mammals, showing human OST4 incorporates into both STT3A- and STT3B-containing complexes and stabilizes them to support co-translational N-glycosylation.\",\n      \"evidence\": \"Co-IP with both STT3 isoforms and ribophorin I, transmembrane mutagenesis, siRNA knockdown with native gel and prosaposin glycosylation assay\",\n      \"pmids\": [\"23606741\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise mammalian subunit interface not resolved at atomic level\", \"Substrate specificity consequences beyond prosaposin not characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a structural mechanism for OST destabilization by the V23D mutation, showing it disrupts hydrophobic packing against Stt3 TM12/TM13 and exposes a buried charge.\",\n      \"evidence\": \"Solution NMR of WT and V23D Ost4 plus MD simulation of the membrane-embedded OST complex\",\n      \"pmids\": [\"33442744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reconstituted activity assay confirming functional disengagement\", \"Simulation-derived, not experimentally resolved complex\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed in a native-like lipid bilayer that the V23D mutation dramatically alters the transmembrane helix environment, validating membrane-context effects beyond micelles.\",\n      \"evidence\": \"Magic-angle-spinning solid-state NMR of Ost4 and Ost4V23D in POPC/POPE bilayers\",\n      \"pmids\": [\"38421550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional activity assay\", \"Effect on intact OST complex not directly observed in bilayer\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified Ost4 as a quality-control substrate, showing that mislocalized Ost4 at mitochondria is extracted by the AAA+ ATPase Msp1 regardless of orientation, linking OST4 biogenesis to membrane-protein surveillance.\",\n      \"evidence\": \"Msp1-protease chimera substrate trapping, mass spectrometry identification, and topology assay of mislocalized Ost4 (preprint)\",\n      \"pmids\": [\"40236206\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Physiological frequency of Ost4 mislocalization unknown\", \"Fate of extracted Ost4 not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Ost4 acts within the catalytic cycle of an intact, reconstituted OST complex and whether its bridging function modulates glycosylation efficiency at the level of enzyme turnover remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reconstituted activity assay isolating Ost4 contribution\", \"No atomic structure of the full Ost4-Stt3-Ost3 interface\", \"Physiological consequences of OST4 loss in mammals beyond cell-based glycosylation assays uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 3, 4, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"complexes\": [\"oligosaccharyltransferase (OST) complex\"],\n    \"partners\": [\"STT3\", \"STT3A\", \"STT3B\", \"OST3\", \"OST6\", \"RPN1\", \"MSP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}