{"gene":"ERG28","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2001,"finding":"ERG28 (yeast Erg28p) is required for sterol C-4 demethylation; deletion of ERG28 in S. cerevisiae causes accumulation of 3-keto and carboxylic acid sterol intermediates identical to those in erg26 and erg27 mutants, suggesting Erg28p facilitates interaction between Erg26p and Erg27p and/or tethers them to the endoplasmic reticulum.","method":"Gas chromatography-mass spectrometry of sterol profiles in ERG28-deleted yeast strains; comparative phenotypic analysis with erg26 and erg27 null strains","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined biochemical phenotype (GC-MS sterol profiling) in deletion strain, but mechanism is inferred from phenotypic similarity rather than direct protein interaction assay in this paper","pmids":["11160377"],"is_preprint":false},{"year":2002,"finding":"Erg28p is a membrane-associated endoplasmic reticulum protein that physically interacts with Erg27p (reciprocal co-IP) and co-precipitates with Erg25p; together Erg25p/Erg26p/Erg27p/Erg28p form a complex of ~66–200 kDa in the ER, consistent with Erg28p functioning as a transmembrane scaffold to tether C-4 demethylation enzymes.","method":"Differential centrifugation (membrane association); GFP-fusion protein localization to ER; co-immunoprecipitation with anti-Erg25p antibody; reciprocal co-IP of Erg28p-Myc and Erg27p-HA; sucrose gradient ultracentrifugation; anti-HA affinity column complex isolation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (fractionation, GFP localization, reciprocal Co-IP, sucrose gradient, affinity purification) in one study establishing scaffold function and ER localization","pmids":["12119386"],"is_preprint":false},{"year":2004,"finding":"Erg28p physically interacts with the downstream ergosterol biosynthetic enzyme Erg6p (C-24 sterol methyltransferase): in erg28 deletion strains, Erg6p protein level in the ER fraction is reduced ~50%; Erg6p and Erg28p reciprocally co-immunoprecipitate; and a split-ubiquitin membrane two-hybrid assay confirms the Erg28p–Erg6p interaction. Erg28p thus acts as a protein bridge between the C-4 demethylation complex and Erg6p.","method":"Western blot quantification of Erg6p in ER fractions of erg28 vs. wild-type strains; reciprocal co-immunoprecipitation (anti-HA, anti-Erg6p); split-ubiquitin yeast membrane two-hybrid assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus orthogonal membrane two-hybrid plus fractionation, single lab","pmids":["15522820"],"is_preprint":false},{"year":2005,"finding":"Using a membrane-protein-specific yeast two-hybrid system (pairwise screen against all 14 sterol biosynthetic proteins), Erg28p was found to interact with Erg27p, Erg25p, Erg26p, Erg6p, Erg11p, and Erg1p; seven of these interactions were confirmed by co-immunoprecipitation. Quantitative reporter gene comparisons showed Erg28p is most closely associated with Erg27p, Erg25p, Erg11p, and Erg6p, suggesting Erg28p organizes a large sterol biosynthetic enzyme complex.","method":"Split-ubiquitin membrane two-hybrid system (two reporter genes + Western blot transcription factor release); co-immunoprecipitation confirmation of seven interactions","journal":"Journal of lipid research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic pairwise two-hybrid screen with multiple reporters confirmed by reciprocal Co-IP, single lab but multiple orthogonal methods","pmids":["15995173"],"is_preprint":false},{"year":2013,"finding":"A two-base deletion in the promoter of yeast ERG28 reduces binding of transcription factors Sok2 and Mot3, abolishing their transcriptional regulation of ERG28 and causing down-regulation of the ergosterol pathway; this cis-regulatory change confers increased resistance to fluconazole.","method":"Promoter deletion mapping; transcription factor binding assays; fluconazole resistance phenotyping in engineered yeast strains","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identified causal promoter mutation and responsible trans-factors by binding and functional assays, single lab","pmids":["24068973"],"is_preprint":false},{"year":2017,"finding":"In C. elegans, ERG-28 (the ERG28 ortholog) is an ER membrane protein that promotes trafficking of SLO-1 BK channels from the ER to the plasma membrane by shielding them from degradation by the aspartic protease DDI-1. Loss of erg-28 results in DDI-1-dependent degradation of SLO-1, markedly reducing SLO-1 expression at presynaptic terminals and suppressing slo-1 gain-of-function phenotypes in locomotion, neurotransmitter release, and AWC olfactory neuron asymmetric differentiation.","method":"Genetic epistasis (erg-28 loss-of-function suppression of slo-1 gain-of-function; erg-28;ddi-1 double mutant); fluorescence microscopy of SLO-1 subcellular localization; behavioral assays (locomotion, ethanol response); neurotransmitter release assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis with defined molecular pathway (ERG-28 → DDI-1-dependent degradation → SLO-1 trafficking), multiple orthogonal phenotypic readouts, localization data","pmids":["28168949"],"is_preprint":false},{"year":2018,"finding":"In S. cerevisiae Erg28p, the region spanning amino acids 63–72 (consensus motif 63LS/QARTFGT/LWT72) within the cytoplasmic loop is a key structural element required for sterol C-4 demethylation. Serial C-terminal truncation mutants and a deletion of residues 175–204 caused accumulation of C-4 methyl sterol intermediates and growth inhibition; homology modeling showed deletion of residues 63–72 disrupts the four-helix bundle of Erg28p required for complex assembly.","method":"Serial truncation mutagenesis; sterol profiling by GC-MS; complementation assays in erg28Δ yeast; homologous sequence alignment; homology modeling of 3D structure","journal":"FEMS microbiology letters","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis with biochemical readout (sterol profiling) and structural modeling, single lab","pmids":["29319811"],"is_preprint":false},{"year":2022,"finding":"Human ERG28 (C14orf1) is transcriptionally regulated by SREBP-2 via sterol-responsive elements in its proximal promoter. ERG28 interacts with itself and with the cholesterol synthesis enzymes NSDHL and SC4MOL (human homologs of Erg26p and Erg25p) as shown by a split-luciferase system. Knockout of ERG28 in Huh7 cells reduces total cholesterol levels under sterol-depleted conditions, reduces the rate of cholesterol synthesis by 60–75% (rescued by ectopic ERG28 re-expression), and impairs SREBP-2 activation under sterol-replete conditions.","method":"Quantitative RT-PCR; luciferase reporter assays; ChIP-seq data analysis; split-luciferase protein–protein interaction assay; CRISPR/Cas9 KO cell line generation; radiolabeled metabolic flux assay; ectopic rescue expression","journal":"Journal of lipid research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (metabolic flux, KO rescue, protein interaction assay, promoter assay) in mammalian cells establishing functional role in cholesterol synthesis","pmids":["36216146"],"is_preprint":false},{"year":2012,"finding":"Human C14orf1/hERG28 was identified as a potential protein-binding partner of CLN8 (an ER-resident NCL protein) using the split-ubiquitin membrane yeast two-hybrid system with a human brain cDNA library, placing hERG28 in an ER protein interaction network relevant to lipid synthesis and transport.","method":"Split-ubiquitin membrane-based yeast two-hybrid screen (full-length human CLN8 as bait, human brain cDNA library as prey)","journal":"Biochimica et biophysica acta","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Y2H screen hit, interaction not independently validated for hERG28 specifically","pmids":["23142642"],"is_preprint":false},{"year":2026,"finding":"In fission yeast (S. pombe), Erg28 is an ER-localized transmembrane protein that physically interacts with the microtubule-nucleating factors Mto1-Mto2 complex and γ-TuSC, and attenuates binding of γ-TuSC to the Mto1-Mto2 complex. In vitro, Erg28 inhibits Mto1-Mto2/γ-TuSC-mediated microtubule assembly. The cytosolic N-terminal region is required for this inhibitory activity. Deletion of erg28 causes excessive microtubule assembly and nuclear shape deformation.","method":"Biochemical interaction assays; in vitro microtubule assembly assay; microscopy (live imaging); deletion mutant analysis; domain mapping (N-terminal cytosolic region)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of inhibitory activity, biochemical binding assays, and genetic KO phenotype with defined molecular mechanism, single study","pmids":["41989917"],"is_preprint":false},{"year":1999,"finding":"The human C14orf1 (ERG28) gene encodes a protein with multiple predicted transmembrane domains, maps to chromosome 14q24, and its mRNA is highly expressed in adult testis and several cancer cell lines, consistent with a conserved membrane protein function.","method":"cDNA cloning; Northern blot / expression analysis; chromosomal mapping; transmembrane domain prediction","journal":"Cytogenetics and cell genetics","confidence":"Low","confidence_rationale":"Tier 4 / Weak — sequence/expression characterization only, no direct functional experiment on the protein mechanism","pmids":["10449901"],"is_preprint":false},{"year":2000,"finding":"The genomic structure of C14orf1 (ERG28) is conserved across eukaryotes (human, S. pombe, Arabidopsis); disruption of the S. cerevisiae ortholog (YER044c/ERG28) causes severe growth defects, and human C14orf1 failed to complement the yeast erg28 mutant when expressed under the yeast ERG28 promoter.","method":"Yeast complementation assay (human C14orf1 under Yer044c promoter in erg28Δ yeast); intron phase comparison across species","journal":"Mammalian genome","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct complementation experiment (negative result: failed to rescue), single lab","pmids":["10967139"],"is_preprint":false}],"current_model":"ERG28 (C14orf1/NET51) encodes a conserved multi-pass ER transmembrane protein that functions as a non-catalytic scaffold: in yeast it tethers the C-4 sterol demethylation complex (Erg25p/Erg26p/Erg27p) and the downstream methyltransferase Erg6p in the ER, with a critical 63–72 amino acid motif required for complex assembly; in mammals (human ERG28/C14orf1), it is transcriptionally driven by SREBP-2, interacts with NSDHL and SC4MOL, and is required for efficient cholesterol synthesis and normal SREBP-2 activation; in C. elegans the ortholog ERG-28 additionally controls BK channel (SLO-1) trafficking from the ER to synaptic membranes by protecting channels from DDI-1-dependent proteasomal degradation; and in fission yeast Erg28 also interacts with the Mto1-Mto2/γ-TuSC microtubule nucleation machinery to restrain microtubule assembly from the ER surface."},"narrative":{"mechanistic_narrative":"ERG28 (C14orf1/NET51) encodes a conserved multi-pass endoplasmic reticulum transmembrane protein that acts as a non-catalytic scaffold organizing sterol biosynthetic enzymes [PMID:12119386, PMID:36216146]. In S. cerevisiae, Erg28p is required for sterol C-4 demethylation, and its deletion causes accumulation of 3-keto and carboxylic acid sterol intermediates that phenocopy erg26 and erg27 mutants [PMID:11160377]. It physically tethers the C-4 demethylation enzymes Erg25p/Erg26p/Erg27p in the ER and bridges them to the downstream C-24 methyltransferase Erg6p, whose ER abundance drops when ERG28 is lost [PMID:12119386, PMID:15522820]; a systematic membrane two-hybrid screen places Erg28p at the center of a broader sterol-enzyme network including Erg11p and Erg1p [PMID:15995173], and a cytoplasmic-loop motif spanning residues 63–72 within a four-helix bundle is required for complex assembly [PMID:29319811]. The human ortholog ERG28 is transcriptionally driven by SREBP-2, self-associates, and interacts with the cholesterol synthesis enzymes NSDHL and SC4MOL; its loss reduces cholesterol synthesis and impairs SREBP-2 activation, establishing a conserved role in cholesterol biosynthesis [PMID:36216146]. Beyond sterol metabolism, ERG28 orthologs perform additional ER-membrane scaffolding functions: in C. elegans, ERG-28 promotes trafficking of SLO-1 BK channels to synaptic membranes by shielding them from DDI-1-dependent proteasomal degradation [PMID:28168949], and in fission yeast Erg28 binds the Mto1-Mto2/γ-TuSC microtubule-nucleation machinery and restrains microtubule assembly from the ER surface via its cytosolic N-terminus [PMID:41989917].","teleology":[{"year":2001,"claim":"Establishing whether ERG28 participates in sterol biosynthesis, the discovery that its deletion blocks C-4 demethylation defined it as a functional component of the ergosterol pathway rather than an incidental ER protein.","evidence":"GC-MS sterol profiling of ERG28-deleted yeast compared to erg26/erg27 nulls","pmids":["11160377"],"confidence":"Medium","gaps":["Mechanism inferred from phenotypic similarity, not direct interaction in this study","Did not establish whether Erg28p is catalytic or scaffolding"]},{"year":2002,"claim":"To resolve how Erg28p acts without enzymatic activity, physical interaction and localization data showed it is an ER membrane protein that binds Erg27p and Erg25p, defining it as a transmembrane scaffold tethering the demethylation complex.","evidence":"Differential centrifugation, GFP localization, reciprocal Co-IP, sucrose gradient and affinity purification in S. cerevisiae","pmids":["12119386"],"confidence":"High","gaps":["Did not map the interacting residues on Erg28p","Stoichiometry of the complex not resolved"]},{"year":2004,"claim":"Extending the scaffold model downstream, Erg28p was shown to bridge the C-4 demethylation complex to Erg6p, explaining how sequential pathway enzymes are coupled in the ER.","evidence":"ER fraction Western blots, reciprocal Co-IP, and split-ubiquitin membrane two-hybrid in erg28 vs. wild-type yeast","pmids":["15522820"],"confidence":"High","gaps":["Whether Erg6p destabilization is direct or secondary to complex disruption not distinguished"]},{"year":2005,"claim":"A systematic pairwise interaction screen tested how broad the scaffold's reach is, revealing Erg28p contacts six sterol enzymes and is most tightly associated with Erg27p/Erg25p/Erg11p/Erg6p, casting it as the organizer of a large sterol biosynthetic enzyme assembly.","evidence":"Split-ubiquitin membrane two-hybrid across all 14 sterol enzymes with Co-IP confirmation","pmids":["15995173"],"confidence":"High","gaps":["Did not establish whether interactions are simultaneous within one complex or mutually exclusive","No structural architecture of the assembly"]},{"year":2013,"claim":"Addressing how ERG28 expression is controlled in yeast, a cis-regulatory promoter mutation was shown to abolish Sok2/Mot3 binding and down-regulate the ergosterol pathway, linking ERG28 dosage to azole drug resistance.","evidence":"Promoter deletion mapping, transcription factor binding, and fluconazole phenotyping in engineered yeast","pmids":["24068973"],"confidence":"Medium","gaps":["Did not test ERG28 protein-level consequences directly","Single-lab regulatory mapping"]},{"year":2018,"claim":"To pinpoint the structural basis of scaffolding, mutagenesis identified a 63–72 cytoplasmic-loop motif within a four-helix bundle as essential for complex assembly and demethylation.","evidence":"Serial truncation mutagenesis, GC-MS sterol profiling, complementation, and homology modeling in yeast","pmids":["29319811"],"confidence":"Medium","gaps":["No experimental structure to confirm the modeled four-helix bundle","Which partner each motif residue contacts not defined"]},{"year":2022,"claim":"Determining whether the yeast scaffold role is conserved in humans, ERG28 was shown to be SREBP-2-driven, to bind NSDHL and SC4MOL, and to be required for efficient cholesterol synthesis and SREBP-2 activation.","evidence":"qRT-PCR, luciferase/ChIP-seq promoter analysis, split-luciferase interaction, CRISPR KO with rescue, and radiolabeled flux in Huh7 cells","pmids":["36216146"],"confidence":"High","gaps":["Whether human ERG28 tethers the full enzyme assembly as in yeast not directly shown","Mechanism of impaired SREBP-2 activation unresolved"]},{"year":2017,"claim":"Revealing a non-sterol scaffolding function, the C. elegans ortholog ERG-28 was shown to protect SLO-1 BK channels from DDI-1-dependent degradation, enabling their ER-to-synapse trafficking.","evidence":"Genetic epistasis (erg-28;ddi-1 double mutants), SLO-1 localization microscopy, and behavioral/neurotransmitter assays in C. elegans","pmids":["28168949"],"confidence":"High","gaps":["Whether ERG-28 directly binds SLO-1 or acts indirectly not established","Relationship to its sterol role in worms not addressed"]},{"year":2026,"claim":"Uncovering a further ER-surface function, fission yeast Erg28 was shown to bind Mto1-Mto2/γ-TuSC and inhibit microtubule nucleation, linking ER membrane scaffolding to cytoskeletal control.","evidence":"Biochemical binding assays, in vitro microtubule assembly reconstitution, domain mapping, and deletion-mutant imaging in S. pombe","pmids":["41989917"],"confidence":"High","gaps":["Whether this microtubule role is conserved in mammals unknown","Mechanistic link between sterol scaffolding and microtubule restraint unresolved"]},{"year":null,"claim":"Whether the divergent ERG28 functions — sterol enzyme scaffolding, BK channel protection, and microtubule restraint — reflect a single unifying biochemical activity or independent moonlighting roles remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of any ERG28 complex","Human ortholog's potential channel-trafficking or microtubule roles untested","Failure of human ERG28 to complement yeast erg28 (#11) suggests functional divergence not yet mechanistically explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,3,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,9]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,5,9]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,7]}],"complexes":["Erg25p/Erg26p/Erg27p C-4 sterol demethylation complex"],"partners":["ERG27","ERG25","ERG26","ERG6","ERG11","NSDHL","SC4MOL","MTO1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UKR5","full_name":"Ergosterol biosynthetic protein 28 homolog","aliases":[],"length_aa":140,"mass_kda":15.9,"function":"","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9UKR5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ERG28","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000133935","cell_line_id":"CID000370","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"er","grade":1}],"interactors":[{"gene":"FUS","stoichiometry":0.2},{"gene":"ZNF703","stoichiometry":0.2},{"gene":"UBAP2L","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000370","total_profiled":1310},"omim":[{"mim_id":"604576","title":"ERGOSTEROL BIOSYNTHESIS 28 HOMOLOG; ERG28","url":"https://www.omim.org/entry/604576"}],"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/ERG28"},"hgnc":{"alias_symbol":["NET51"],"prev_symbol":["C14orf1"]},"alphafold":{"accession":"Q9UKR5","domains":[{"cath_id":"-","chopping":"1-140","consensus_level":"medium","plddt":93.1867,"start":1,"end":140}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UKR5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UKR5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UKR5-F1-predicted_aligned_error_v6.png","plddt_mean":93.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ERG28","jax_strain_url":"https://www.jax.org/strain/search?query=ERG28"},"sequence":{"accession":"Q9UKR5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UKR5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UKR5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UKR5"}},"corpus_meta":[{"pmid":"12702675","id":"PMC_12702675","title":"Mode of selection and experimental 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reports","url":"https://pubmed.ncbi.nlm.nih.gov/41989917","citation_count":0,"is_preprint":false},{"pmid":"40467731","id":"PMC_40467731","title":"Comparative transcriptome analysis reveals genes associated with variation in liver copper concentration in Polish Merino sheep.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40467731","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16316,"output_tokens":3722,"usd":0.052389,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11293,"output_tokens":3402,"usd":0.070758,"stage2_stop_reason":"end_turn"},"total_usd":0.123147,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"ERG28 (yeast Erg28p) is required for sterol C-4 demethylation; deletion of ERG28 in S. cerevisiae causes accumulation of 3-keto and carboxylic acid sterol intermediates identical to those in erg26 and erg27 mutants, suggesting Erg28p facilitates interaction between Erg26p and Erg27p and/or tethers them to the endoplasmic reticulum.\",\n      \"method\": \"Gas chromatography-mass spectrometry of sterol profiles in ERG28-deleted yeast strains; comparative phenotypic analysis with erg26 and erg27 null strains\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined biochemical phenotype (GC-MS sterol profiling) in deletion strain, but mechanism is inferred from phenotypic similarity rather than direct protein interaction assay in this paper\",\n      \"pmids\": [\"11160377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Erg28p is a membrane-associated endoplasmic reticulum protein that physically interacts with Erg27p (reciprocal co-IP) and co-precipitates with Erg25p; together Erg25p/Erg26p/Erg27p/Erg28p form a complex of ~66–200 kDa in the ER, consistent with Erg28p functioning as a transmembrane scaffold to tether C-4 demethylation enzymes.\",\n      \"method\": \"Differential centrifugation (membrane association); GFP-fusion protein localization to ER; co-immunoprecipitation with anti-Erg25p antibody; reciprocal co-IP of Erg28p-Myc and Erg27p-HA; sucrose gradient ultracentrifugation; anti-HA affinity column complex isolation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (fractionation, GFP localization, reciprocal Co-IP, sucrose gradient, affinity purification) in one study establishing scaffold function and ER localization\",\n      \"pmids\": [\"12119386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Erg28p physically interacts with the downstream ergosterol biosynthetic enzyme Erg6p (C-24 sterol methyltransferase): in erg28 deletion strains, Erg6p protein level in the ER fraction is reduced ~50%; Erg6p and Erg28p reciprocally co-immunoprecipitate; and a split-ubiquitin membrane two-hybrid assay confirms the Erg28p–Erg6p interaction. Erg28p thus acts as a protein bridge between the C-4 demethylation complex and Erg6p.\",\n      \"method\": \"Western blot quantification of Erg6p in ER fractions of erg28 vs. wild-type strains; reciprocal co-immunoprecipitation (anti-HA, anti-Erg6p); split-ubiquitin yeast membrane two-hybrid assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus orthogonal membrane two-hybrid plus fractionation, single lab\",\n      \"pmids\": [\"15522820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Using a membrane-protein-specific yeast two-hybrid system (pairwise screen against all 14 sterol biosynthetic proteins), Erg28p was found to interact with Erg27p, Erg25p, Erg26p, Erg6p, Erg11p, and Erg1p; seven of these interactions were confirmed by co-immunoprecipitation. Quantitative reporter gene comparisons showed Erg28p is most closely associated with Erg27p, Erg25p, Erg11p, and Erg6p, suggesting Erg28p organizes a large sterol biosynthetic enzyme complex.\",\n      \"method\": \"Split-ubiquitin membrane two-hybrid system (two reporter genes + Western blot transcription factor release); co-immunoprecipitation confirmation of seven interactions\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic pairwise two-hybrid screen with multiple reporters confirmed by reciprocal Co-IP, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15995173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A two-base deletion in the promoter of yeast ERG28 reduces binding of transcription factors Sok2 and Mot3, abolishing their transcriptional regulation of ERG28 and causing down-regulation of the ergosterol pathway; this cis-regulatory change confers increased resistance to fluconazole.\",\n      \"method\": \"Promoter deletion mapping; transcription factor binding assays; fluconazole resistance phenotyping in engineered yeast strains\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identified causal promoter mutation and responsible trans-factors by binding and functional assays, single lab\",\n      \"pmids\": [\"24068973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In C. elegans, ERG-28 (the ERG28 ortholog) is an ER membrane protein that promotes trafficking of SLO-1 BK channels from the ER to the plasma membrane by shielding them from degradation by the aspartic protease DDI-1. Loss of erg-28 results in DDI-1-dependent degradation of SLO-1, markedly reducing SLO-1 expression at presynaptic terminals and suppressing slo-1 gain-of-function phenotypes in locomotion, neurotransmitter release, and AWC olfactory neuron asymmetric differentiation.\",\n      \"method\": \"Genetic epistasis (erg-28 loss-of-function suppression of slo-1 gain-of-function; erg-28;ddi-1 double mutant); fluorescence microscopy of SLO-1 subcellular localization; behavioral assays (locomotion, ethanol response); neurotransmitter release assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with defined molecular pathway (ERG-28 → DDI-1-dependent degradation → SLO-1 trafficking), multiple orthogonal phenotypic readouts, localization data\",\n      \"pmids\": [\"28168949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In S. cerevisiae Erg28p, the region spanning amino acids 63–72 (consensus motif 63LS/QARTFGT/LWT72) within the cytoplasmic loop is a key structural element required for sterol C-4 demethylation. Serial C-terminal truncation mutants and a deletion of residues 175–204 caused accumulation of C-4 methyl sterol intermediates and growth inhibition; homology modeling showed deletion of residues 63–72 disrupts the four-helix bundle of Erg28p required for complex assembly.\",\n      \"method\": \"Serial truncation mutagenesis; sterol profiling by GC-MS; complementation assays in erg28Δ yeast; homologous sequence alignment; homology modeling of 3D structure\",\n      \"journal\": \"FEMS microbiology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis with biochemical readout (sterol profiling) and structural modeling, single lab\",\n      \"pmids\": [\"29319811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human ERG28 (C14orf1) is transcriptionally regulated by SREBP-2 via sterol-responsive elements in its proximal promoter. ERG28 interacts with itself and with the cholesterol synthesis enzymes NSDHL and SC4MOL (human homologs of Erg26p and Erg25p) as shown by a split-luciferase system. Knockout of ERG28 in Huh7 cells reduces total cholesterol levels under sterol-depleted conditions, reduces the rate of cholesterol synthesis by 60–75% (rescued by ectopic ERG28 re-expression), and impairs SREBP-2 activation under sterol-replete conditions.\",\n      \"method\": \"Quantitative RT-PCR; luciferase reporter assays; ChIP-seq data analysis; split-luciferase protein–protein interaction assay; CRISPR/Cas9 KO cell line generation; radiolabeled metabolic flux assay; ectopic rescue expression\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (metabolic flux, KO rescue, protein interaction assay, promoter assay) in mammalian cells establishing functional role in cholesterol synthesis\",\n      \"pmids\": [\"36216146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human C14orf1/hERG28 was identified as a potential protein-binding partner of CLN8 (an ER-resident NCL protein) using the split-ubiquitin membrane yeast two-hybrid system with a human brain cDNA library, placing hERG28 in an ER protein interaction network relevant to lipid synthesis and transport.\",\n      \"method\": \"Split-ubiquitin membrane-based yeast two-hybrid screen (full-length human CLN8 as bait, human brain cDNA library as prey)\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Y2H screen hit, interaction not independently validated for hERG28 specifically\",\n      \"pmids\": [\"23142642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In fission yeast (S. pombe), Erg28 is an ER-localized transmembrane protein that physically interacts with the microtubule-nucleating factors Mto1-Mto2 complex and γ-TuSC, and attenuates binding of γ-TuSC to the Mto1-Mto2 complex. In vitro, Erg28 inhibits Mto1-Mto2/γ-TuSC-mediated microtubule assembly. The cytosolic N-terminal region is required for this inhibitory activity. Deletion of erg28 causes excessive microtubule assembly and nuclear shape deformation.\",\n      \"method\": \"Biochemical interaction assays; in vitro microtubule assembly assay; microscopy (live imaging); deletion mutant analysis; domain mapping (N-terminal cytosolic region)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of inhibitory activity, biochemical binding assays, and genetic KO phenotype with defined molecular mechanism, single study\",\n      \"pmids\": [\"41989917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The human C14orf1 (ERG28) gene encodes a protein with multiple predicted transmembrane domains, maps to chromosome 14q24, and its mRNA is highly expressed in adult testis and several cancer cell lines, consistent with a conserved membrane protein function.\",\n      \"method\": \"cDNA cloning; Northern blot / expression analysis; chromosomal mapping; transmembrane domain prediction\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — sequence/expression characterization only, no direct functional experiment on the protein mechanism\",\n      \"pmids\": [\"10449901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The genomic structure of C14orf1 (ERG28) is conserved across eukaryotes (human, S. pombe, Arabidopsis); disruption of the S. cerevisiae ortholog (YER044c/ERG28) causes severe growth defects, and human C14orf1 failed to complement the yeast erg28 mutant when expressed under the yeast ERG28 promoter.\",\n      \"method\": \"Yeast complementation assay (human C14orf1 under Yer044c promoter in erg28Δ yeast); intron phase comparison across species\",\n      \"journal\": \"Mammalian genome\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct complementation experiment (negative result: failed to rescue), single lab\",\n      \"pmids\": [\"10967139\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ERG28 (C14orf1/NET51) encodes a conserved multi-pass ER transmembrane protein that functions as a non-catalytic scaffold: in yeast it tethers the C-4 sterol demethylation complex (Erg25p/Erg26p/Erg27p) and the downstream methyltransferase Erg6p in the ER, with a critical 63–72 amino acid motif required for complex assembly; in mammals (human ERG28/C14orf1), it is transcriptionally driven by SREBP-2, interacts with NSDHL and SC4MOL, and is required for efficient cholesterol synthesis and normal SREBP-2 activation; in C. elegans the ortholog ERG-28 additionally controls BK channel (SLO-1) trafficking from the ER to synaptic membranes by protecting channels from DDI-1-dependent proteasomal degradation; and in fission yeast Erg28 also interacts with the Mto1-Mto2/γ-TuSC microtubule nucleation machinery to restrain microtubule assembly from the ER surface.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ERG28 (C14orf1/NET51) encodes a conserved multi-pass endoplasmic reticulum transmembrane protein that acts as a non-catalytic scaffold organizing sterol biosynthetic enzymes [#1, #7]. In S. cerevisiae, Erg28p is required for sterol C-4 demethylation, and its deletion causes accumulation of 3-keto and carboxylic acid sterol intermediates that phenocopy erg26 and erg27 mutants [#0]. It physically tethers the C-4 demethylation enzymes Erg25p/Erg26p/Erg27p in the ER and bridges them to the downstream C-24 methyltransferase Erg6p, whose ER abundance drops when ERG28 is lost [#1, #2]; a systematic membrane two-hybrid screen places Erg28p at the center of a broader sterol-enzyme network including Erg11p and Erg1p [#3], and a cytoplasmic-loop motif spanning residues 63\\u201372 within a four-helix bundle is required for complex assembly [#6]. The human ortholog ERG28 is transcriptionally driven by SREBP-2, self-associates, and interacts with the cholesterol synthesis enzymes NSDHL and SC4MOL; its loss reduces cholesterol synthesis and impairs SREBP-2 activation, establishing a conserved role in cholesterol biosynthesis [#7]. Beyond sterol metabolism, ERG28 orthologs perform additional ER-membrane scaffolding functions: in C. elegans, ERG-28 promotes trafficking of SLO-1 BK channels to synaptic membranes by shielding them from DDI-1-dependent proteasomal degradation [#5], and in fission yeast Erg28 binds the Mto1-Mto2/\\u03b3-TuSC microtubule-nucleation machinery and restrains microtubule assembly from the ER surface via its cytosolic N-terminus [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing whether ERG28 participates in sterol biosynthesis, the discovery that its deletion blocks C-4 demethylation defined it as a functional component of the ergosterol pathway rather than an incidental ER protein.\",\n      \"evidence\": \"GC-MS sterol profiling of ERG28-deleted yeast compared to erg26/erg27 nulls\",\n      \"pmids\": [\"11160377\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism inferred from phenotypic similarity, not direct interaction in this study\", \"Did not establish whether Erg28p is catalytic or scaffolding\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"To resolve how Erg28p acts without enzymatic activity, physical interaction and localization data showed it is an ER membrane protein that binds Erg27p and Erg25p, defining it as a transmembrane scaffold tethering the demethylation complex.\",\n      \"evidence\": \"Differential centrifugation, GFP localization, reciprocal Co-IP, sucrose gradient and affinity purification in S. cerevisiae\",\n      \"pmids\": [\"12119386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the interacting residues on Erg28p\", \"Stoichiometry of the complex not resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Extending the scaffold model downstream, Erg28p was shown to bridge the C-4 demethylation complex to Erg6p, explaining how sequential pathway enzymes are coupled in the ER.\",\n      \"evidence\": \"ER fraction Western blots, reciprocal Co-IP, and split-ubiquitin membrane two-hybrid in erg28 vs. wild-type yeast\",\n      \"pmids\": [\"15522820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Erg6p destabilization is direct or secondary to complex disruption not distinguished\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"A systematic pairwise interaction screen tested how broad the scaffold's reach is, revealing Erg28p contacts six sterol enzymes and is most tightly associated with Erg27p/Erg25p/Erg11p/Erg6p, casting it as the organizer of a large sterol biosynthetic enzyme assembly.\",\n      \"evidence\": \"Split-ubiquitin membrane two-hybrid across all 14 sterol enzymes with Co-IP confirmation\",\n      \"pmids\": [\"15995173\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether interactions are simultaneous within one complex or mutually exclusive\", \"No structural architecture of the assembly\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Addressing how ERG28 expression is controlled in yeast, a cis-regulatory promoter mutation was shown to abolish Sok2/Mot3 binding and down-regulate the ergosterol pathway, linking ERG28 dosage to azole drug resistance.\",\n      \"evidence\": \"Promoter deletion mapping, transcription factor binding, and fluconazole phenotyping in engineered yeast\",\n      \"pmids\": [\"24068973\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not test ERG28 protein-level consequences directly\", \"Single-lab regulatory mapping\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"To pinpoint the structural basis of scaffolding, mutagenesis identified a 63\\u201372 cytoplasmic-loop motif within a four-helix bundle as essential for complex assembly and demethylation.\",\n      \"evidence\": \"Serial truncation mutagenesis, GC-MS sterol profiling, complementation, and homology modeling in yeast\",\n      \"pmids\": [\"29319811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure to confirm the modeled four-helix bundle\", \"Which partner each motif residue contacts not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Determining whether the yeast scaffold role is conserved in humans, ERG28 was shown to be SREBP-2-driven, to bind NSDHL and SC4MOL, and to be required for efficient cholesterol synthesis and SREBP-2 activation.\",\n      \"evidence\": \"qRT-PCR, luciferase/ChIP-seq promoter analysis, split-luciferase interaction, CRISPR KO with rescue, and radiolabeled flux in Huh7 cells\",\n      \"pmids\": [\"36216146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human ERG28 tethers the full enzyme assembly as in yeast not directly shown\", \"Mechanism of impaired SREBP-2 activation unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealing a non-sterol scaffolding function, the C. elegans ortholog ERG-28 was shown to protect SLO-1 BK channels from DDI-1-dependent degradation, enabling their ER-to-synapse trafficking.\",\n      \"evidence\": \"Genetic epistasis (erg-28;ddi-1 double mutants), SLO-1 localization microscopy, and behavioral/neurotransmitter assays in C. elegans\",\n      \"pmids\": [\"28168949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ERG-28 directly binds SLO-1 or acts indirectly not established\", \"Relationship to its sterol role in worms not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Uncovering a further ER-surface function, fission yeast Erg28 was shown to bind Mto1-Mto2/\\u03b3-TuSC and inhibit microtubule nucleation, linking ER membrane scaffolding to cytoskeletal control.\",\n      \"evidence\": \"Biochemical binding assays, in vitro microtubule assembly reconstitution, domain mapping, and deletion-mutant imaging in S. pombe\",\n      \"pmids\": [\"41989917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this microtubule role is conserved in mammals unknown\", \"Mechanistic link between sterol scaffolding and microtubule restraint unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether the divergent ERG28 functions \\u2014 sterol enzyme scaffolding, BK channel protection, and microtubule restraint \\u2014 reflect a single unifying biochemical activity or independent moonlighting roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure of any ERG28 complex\", \"Human ortholog's potential channel-trafficking or microtubule roles untested\", \"Failure of human ERG28 to complement yeast erg28 (#11) suggests functional divergence not yet mechanistically explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"complexes\": [\"Erg25p/Erg26p/Erg27p C-4 sterol demethylation complex\"],\n    \"partners\": [\"ERG27\", \"ERG25\", \"ERG26\", \"ERG6\", \"ERG11\", \"NSDHL\", \"SC4MOL\", \"MTO1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}