{"gene":"NCLN","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2004,"finding":"Nicalin forms a protein complex with NOMO (Nodal modulator) at the membrane, and this Nicalin/NOMO complex antagonizes Nodal/TGFbeta signaling during mesendodermal patterning. Ectopic expression of both proteins in zebrafish causes cyclopia, and Nodal- and Activin-induced signaling was inhibited by Nicalin and NOMO in a cell-based reporter assay.","method":"Zebrafish ectopic expression, morpholino knockdown, cell-based Nodal/Activin reporter assay, co-immunoprecipitation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional experiments in vivo (zebrafish) and in vitro (reporter assay), replicated across multiple experimental approaches in one study and confirmed in subsequent papers","pmids":["15257293"],"is_preprint":false},{"year":2007,"finding":"Nicalin regulates assembly and stability of the Nicalin-NOMO complex in the ER: Nicalin and NOMO are mutually stabilizing (each becomes unstable without the other), Nicalin is the limiting factor controlling complex assembly rate, and NOMO is produced in excess and either stabilized by Nicalin or rapidly degraded. These effects occur post-transcriptionally.","method":"RNA interference knockdown, overexpression, protein stability assays (post-transcriptional level confirmed by mRNA analysis)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi and overexpression with multiple readouts in a single lab study","pmids":["17261586"],"is_preprint":false},{"year":2010,"finding":"TMEM147, a ~22 kDa transmembrane protein, is a third core component of the Nicalin-NOMO complex. Complex assembly is hierarchical: a Nicalin-NOMO intermediate forms first, then TMEM147 joins. Nicalin acts as the limiting/stabilizing factor for all three components. All three proteins localize to the ER.","method":"Affinity purification of native complex, mass spectrometry identification of TMEM147, overexpression and RNAi knockdown in cultured cells, ER localization by immunofluorescence, zebrafish expression analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mass spectrometry identification of endogenous complex component, validated by overexpression and RNAi, localization confirmed, consistent with prior mechanistic framework","pmids":["20538592"],"is_preprint":false},{"year":2020,"finding":"Nicalin is a core component of a ~360 kDa ribosome-associated multipass translocon (MPT) complex at the ER that also includes Sec61, TMCO1, CCDC47, TMEM147, and NOMO. Cryo-EM reveals this assembly at the ribosome exit tunnel organized around a central membrane cavity, with a luminal funnel in TMEM147 suggesting a route into the cavity. Cells lacking accessory components (including those of the Nicalin-TMEM147-NOMO sub-complex) show reduced levels of the multi-pass membrane protein client EAAT1.","method":"Cryo-electron microscopy, high-throughput mRNA sequencing (ribosome profiling), genetic knockout of accessory components with EAAT1 protein-level readout","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with genetic loss-of-function and client protein assay, multiple orthogonal methods in one rigorous study","pmids":["32820719"],"is_preprint":false},{"year":2014,"finding":"The C. elegans nicalin homolog NRA-2 controls the distribution of DEG/ENaC channel MEC-10(d) between the ER and the cell surface, acting as a quality control mechanism to retain mutant channels in the ER. NRA-2 knockout enhanced MEC-10(d)-induced neuronal death. Electrophysiology in Xenopus oocytes showed that NRA-2's control of channel distribution is dependent on subunit composition.","method":"C. elegans RNAi and knockout genetics, cell biological localization assays in neurons, two-electrode voltage clamp electrophysiology in Xenopus oocytes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined cellular phenotype and electrophysiological validation, ortholog (C. elegans NRA-2) studied in single lab","pmids":["24567339"],"is_preprint":false},{"year":2014,"finding":"Nicalin (the transmembrane protein) was identified as a ganglioside GT1b-binding protein in rat cerebellar granule neurons and associates with the AMPA receptor trafficking complex containing Thorase and NSF. Addition of non-cleavable ATP (ATPγS) enhanced AMPAR association with Nicalin.","method":"Affinity capture with ganglioside GT1b, quantitative proteomic mass spectrometry, ATPγS competition assay","journal":"The Journal of neuroscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single affinity-capture/MS experiment identifying Nicalin as a GT1b-binding protein; no functional follow-up specific to Nicalin's role","pmids":["25253868"],"is_preprint":false},{"year":2017,"finding":"NCLN ortholog is indispensable for enteric nervous system (ENS) development in zebrafish, as shown by both morpholino knockdown and CRISPR knockout resulting in ENS defects.","method":"Morpholino knockdown, CRISPR knockout in zebrafish, ENS phenotype analysis","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two complementary loss-of-function approaches (morpholino + CRISPR) with defined developmental phenotype, single study","pmids":["28274275"],"is_preprint":false},{"year":2020,"finding":"Nicalin interacts with soluble EphA7 (sEphA7) and co-localizes with it predominantly in the ER. Nicalin reduces sEphA7 in the membranous fraction and increases it in the insoluble cytoplasmic fraction with reduced molecular weight, suggesting Nicalin restricts sEphA7 entry into the ER for further modification and limits sEphA7 secretion, thereby preventing formation of the EphA7 complex.","method":"Immunoprecipitation/mass spectrometry identification, co-localization by immunofluorescence in HEK293 cells, subcellular fractionation","journal":"Molecular and cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single IP/MS identification and fractionation without mutagenesis or rescue experiments","pmids":["32914261"],"is_preprint":false},{"year":2024,"finding":"In the native ER membrane environment, the Nicalin-TMEM147-NOMO sub-complex is part of the multipass translocon (MPT). Cryo-electron tomography and subtomogram averaging reveal that NOMO bridges the luminal domains of Nicalin and TRAPα, forming intermolecular contacts. The MPT composition varies with ribosome translational activity.","method":"Cryo-electron tomography, subtomogram averaging, AlphaFold structural modeling","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-ET with subtomogram averaging in native membranes plus AlphaFold modeling, single study without mutagenesis validation","pmids":["38866426"],"is_preprint":false},{"year":2025,"finding":"SigmaR1 directly interacts with Nicalin as a component of the translocon complex at the ER, as demonstrated by biochemical co-immunoprecipitation. Phosphatidylcholine (PC) binding by SigmaR1 strengthens its association with the translocon complex including Nicalin.","method":"Endogenous tagging, cell fractionation, co-immunoprecipitation, lipid-binding assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical co-IP with endogenous tagging and lipid-binding assay, single lab study","pmids":["41476177"],"is_preprint":false}],"current_model":"Nicalin (NCLN) is a core ER-resident transmembrane protein that forms a hierarchical complex with NOMO and TMEM147 (the Nicalin-TMEM147-NOMO sub-complex), which in turn is embedded within a larger ~360 kDa ribosome-associated multipass translocon (MPT) alongside Sec61, TMCO1, and CCDC47; this MPT complex facilitates biogenesis of multi-pass membrane proteins, while the Nicalin-NOMO complex also antagonizes Nodal/TGFbeta signaling during vertebrate embryonic development, and Nicalin additionally functions in ER quality control by regulating surface localization of client proteins such as DEG/ENaC channels."},"narrative":{"mechanistic_narrative":"Nicalin (NCLN) is a core ER-resident transmembrane protein that organizes a hierarchical sub-complex with NOMO and TMEM147 embedded within a ribosome-associated multipass translocon that supports the biogenesis of multi-pass membrane proteins [PMID:20538592, PMID:32820719]. Nicalin acts as the limiting, stabilizing factor for the sub-complex: it first forms a mutually stabilizing intermediate with NOMO, after which TMEM147 joins, with each partner becoming unstable in the absence of Nicalin [PMID:17261586, PMID:20538592]. Within the native ER membrane, this Nicalin-TMEM147-NOMO module assembles into a ~360 kDa multipass translocon together with Sec61, TMCO1, and CCDC47, organized around a central membrane cavity at the ribosome exit tunnel; NOMO bridges the luminal domains of Nicalin and TRAPalpha, and loss of accessory components reduces levels of the multi-pass client EAAT1 [PMID:32820719, PMID:38866426]. The translocon-associated chaperone SigmaR1 directly binds Nicalin, an interaction strengthened by phosphatidylcholine binding [PMID:41476177]. Independent of its translocon role, the Nicalin-NOMO complex antagonizes Nodal/Activin (TGFbeta) signaling during vertebrate mesendodermal patterning, and the Nicalin ortholog is required for enteric nervous system development in zebrafish [PMID:15257293, PMID:28274275]. Nicalin also participates in ER quality control of membrane clients, governing the ER-versus-surface distribution of DEG/ENaC channels through its C. elegans ortholog NRA-2 [PMID:24567339].","teleology":[{"year":2004,"claim":"Established Nicalin's first biological function by showing it partners with NOMO to antagonize Nodal/TGFbeta signaling during embryonic patterning.","evidence":"Zebrafish ectopic expression and morpholino knockdown, cell-based Nodal/Activin reporter assay, and co-immunoprecipitation","pmids":["15257293"],"confidence":"High","gaps":["Molecular mechanism by which the Nicalin/NOMO complex inhibits Nodal signaling not defined","Relationship between this signaling role and ER residence unresolved"]},{"year":2007,"claim":"Defined Nicalin as the assembly-limiting and stabilizing subunit of the Nicalin-NOMO complex, explaining how complex stoichiometry is controlled post-transcriptionally.","evidence":"RNAi knockdown, overexpression, and protein stability assays with mRNA controls in cultured cells","pmids":["17261586"],"confidence":"Medium","gaps":["Degradation pathway for excess NOMO not identified","Structural basis of mutual stabilization unknown"]},{"year":2010,"claim":"Extended the complex to a third subunit by identifying TMEM147 and showing hierarchical assembly (Nicalin-NOMO first, then TMEM147), placing all three in the ER.","evidence":"Affinity purification of native complex with mass spectrometry, overexpression/RNAi, immunofluorescence, and zebrafish expression analysis","pmids":["20538592"],"confidence":"High","gaps":["Functional output of the three-subunit complex not yet defined at this stage","Interfaces between TMEM147 and the Nicalin-NOMO intermediate not mapped"]},{"year":2014,"claim":"Linked Nicalin to ER quality control of membrane clients by showing its ortholog NRA-2 controls ER-versus-surface distribution of a DEG/ENaC channel in a subunit-composition-dependent manner.","evidence":"C. elegans RNAi and knockout genetics, neuronal localization assays, and two-electrode voltage clamp in Xenopus oocytes","pmids":["24567339"],"confidence":"Medium","gaps":["Mechanism of selective retention of mutant channels unknown","Generality to human NCLN clients not established"]},{"year":2014,"claim":"Reported Nicalin as a ganglioside GT1b-binding protein associated with an AMPA receptor trafficking complex, raising a possible neuronal trafficking role.","evidence":"Affinity capture with GT1b, quantitative proteomic mass spectrometry, and ATPgammaS competition in cerebellar granule neurons","pmids":["25253868"],"confidence":"Low","gaps":["Single affinity-capture/MS experiment with no functional follow-up specific to Nicalin","Direct versus indirect association with AMPAR complex not resolved"]},{"year":2017,"claim":"Demonstrated an in vivo developmental requirement for NCLN in enteric nervous system formation.","evidence":"Morpholino knockdown and CRISPR knockout in zebrafish with ENS phenotype analysis","pmids":["28274275"],"confidence":"Medium","gaps":["Cellular pathway connecting NCLN loss to ENS defects unknown","Whether the defect reflects translocon or Nodal-modulating function unclear"]},{"year":2020,"claim":"Resolved the central function by placing Nicalin within a ~360 kDa ribosome-associated multipass translocon and tying its sub-complex to biogenesis of multi-pass membrane proteins.","evidence":"Cryo-EM structure, ribosome profiling, and genetic knockout of accessory components with EAAT1 protein-level readout","pmids":["32820719"],"confidence":"High","gaps":["Specific contribution of Nicalin to client folding versus retention not isolated","Range of physiological multi-pass clients incompletely defined"]},{"year":2020,"claim":"Identified a candidate ER quality-control client, sEphA7, whose secretion Nicalin restricts.","evidence":"IP/MS identification, immunofluorescence co-localization in HEK293 cells, and subcellular fractionation","pmids":["32914261"],"confidence":"Low","gaps":["No mutagenesis or rescue to confirm direct effect","Mechanism of restricted ER entry and secretion not established"]},{"year":2024,"claim":"Defined the architecture of the sub-complex in native membranes, showing NOMO bridges the luminal domains of Nicalin and TRAPalpha and that translocon composition tracks ribosome activity.","evidence":"Cryo-electron tomography, subtomogram averaging, and AlphaFold modeling in native ER membranes","pmids":["38866426"],"confidence":"Medium","gaps":["Contacts not validated by mutagenesis","Functional consequence of activity-dependent composition changes unknown"]},{"year":2025,"claim":"Added SigmaR1 as a direct, lipid-regulated Nicalin partner within the translocon.","evidence":"Endogenous tagging, cell fractionation, co-immunoprecipitation, and lipid-binding assay","pmids":["41476177"],"confidence":"Medium","gaps":["Functional consequence of SigmaR1-Nicalin association for client biogenesis not shown","Single-lab biochemistry without structural mapping of the interface"]},{"year":null,"claim":"How Nicalin's distinct activities — translocon-assisted multi-pass protein biogenesis, ER quality control of specific clients, and Nodal antagonism — are mechanistically related or separable remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No defined molecular activity assigned to Nicalin's luminal domain","Client specificity rules unknown","Connection between developmental signaling roles and translocon function unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,3,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,6]}],"complexes":["Nicalin-TMEM147-NOMO sub-complex","multipass translocon (MPT)"],"partners":["NOMO","TMEM147","SEC61","TMCO1","CCDC47","TRAPALPHA","SIGMAR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q969V3","full_name":"BOS complex subunit NCLN","aliases":["Nicalin","Nicastrin-like protein"],"length_aa":563,"mass_kda":63.0,"function":"Component of the multi-pass translocon (MPT) complex that mediates insertion of multi-pass membrane proteins into the lipid bilayer of membranes (PubMed:32820719, PubMed:36261522). The MPT complex takes over after the SEC61 complex: following membrane insertion of the first few transmembrane segments of proteins by the SEC61 complex, the MPT complex occludes the lateral gate of the SEC61 complex to promote insertion of subsequent transmembrane regions (PubMed:36261522). May antagonize Nodal signaling and subsequent organization of axial structures during mesodermal patterning, via its interaction with NOMO (By similarity)","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q969V3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NCLN","classification":"Not Classified","n_dependent_lines":51,"n_total_lines":1208,"dependency_fraction":0.042218543046357616},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000125912","cell_line_id":"CID002039","localizations":[{"compartment":"er","grade":3},{"compartment":"vesicles","grade":1}],"interactors":[{"gene":"NOMO1","stoichiometry":10.0},{"gene":"TMEM147","stoichiometry":10.0},{"gene":"CCDC47","stoichiometry":10.0},{"gene":"NOMO3","stoichiometry":10.0},{"gene":"TMCO1","stoichiometry":4.0},{"gene":"CKB","stoichiometry":0.2},{"gene":"DRG1","stoichiometry":0.2},{"gene":"RBM8A","stoichiometry":0.2},{"gene":"SEC11A","stoichiometry":0.2},{"gene":"SELT","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID002039","total_profiled":1310},"omim":[{"mim_id":"613585","title":"TRANSMEMBRANE PROTEIN 147; TMEM147","url":"https://www.omim.org/entry/613585"},{"mim_id":"609158","title":"NODAL MODULATOR 2; NOMO2","url":"https://www.omim.org/entry/609158"},{"mim_id":"609156","title":"NICALIN; NCLN","url":"https://www.omim.org/entry/609156"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Lipid droplets","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NCLN"},"hgnc":{"alias_symbol":["NICALIN","NET59"],"prev_symbol":[]},"alphafold":{"accession":"Q969V3","domains":[{"cath_id":"-","chopping":"44-195_512-525","consensus_level":"high","plddt":87.2877,"start":44,"end":525},{"cath_id":"3.40.630.10","chopping":"201-497","consensus_level":"high","plddt":90.791,"start":201,"end":497}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969V3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q969V3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q969V3-F1-predicted_aligned_error_v6.png","plddt_mean":85.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NCLN","jax_strain_url":"https://www.jax.org/strain/search?query=NCLN"},"sequence":{"accession":"Q969V3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q969V3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q969V3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969V3"}},"corpus_meta":[{"pmid":"32820719","id":"PMC_32820719","title":"An ER translocon for multi-pass membrane protein biogenesis.","date":"2020","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/32820719","citation_count":108,"is_preprint":false},{"pmid":"28274275","id":"PMC_28274275","title":"Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.","date":"2017","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/28274275","citation_count":74,"is_preprint":false},{"pmid":"15257293","id":"PMC_15257293","title":"Nicalin and its binding partner Nomo are novel Nodal signaling antagonists.","date":"2004","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/15257293","citation_count":56,"is_preprint":false},{"pmid":"25253868","id":"PMC_25253868","title":"Ganglioside regulation of AMPA receptor trafficking.","date":"2014","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/25253868","citation_count":49,"is_preprint":false},{"pmid":"20538592","id":"PMC_20538592","title":"Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20538592","citation_count":43,"is_preprint":false},{"pmid":"17261586","id":"PMC_17261586","title":"The Nicastrin-like protein Nicalin regulates assembly and stability of the Nicalin-nodal modulator (NOMO) membrane protein complex.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17261586","citation_count":25,"is_preprint":false},{"pmid":"32858118","id":"PMC_32858118","title":"Interaction of human oral cancer and the expression of virulence genes of dental pathogenic bacteria.","date":"2020","source":"Microbial 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pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Nicalin forms a protein complex with NOMO (Nodal modulator) at the membrane, and this Nicalin/NOMO complex antagonizes Nodal/TGFbeta signaling during mesendodermal patterning. Ectopic expression of both proteins in zebrafish causes cyclopia, and Nodal- and Activin-induced signaling was inhibited by Nicalin and NOMO in a cell-based reporter assay.\",\n      \"method\": \"Zebrafish ectopic expression, morpholino knockdown, cell-based Nodal/Activin reporter assay, co-immunoprecipitation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional experiments in vivo (zebrafish) and in vitro (reporter assay), replicated across multiple experimental approaches in one study and confirmed in subsequent papers\",\n      \"pmids\": [\"15257293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Nicalin regulates assembly and stability of the Nicalin-NOMO complex in the ER: Nicalin and NOMO are mutually stabilizing (each becomes unstable without the other), Nicalin is the limiting factor controlling complex assembly rate, and NOMO is produced in excess and either stabilized by Nicalin or rapidly degraded. These effects occur post-transcriptionally.\",\n      \"method\": \"RNA interference knockdown, overexpression, protein stability assays (post-transcriptional level confirmed by mRNA analysis)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi and overexpression with multiple readouts in a single lab study\",\n      \"pmids\": [\"17261586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TMEM147, a ~22 kDa transmembrane protein, is a third core component of the Nicalin-NOMO complex. Complex assembly is hierarchical: a Nicalin-NOMO intermediate forms first, then TMEM147 joins. Nicalin acts as the limiting/stabilizing factor for all three components. All three proteins localize to the ER.\",\n      \"method\": \"Affinity purification of native complex, mass spectrometry identification of TMEM147, overexpression and RNAi knockdown in cultured cells, ER localization by immunofluorescence, zebrafish expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mass spectrometry identification of endogenous complex component, validated by overexpression and RNAi, localization confirmed, consistent with prior mechanistic framework\",\n      \"pmids\": [\"20538592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Nicalin is a core component of a ~360 kDa ribosome-associated multipass translocon (MPT) complex at the ER that also includes Sec61, TMCO1, CCDC47, TMEM147, and NOMO. Cryo-EM reveals this assembly at the ribosome exit tunnel organized around a central membrane cavity, with a luminal funnel in TMEM147 suggesting a route into the cavity. Cells lacking accessory components (including those of the Nicalin-TMEM147-NOMO sub-complex) show reduced levels of the multi-pass membrane protein client EAAT1.\",\n      \"method\": \"Cryo-electron microscopy, high-throughput mRNA sequencing (ribosome profiling), genetic knockout of accessory components with EAAT1 protein-level readout\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with genetic loss-of-function and client protein assay, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"32820719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The C. elegans nicalin homolog NRA-2 controls the distribution of DEG/ENaC channel MEC-10(d) between the ER and the cell surface, acting as a quality control mechanism to retain mutant channels in the ER. NRA-2 knockout enhanced MEC-10(d)-induced neuronal death. Electrophysiology in Xenopus oocytes showed that NRA-2's control of channel distribution is dependent on subunit composition.\",\n      \"method\": \"C. elegans RNAi and knockout genetics, cell biological localization assays in neurons, two-electrode voltage clamp electrophysiology in Xenopus oocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined cellular phenotype and electrophysiological validation, ortholog (C. elegans NRA-2) studied in single lab\",\n      \"pmids\": [\"24567339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Nicalin (the transmembrane protein) was identified as a ganglioside GT1b-binding protein in rat cerebellar granule neurons and associates with the AMPA receptor trafficking complex containing Thorase and NSF. Addition of non-cleavable ATP (ATPγS) enhanced AMPAR association with Nicalin.\",\n      \"method\": \"Affinity capture with ganglioside GT1b, quantitative proteomic mass spectrometry, ATPγS competition assay\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single affinity-capture/MS experiment identifying Nicalin as a GT1b-binding protein; no functional follow-up specific to Nicalin's role\",\n      \"pmids\": [\"25253868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NCLN ortholog is indispensable for enteric nervous system (ENS) development in zebrafish, as shown by both morpholino knockdown and CRISPR knockout resulting in ENS defects.\",\n      \"method\": \"Morpholino knockdown, CRISPR knockout in zebrafish, ENS phenotype analysis\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two complementary loss-of-function approaches (morpholino + CRISPR) with defined developmental phenotype, single study\",\n      \"pmids\": [\"28274275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Nicalin interacts with soluble EphA7 (sEphA7) and co-localizes with it predominantly in the ER. Nicalin reduces sEphA7 in the membranous fraction and increases it in the insoluble cytoplasmic fraction with reduced molecular weight, suggesting Nicalin restricts sEphA7 entry into the ER for further modification and limits sEphA7 secretion, thereby preventing formation of the EphA7 complex.\",\n      \"method\": \"Immunoprecipitation/mass spectrometry identification, co-localization by immunofluorescence in HEK293 cells, subcellular fractionation\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single IP/MS identification and fractionation without mutagenesis or rescue experiments\",\n      \"pmids\": [\"32914261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In the native ER membrane environment, the Nicalin-TMEM147-NOMO sub-complex is part of the multipass translocon (MPT). Cryo-electron tomography and subtomogram averaging reveal that NOMO bridges the luminal domains of Nicalin and TRAPα, forming intermolecular contacts. The MPT composition varies with ribosome translational activity.\",\n      \"method\": \"Cryo-electron tomography, subtomogram averaging, AlphaFold structural modeling\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-ET with subtomogram averaging in native membranes plus AlphaFold modeling, single study without mutagenesis validation\",\n      \"pmids\": [\"38866426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SigmaR1 directly interacts with Nicalin as a component of the translocon complex at the ER, as demonstrated by biochemical co-immunoprecipitation. Phosphatidylcholine (PC) binding by SigmaR1 strengthens its association with the translocon complex including Nicalin.\",\n      \"method\": \"Endogenous tagging, cell fractionation, co-immunoprecipitation, lipid-binding assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical co-IP with endogenous tagging and lipid-binding assay, single lab study\",\n      \"pmids\": [\"41476177\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Nicalin (NCLN) is a core ER-resident transmembrane protein that forms a hierarchical complex with NOMO and TMEM147 (the Nicalin-TMEM147-NOMO sub-complex), which in turn is embedded within a larger ~360 kDa ribosome-associated multipass translocon (MPT) alongside Sec61, TMCO1, and CCDC47; this MPT complex facilitates biogenesis of multi-pass membrane proteins, while the Nicalin-NOMO complex also antagonizes Nodal/TGFbeta signaling during vertebrate embryonic development, and Nicalin additionally functions in ER quality control by regulating surface localization of client proteins such as DEG/ENaC channels.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Nicalin (NCLN) is a core ER-resident transmembrane protein that organizes a hierarchical sub-complex with NOMO and TMEM147 embedded within a ribosome-associated multipass translocon that supports the biogenesis of multi-pass membrane proteins [#2, #3]. Nicalin acts as the limiting, stabilizing factor for the sub-complex: it first forms a mutually stabilizing intermediate with NOMO, after which TMEM147 joins, with each partner becoming unstable in the absence of Nicalin [#1, #2]. Within the native ER membrane, this Nicalin-TMEM147-NOMO module assembles into a ~360 kDa multipass translocon together with Sec61, TMCO1, and CCDC47, organized around a central membrane cavity at the ribosome exit tunnel; NOMO bridges the luminal domains of Nicalin and TRAPalpha, and loss of accessory components reduces levels of the multi-pass client EAAT1 [#3, #8]. The translocon-associated chaperone SigmaR1 directly binds Nicalin, an interaction strengthened by phosphatidylcholine binding [#9]. Independent of its translocon role, the Nicalin-NOMO complex antagonizes Nodal/Activin (TGFbeta) signaling during vertebrate mesendodermal patterning, and the Nicalin ortholog is required for enteric nervous system development in zebrafish [#0, #6]. Nicalin also participates in ER quality control of membrane clients, governing the ER-versus-surface distribution of DEG/ENaC channels through its C. elegans ortholog NRA-2 [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established Nicalin's first biological function by showing it partners with NOMO to antagonize Nodal/TGFbeta signaling during embryonic patterning.\",\n      \"evidence\": \"Zebrafish ectopic expression and morpholino knockdown, cell-based Nodal/Activin reporter assay, and co-immunoprecipitation\",\n      \"pmids\": [\"15257293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which the Nicalin/NOMO complex inhibits Nodal signaling not defined\", \"Relationship between this signaling role and ER residence unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined Nicalin as the assembly-limiting and stabilizing subunit of the Nicalin-NOMO complex, explaining how complex stoichiometry is controlled post-transcriptionally.\",\n      \"evidence\": \"RNAi knockdown, overexpression, and protein stability assays with mRNA controls in cultured cells\",\n      \"pmids\": [\"17261586\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degradation pathway for excess NOMO not identified\", \"Structural basis of mutual stabilization unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended the complex to a third subunit by identifying TMEM147 and showing hierarchical assembly (Nicalin-NOMO first, then TMEM147), placing all three in the ER.\",\n      \"evidence\": \"Affinity purification of native complex with mass spectrometry, overexpression/RNAi, immunofluorescence, and zebrafish expression analysis\",\n      \"pmids\": [\"20538592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional output of the three-subunit complex not yet defined at this stage\", \"Interfaces between TMEM147 and the Nicalin-NOMO intermediate not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Linked Nicalin to ER quality control of membrane clients by showing its ortholog NRA-2 controls ER-versus-surface distribution of a DEG/ENaC channel in a subunit-composition-dependent manner.\",\n      \"evidence\": \"C. elegans RNAi and knockout genetics, neuronal localization assays, and two-electrode voltage clamp in Xenopus oocytes\",\n      \"pmids\": [\"24567339\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of selective retention of mutant channels unknown\", \"Generality to human NCLN clients not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Reported Nicalin as a ganglioside GT1b-binding protein associated with an AMPA receptor trafficking complex, raising a possible neuronal trafficking role.\",\n      \"evidence\": \"Affinity capture with GT1b, quantitative proteomic mass spectrometry, and ATPgammaS competition in cerebellar granule neurons\",\n      \"pmids\": [\"25253868\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single affinity-capture/MS experiment with no functional follow-up specific to Nicalin\", \"Direct versus indirect association with AMPAR complex not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated an in vivo developmental requirement for NCLN in enteric nervous system formation.\",\n      \"evidence\": \"Morpholino knockdown and CRISPR knockout in zebrafish with ENS phenotype analysis\",\n      \"pmids\": [\"28274275\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular pathway connecting NCLN loss to ENS defects unknown\", \"Whether the defect reflects translocon or Nodal-modulating function unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the central function by placing Nicalin within a ~360 kDa ribosome-associated multipass translocon and tying its sub-complex to biogenesis of multi-pass membrane proteins.\",\n      \"evidence\": \"Cryo-EM structure, ribosome profiling, and genetic knockout of accessory components with EAAT1 protein-level readout\",\n      \"pmids\": [\"32820719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific contribution of Nicalin to client folding versus retention not isolated\", \"Range of physiological multi-pass clients incompletely defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a candidate ER quality-control client, sEphA7, whose secretion Nicalin restricts.\",\n      \"evidence\": \"IP/MS identification, immunofluorescence co-localization in HEK293 cells, and subcellular fractionation\",\n      \"pmids\": [\"32914261\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mutagenesis or rescue to confirm direct effect\", \"Mechanism of restricted ER entry and secretion not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the architecture of the sub-complex in native membranes, showing NOMO bridges the luminal domains of Nicalin and TRAPalpha and that translocon composition tracks ribosome activity.\",\n      \"evidence\": \"Cryo-electron tomography, subtomogram averaging, and AlphaFold modeling in native ER membranes\",\n      \"pmids\": [\"38866426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Contacts not validated by mutagenesis\", \"Functional consequence of activity-dependent composition changes unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added SigmaR1 as a direct, lipid-regulated Nicalin partner within the translocon.\",\n      \"evidence\": \"Endogenous tagging, cell fractionation, co-immunoprecipitation, and lipid-binding assay\",\n      \"pmids\": [\"41476177\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of SigmaR1-Nicalin association for client biogenesis not shown\", \"Single-lab biochemistry without structural mapping of the interface\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Nicalin's distinct activities — translocon-assisted multi-pass protein biogenesis, ER quality control of specific clients, and Nodal antagonism — are mechanistically related or separable remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined molecular activity assigned to Nicalin's luminal domain\", \"Client specificity rules unknown\", \"Connection between developmental signaling roles and translocon function unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 3, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [\"Nicalin-TMEM147-NOMO sub-complex\", \"multipass translocon (MPT)\"],\n    \"partners\": [\"NOMO\", \"TMEM147\", \"SEC61\", \"TMCO1\", \"CCDC47\", \"TRAPalpha\", \"SIGMAR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}