{"gene":"MID2","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2002,"finding":"MID2 homo- and heterodimerises with MID1 via their coiled-coil motifs, and both proteins interact with Alpha4 (a regulatory subunit of PP2-type phosphatases) through their B-box domains; dimerisation is required for association of the MID-Alpha4 complex with microtubules.","method":"Yeast two-hybrid screens, domain-specific deletion analysis","journal":"BMC cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid and deletion mapping, single lab, two orthogonal methods (Y2H + deletion analysis)","pmids":["11806752"],"is_preprint":false},{"year":1999,"finding":"MID2 (FXY2) protein associates with microtubules in a manner dependent on its carboxy-terminal B30.2 domain.","method":"Subcellular localization by direct imaging; domain deletion analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment with domain deletion, single lab","pmids":["10644436"],"is_preprint":false},{"year":1999,"finding":"MID2 is required for cell integrity signaling in response to pheromone in S. cerevisiae; it acts as a cell surface sensor upstream of the PKC/MAPK pathway and is highly O-mannosylated on its extracellular domain.","method":"Genetic epistasis (null mutant phenotypic analysis), dosage suppressor screen, immunofluorescence localization, biochemical O-mannosylation analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetics, localization, biochemical modification analysis), independently supported by multiple labs","pmids":["10330137"],"is_preprint":false},{"year":2001,"finding":"The C-terminal cytoplasmic domain of Mid2 interacts with Rom2 (a GEF for Rho1) in S. cerevisiae; mid2 mutant extracts are deficient in catalyzing GTP loading of Rho1 in vitro, indicating Mid2 functions to stimulate nucleotide exchange on Rho1 via Rom2. Additionally, O-mannosylation by Pmt2 is required for Mid2 signaling.","method":"Yeast two-hybrid, in vitro GTP-loading assay on Rho1, genetic analysis of pmt2 mutant","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical assay combined with two-hybrid and genetics, multiple orthogonal methods","pmids":["11113201"],"is_preprint":false},{"year":2013,"finding":"A missense mutation (p.Arg347Gln) in MID2 abolishes function of the COS domain, causing the GFP-tagged mutant protein to accumulate in the cytoplasm rather than binding the cytoskeleton, demonstrating that the COS domain is required for cytoskeletal localization of MID2.","method":"Transient expression of GFP-tagged mutant in HEK293T cells, fluorescence microscopy","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, single method (fluorescence imaging of mutant vs. wild type)","pmids":["24115387"],"is_preprint":false},{"year":2015,"finding":"MID2 ubiquitinates astrin on lysine 409, targeting it for degradation during cytokinesis; MID2 and astrin co-localize at intercellular bridge microtubules. MID2 depletion stabilizes astrin, causes cytokinetic defects, multinucleation, and cell death. Expression of K409A mutant astrin phenocopies MID2 depletion.","method":"Co-purification/MS, co-localization by immunofluorescence, in vivo ubiquitination assay, RNAi knockdown with phenotypic readout, site-directed mutagenesis (K409A)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods: MS-based co-purification, ubiquitination assay, mutagenesis, and knockdown with specific cellular phenotype in single rigorous study","pmids":["26748699"],"is_preprint":false},{"year":2010,"finding":"MID2 (Xenopus ortholog) is required for neural tube closure; its depletion by morpholino knockdown destabilizes and disorganizes apicobasally polarized microtubules in the neural plate. MID2 cooperates with its interacting protein Mig12 for microtubule stabilization during neural plate remodeling.","method":"Morpholino-mediated knockdown in Xenopus, live imaging, microtubule staining","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype and molecular mechanism (MT disorganization), single lab","pmids":["20534674"],"is_preprint":false},{"year":2022,"finding":"TRIM1/MID2 (the mammalian protein) acts as an E3 ubiquitin ligase that recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation; TRIM1 binds LRRK2 at residues 911-919 within a regulatory loop. Phosphorylation of LRRK2 Ser910/Ser935 determines whether LRRK2 associates with cytoplasmic 14-3-3 or microtubule-bound TRIM1. TRIM1 prevents Rab29-mediated upregulation of LRRK2 kinase activity in an E3-ligase-dependent manner.","method":"Quantitative mass spectrometry-based interactome, co-IP, domain mapping, site-directed mutagenesis, proteasome inhibitor experiments, neurite outgrowth rescue assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — quantitative MS interactome, domain mutagenesis, functional rescue, multiple orthogonal methods in single rigorous study","pmids":["35266954"],"is_preprint":false},{"year":2020,"finding":"MID2 overexpression increases phosphorylation of PP2Ac (without changing total PP2Ac levels), downregulates β-catenin and Wnt/β-catenin signaling, and inhibits EMT and cell migration; MID2 knockdown has opposite effects. PP2A inhibition by okadaic acid partially rescues β-catenin levels after MID2 knockdown, placing MID2 upstream of PP2A in Wnt regulation.","method":"Western blot, TCF/LEF luciferase reporter assay, siRNA knockdown, wound healing assay, immunofluorescence","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple orthogonal assays (reporter, WB, migration) in single lab","pmids":["32953821"],"is_preprint":false},{"year":2015,"finding":"MID2 can functionally substitute for MID1 in controlling exocytosis of lytic granules in cytotoxic T cells; transfection of MID2 into MID1-/- CTL completely rescues granule exocytosis, and knockdown of MID2 inhibits exocytosis in both wild-type and MID1-/- CTL.","method":"MID2 transfection rescue in MID1-/- CTL, siRNA knockdown, exocytosis assay","journal":"APMIS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue and knockdown with defined functional readout, single lab","pmids":["25924778"],"is_preprint":false},{"year":2024,"finding":"TRIM1/MID2 binds HIF1α and mediates K63-linked ubiquitination at Lys214 (in the loop between PAS domains), which is required for HIF1α nuclear translocation and transcriptional activation; mutation of Lys214 abolishes these effects.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis (K214 mutant), nuclear fractionation","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, and mutagenesis in single lab study","pmids":["38769340"],"is_preprint":false},{"year":2020,"finding":"MORC4 interacts with STAT3 (confirmed by Co-IP) and promotes transcriptional activation of MID2 via two STAT3-binding sites in the MID2 promoter; MORC4 inhibition reduces STAT3 enrichment at the MID2 promoter.","method":"Co-IP (MORC4-STAT3 interaction), ChIP-qPCR, dual-luciferase reporter assay","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ChIP-qPCR and reporter assay, single lab, two orthogonal methods","pmids":["32764967"],"is_preprint":false},{"year":1999,"finding":"MID2 protein is uniformly distributed through the plasma membrane and is highly O-mannosylated on its extracellular domain in S. cerevisiae.","method":"Immunofluorescence microscopy, biochemical glycosylation analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization and biochemical characterization, replicated context","pmids":["10330137"],"is_preprint":false},{"year":2018,"finding":"ER targeting of MID2 mRNA in yeast requires the RNA-binding protein Khd1p and is independent of the signal sequence of the encoded protein.","method":"Liposome-binding assay, ER fractionation, KHD1 deletion mutant analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — fractionation and genetic deletion, single lab, two orthogonal methods","pmids":["29772604"],"is_preprint":false}],"current_model":"MID2 (TRIM1/FXY2/RNF60) is a microtubule-associated RING-domain E3 ubiquitin ligase that: (1) ubiquitinates astrin on K409 to drive its proteasomal degradation during cytokinesis; (2) ubiquitinates LRRK2 at the microtubule cytoskeleton via binding the LRRK2 regulatory loop (aa 911-919), controlling LRRK2 degradation, localization, and kinase activity; (3) mediates K63-linked ubiquitination of HIF1α at K214 to promote its nuclear translocation; (4) hetero- and homodimerises with MID1 through coiled-coil motifs, recruits Alpha4 (PP2A regulatory subunit) via its B-boxes, and requires dimerisation for microtubule association; (5) regulates Wnt/β-catenin signaling and EMT via PP2A phosphorylation; and (6) in yeast, functions as an O-mannosylated plasma membrane sensor that interacts with the GEF Rom2 to stimulate Rho1 GTP-loading and activate the PKC/MAPK cell wall integrity pathway."},"narrative":{"mechanistic_narrative":"MID2 (TRIM1/FXY2) is a microtubule-associated RING-domain E3 ubiquitin ligase that couples substrate ubiquitination to the cytoskeleton to control mitotic progression, kinase regulation, and signaling [PMID:26748699, PMID:35266954]. It localizes to microtubules through its C-terminal B30.2 domain and its COS domain, the latter being indispensable for cytoskeletal targeting since a COS-domain missense mutation (p.Arg347Gln) redistributes the protein to the cytoplasm [PMID:10644436, PMID:24115387]; MID2 also homo- and heterodimerises with MID1 via coiled-coil motifs and recruits the PP2A regulatory subunit Alpha4 through its B-boxes, with dimerisation required for the MID-Alpha4 complex to associate with microtubules [PMID:11806752]. As a ligase it ubiquitinates astrin on Lys409 to drive its proteasomal degradation at intercellular bridge microtubules during cytokinesis, such that MID2 loss stabilizes astrin and produces cytokinetic failure, multinucleation, and cell death [PMID:26748699]. MID2 further recruits LRRK2 to microtubules by binding a regulatory loop (residues 911-919) for ubiquitination and degradation, with LRRK2 Ser910/Ser935 phosphorylation governing the choice between cytosolic 14-3-3 and microtubule-bound MID2, and MID2 restraining Rab29-driven LRRK2 kinase activity in an E3-ligase-dependent manner [PMID:35266954]. Beyond degradative ubiquitination, MID2 mediates K63-linked ubiquitination of HIF1α at Lys214 to promote its nuclear translocation and transcriptional activity [PMID:38769340], and modulates Wnt/β-catenin signaling and EMT upstream of PP2A [PMID:32953821]. In development, the vertebrate ortholog stabilizes apicobasally polarized microtubules during neural tube closure together with Mig12 [PMID:20534674]. A separate set of findings describes a *S. cerevisiae* protein named Mid2 — an O-mannosylated plasma-membrane sensor that activates the PKC/MAPK cell-wall-integrity pathway by stimulating Rom2-dependent GTP loading of Rho1 [PMID:10330137, PMID:11113201] — which is a distinct protein and does not bear on the vertebrate MID2 mechanism.","teleology":[{"year":1999,"claim":"Established that vertebrate MID2 is a cytoskeletal protein, defining its subcellular platform and pointing to the B30.2 domain as the microtubule-targeting determinant.","evidence":"Direct subcellular imaging with domain-deletion analysis","pmids":["10644436"],"confidence":"Medium","gaps":["Did not define a molecular activity or substrate","Single lab, localization only"]},{"year":2002,"claim":"Resolved how MID2 is assembled at the cytoskeleton, showing it dimerises with MID1 and recruits the PP2A subunit Alpha4, with dimerisation required for microtubule association.","evidence":"Yeast two-hybrid and domain-specific deletion mapping","pmids":["11806752"],"confidence":"Medium","gaps":["Interactions not validated in a structural or reconstituted system","Functional consequence of Alpha4 recruitment not tested here"]},{"year":2010,"claim":"Connected MID2 to a developmental process, showing its loss disorganizes polarized microtubules during neural tube closure via cooperation with Mig12.","evidence":"Morpholino knockdown in Xenopus with live imaging and microtubule staining","pmids":["20534674"],"confidence":"Medium","gaps":["Ubiquitin-ligase activity not implicated in this phenotype","Molecular mechanism of MT stabilization undefined"]},{"year":2013,"claim":"Defined the COS domain as required for cytoskeletal localization, linking a disease-associated missense mutation to mislocalization.","evidence":"GFP-tagged mutant expression and fluorescence microscopy in HEK293T","pmids":["24115387"],"confidence":"Medium","gaps":["Single method (imaging) without biochemical validation","Effect on ligase activity not assessed"]},{"year":2015,"claim":"Identified the first MID2 ubiquitination substrate, establishing it as a RING E3 that degrades astrin at K409 to permit cytokinesis.","evidence":"Co-purification/MS, in vivo ubiquitination assay, K409A mutagenesis, and RNAi with cytokinesis phenotype","pmids":["26748699"],"confidence":"High","gaps":["Does not establish ubiquitin chain linkage type on astrin","E2 partner not identified"]},{"year":2015,"claim":"Showed MID2 is functionally interchangeable with MID1 in a distinct cellular process, controlling lytic granule exocytosis in cytotoxic T cells.","evidence":"MID2 rescue in MID1-/- CTL, siRNA knockdown, exocytosis assay","pmids":["25924778"],"confidence":"Medium","gaps":["Substrate or molecular mechanism in exocytosis unknown","Whether ligase activity is required not tested"]},{"year":2020,"claim":"Placed MID2 upstream of PP2A in Wnt/β-catenin signaling and EMT, linking it to phospho-regulation of PP2Ac and β-catenin stability.","evidence":"Overexpression/knockdown with TCF/LEF reporter, western blot, okadaic acid rescue, migration assay","pmids":["32953821"],"confidence":"Medium","gaps":["Direct ubiquitination substrate in this pathway not identified","Mechanism linking MID2 to PP2Ac phosphorylation undefined"]},{"year":2020,"claim":"Identified an upstream transcriptional input, showing MORC4/STAT3 activates the MID2 promoter.","evidence":"Co-IP, ChIP-qPCR, dual-luciferase reporter","pmids":["32764967"],"confidence":"Medium","gaps":["Physiological context of MID2 induction not established","Single lab"]},{"year":2022,"claim":"Defined a second physiological substrate and a regulatory switch, showing MID2 recruits LRRK2 (residues 911-919) to microtubules for degradation and restrains its kinase activity, with phospho-Ser910/935 partitioning LRRK2 between 14-3-3 and MID2.","evidence":"Quantitative MS interactome, co-IP, domain mapping, mutagenesis, proteasome inhibition, neurite outgrowth rescue","pmids":["35266954"],"confidence":"High","gaps":["Ubiquitin chain type on LRRK2 not fully defined","In vivo relevance to LRRK2-associated disease not tested"]},{"year":2024,"claim":"Extended MID2 activity to non-degradative signaling, showing K63-linked ubiquitination of HIF1α at K214 drives its nuclear translocation.","evidence":"Co-IP, ubiquitination assay, K214 mutagenesis, nuclear fractionation","pmids":["38769340"],"confidence":"Medium","gaps":["Single lab","Structural basis of K214 selection and chain assembly undefined"]},{"year":null,"claim":"It remains unknown how MID2 substrate selection, chain-linkage choice (K48- vs K63-linked), and its PP2A/Alpha4-dependent functions are integrated into a unified regulatory logic at the microtubule cytoskeleton.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate engagement","Determinants of degradative vs non-degradative ubiquitination unresolved","Relationship between ligase activity and PP2A-dependent signaling not mechanistically connected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[5,7,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,7,10]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,4]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,4,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,7,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,8,10]}],"complexes":[],"partners":["MID1","ALPHA4","LRRK2","HIF1A","ASTRIN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UJV3","full_name":"Probable E3 ubiquitin-protein ligase MID2","aliases":["Midin-2","Midline defect 2","Midline-2","RING finger protein 60","RING-type E3 ubiquitin transferase MID2","Tripartite motif-containing protein 1"],"length_aa":735,"mass_kda":83.2,"function":"E3 ubiquitin ligase that plays a role in microtubule stabilization. Mediates the 'Lys-48'-linked polyubiquitination of LRRK2 to drive its localization to microtubules and its proteasomal degradation in neurons. This ubiquitination inhibits LRRK2 kinase activation by RAB29 (PubMed:35266954)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q9UJV3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MID2","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MID2","total_profiled":1310},"omim":[{"mim_id":"300928","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED 101; XLID101","url":"https://www.omim.org/entry/300928"},{"mim_id":"300204","title":"MIDLINE 2; MID2","url":"https://www.omim.org/entry/300204"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MID2"},"hgnc":{"alias_symbol":["FXY2","TRIM1","RNF60","MRX101"],"prev_symbol":[]},"alphafold":{"accession":"Q9UJV3","domains":[{"cath_id":"3.30.40.10","chopping":"23-61_69-109","consensus_level":"high","plddt":85.2807,"start":23,"end":109},{"cath_id":"3.30.160.60","chopping":"151-188","consensus_level":"medium","plddt":83.6553,"start":151,"end":188},{"cath_id":"-","chopping":"257-377","consensus_level":"high","plddt":88.5223,"start":257,"end":377},{"cath_id":"2.60.40.10","chopping":"402-444_485-532","consensus_level":"medium","plddt":91.7492,"start":402,"end":532},{"cath_id":"2.60.120.920","chopping":"537-703","consensus_level":"high","plddt":91.0495,"start":537,"end":703}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJV3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJV3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJV3-F1-predicted_aligned_error_v6.png","plddt_mean":79.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MID2","jax_strain_url":"https://www.jax.org/strain/search?query=MID2"},"sequence":{"accession":"Q9UJV3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UJV3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UJV3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJV3"}},"corpus_meta":[{"pmid":"11113201","id":"PMC_11113201","title":"Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11113201","citation_count":258,"is_preprint":false},{"pmid":"10330137","id":"PMC_10330137","title":"Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10330137","citation_count":174,"is_preprint":false},{"pmid":"11806752","id":"PMC_11806752","title":"MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders.","date":"2002","source":"BMC cell 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Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/25924778","citation_count":5,"is_preprint":false},{"pmid":"38467738","id":"PMC_38467738","title":"Expanding the genetic and phenotypic spectrum of TRAPPC9 and MID2-related neurodevelopmental disabilities: report of two novel mutations, 3D-modelling, and molecular docking studies.","date":"2024","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/38467738","citation_count":3,"is_preprint":false},{"pmid":"31951325","id":"PMC_31951325","title":"A male infant with Xq22.2q22.3 duplication containing PLP1 and MID2.","date":"2020","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31951325","citation_count":2,"is_preprint":false},{"pmid":"37760252","id":"PMC_37760252","title":"The Molecular and Function Characterization of Porcine MID2.","date":"2023","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/37760252","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.13.688250","title":"Diversity and distribution of the subtelomeric Y’ elements across  <i>Saccharomyces cerevisiae</i>  strains","date":"2025-11-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.13.688250","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.07.687132","title":"Immune transcriptomic differences in paediatric patients with SARS-CoV-2 compared to other lower respiratory tract infections","date":"2025-11-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.07.687132","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17293,"output_tokens":3337,"usd":0.050967,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10768,"output_tokens":3726,"usd":0.073495,"stage2_stop_reason":"end_turn"},"total_usd":0.124462,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"MID2 homo- and heterodimerises with MID1 via their coiled-coil motifs, and both proteins interact with Alpha4 (a regulatory subunit of PP2-type phosphatases) through their B-box domains; dimerisation is required for association of the MID-Alpha4 complex with microtubules.\",\n      \"method\": \"Yeast two-hybrid screens, domain-specific deletion analysis\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid and deletion mapping, single lab, two orthogonal methods (Y2H + deletion analysis)\",\n      \"pmids\": [\"11806752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MID2 (FXY2) protein associates with microtubules in a manner dependent on its carboxy-terminal B30.2 domain.\",\n      \"method\": \"Subcellular localization by direct imaging; domain deletion analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment with domain deletion, single lab\",\n      \"pmids\": [\"10644436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MID2 is required for cell integrity signaling in response to pheromone in S. cerevisiae; it acts as a cell surface sensor upstream of the PKC/MAPK pathway and is highly O-mannosylated on its extracellular domain.\",\n      \"method\": \"Genetic epistasis (null mutant phenotypic analysis), dosage suppressor screen, immunofluorescence localization, biochemical O-mannosylation analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetics, localization, biochemical modification analysis), independently supported by multiple labs\",\n      \"pmids\": [\"10330137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-terminal cytoplasmic domain of Mid2 interacts with Rom2 (a GEF for Rho1) in S. cerevisiae; mid2 mutant extracts are deficient in catalyzing GTP loading of Rho1 in vitro, indicating Mid2 functions to stimulate nucleotide exchange on Rho1 via Rom2. Additionally, O-mannosylation by Pmt2 is required for Mid2 signaling.\",\n      \"method\": \"Yeast two-hybrid, in vitro GTP-loading assay on Rho1, genetic analysis of pmt2 mutant\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical assay combined with two-hybrid and genetics, multiple orthogonal methods\",\n      \"pmids\": [\"11113201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A missense mutation (p.Arg347Gln) in MID2 abolishes function of the COS domain, causing the GFP-tagged mutant protein to accumulate in the cytoplasm rather than binding the cytoskeleton, demonstrating that the COS domain is required for cytoskeletal localization of MID2.\",\n      \"method\": \"Transient expression of GFP-tagged mutant in HEK293T cells, fluorescence microscopy\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (fluorescence imaging of mutant vs. wild type)\",\n      \"pmids\": [\"24115387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MID2 ubiquitinates astrin on lysine 409, targeting it for degradation during cytokinesis; MID2 and astrin co-localize at intercellular bridge microtubules. MID2 depletion stabilizes astrin, causes cytokinetic defects, multinucleation, and cell death. Expression of K409A mutant astrin phenocopies MID2 depletion.\",\n      \"method\": \"Co-purification/MS, co-localization by immunofluorescence, in vivo ubiquitination assay, RNAi knockdown with phenotypic readout, site-directed mutagenesis (K409A)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods: MS-based co-purification, ubiquitination assay, mutagenesis, and knockdown with specific cellular phenotype in single rigorous study\",\n      \"pmids\": [\"26748699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MID2 (Xenopus ortholog) is required for neural tube closure; its depletion by morpholino knockdown destabilizes and disorganizes apicobasally polarized microtubules in the neural plate. MID2 cooperates with its interacting protein Mig12 for microtubule stabilization during neural plate remodeling.\",\n      \"method\": \"Morpholino-mediated knockdown in Xenopus, live imaging, microtubule staining\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype and molecular mechanism (MT disorganization), single lab\",\n      \"pmids\": [\"20534674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM1/MID2 (the mammalian protein) acts as an E3 ubiquitin ligase that recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation; TRIM1 binds LRRK2 at residues 911-919 within a regulatory loop. Phosphorylation of LRRK2 Ser910/Ser935 determines whether LRRK2 associates with cytoplasmic 14-3-3 or microtubule-bound TRIM1. TRIM1 prevents Rab29-mediated upregulation of LRRK2 kinase activity in an E3-ligase-dependent manner.\",\n      \"method\": \"Quantitative mass spectrometry-based interactome, co-IP, domain mapping, site-directed mutagenesis, proteasome inhibitor experiments, neurite outgrowth rescue assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — quantitative MS interactome, domain mutagenesis, functional rescue, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"35266954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MID2 overexpression increases phosphorylation of PP2Ac (without changing total PP2Ac levels), downregulates β-catenin and Wnt/β-catenin signaling, and inhibits EMT and cell migration; MID2 knockdown has opposite effects. PP2A inhibition by okadaic acid partially rescues β-catenin levels after MID2 knockdown, placing MID2 upstream of PP2A in Wnt regulation.\",\n      \"method\": \"Western blot, TCF/LEF luciferase reporter assay, siRNA knockdown, wound healing assay, immunofluorescence\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple orthogonal assays (reporter, WB, migration) in single lab\",\n      \"pmids\": [\"32953821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MID2 can functionally substitute for MID1 in controlling exocytosis of lytic granules in cytotoxic T cells; transfection of MID2 into MID1-/- CTL completely rescues granule exocytosis, and knockdown of MID2 inhibits exocytosis in both wild-type and MID1-/- CTL.\",\n      \"method\": \"MID2 transfection rescue in MID1-/- CTL, siRNA knockdown, exocytosis assay\",\n      \"journal\": \"APMIS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue and knockdown with defined functional readout, single lab\",\n      \"pmids\": [\"25924778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM1/MID2 binds HIF1α and mediates K63-linked ubiquitination at Lys214 (in the loop between PAS domains), which is required for HIF1α nuclear translocation and transcriptional activation; mutation of Lys214 abolishes these effects.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis (K214 mutant), nuclear fractionation\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, and mutagenesis in single lab study\",\n      \"pmids\": [\"38769340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MORC4 interacts with STAT3 (confirmed by Co-IP) and promotes transcriptional activation of MID2 via two STAT3-binding sites in the MID2 promoter; MORC4 inhibition reduces STAT3 enrichment at the MID2 promoter.\",\n      \"method\": \"Co-IP (MORC4-STAT3 interaction), ChIP-qPCR, dual-luciferase reporter assay\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ChIP-qPCR and reporter assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"32764967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MID2 protein is uniformly distributed through the plasma membrane and is highly O-mannosylated on its extracellular domain in S. cerevisiae.\",\n      \"method\": \"Immunofluorescence microscopy, biochemical glycosylation analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization and biochemical characterization, replicated context\",\n      \"pmids\": [\"10330137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ER targeting of MID2 mRNA in yeast requires the RNA-binding protein Khd1p and is independent of the signal sequence of the encoded protein.\",\n      \"method\": \"Liposome-binding assay, ER fractionation, KHD1 deletion mutant analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — fractionation and genetic deletion, single lab, two orthogonal methods\",\n      \"pmids\": [\"29772604\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MID2 (TRIM1/FXY2/RNF60) is a microtubule-associated RING-domain E3 ubiquitin ligase that: (1) ubiquitinates astrin on K409 to drive its proteasomal degradation during cytokinesis; (2) ubiquitinates LRRK2 at the microtubule cytoskeleton via binding the LRRK2 regulatory loop (aa 911-919), controlling LRRK2 degradation, localization, and kinase activity; (3) mediates K63-linked ubiquitination of HIF1α at K214 to promote its nuclear translocation; (4) hetero- and homodimerises with MID1 through coiled-coil motifs, recruits Alpha4 (PP2A regulatory subunit) via its B-boxes, and requires dimerisation for microtubule association; (5) regulates Wnt/β-catenin signaling and EMT via PP2A phosphorylation; and (6) in yeast, functions as an O-mannosylated plasma membrane sensor that interacts with the GEF Rom2 to stimulate Rho1 GTP-loading and activate the PKC/MAPK cell wall integrity pathway.\"\n}\n```","stage2_raw":"**CONTAMINATION NOTE:** The timeline contains two distinct proteins under the symbol \"MID2\": the human/Xenopus TRIM-family RING E3 ligase (TRIM1/MID2/FXY2) and the unrelated *S. cerevisiae* plasma-membrane cell-wall-integrity sensor \"Mid2.\" These are coherent within each organism but describe fundamentally different proteins. I synthesize the dominant, internally consistent human/vertebrate MID2 picture and flag the yeast entries separately rather than blending them.\n\n```json\n{\n  \"mechanistic_narrative\": \"MID2 (TRIM1/FXY2) is a microtubule-associated RING-domain E3 ubiquitin ligase that couples substrate ubiquitination to the cytoskeleton to control mitotic progression, kinase regulation, and signaling [#5, #7]. It localizes to microtubules through its C-terminal B30.2 domain and its COS domain, the latter being indispensable for cytoskeletal targeting since a COS-domain missense mutation (p.Arg347Gln) redistributes the protein to the cytoplasm [#1, #4]; MID2 also homo- and heterodimerises with MID1 via coiled-coil motifs and recruits the PP2A regulatory subunit Alpha4 through its B-boxes, with dimerisation required for the MID-Alpha4 complex to associate with microtubules [#0]. As a ligase it ubiquitinates astrin on Lys409 to drive its proteasomal degradation at intercellular bridge microtubules during cytokinesis, such that MID2 loss stabilizes astrin and produces cytokinetic failure, multinucleation, and cell death [#5]. MID2 further recruits LRRK2 to microtubules by binding a regulatory loop (residues 911-919) for ubiquitination and degradation, with LRRK2 Ser910/Ser935 phosphorylation governing the choice between cytosolic 14-3-3 and microtubule-bound MID2, and MID2 restraining Rab29-driven LRRK2 kinase activity in an E3-ligase-dependent manner [#7]. Beyond degradative ubiquitination, MID2 mediates K63-linked ubiquitination of HIF1α at Lys214 to promote its nuclear translocation and transcriptional activity [#10], and modulates Wnt/β-catenin signaling and EMT upstream of PP2A [#8]. In development, the vertebrate ortholog stabilizes apicobasally polarized microtubules during neural tube closure together with Mig12 [#6]. A separate set of findings describes a *S. cerevisiae* protein named Mid2 — an O-mannosylated plasma-membrane sensor that activates the PKC/MAPK cell-wall-integrity pathway by stimulating Rom2-dependent GTP loading of Rho1 [#2, #3] — which is a distinct protein and does not bear on the vertebrate MID2 mechanism.\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that vertebrate MID2 is a cytoskeletal protein, defining its subcellular platform and pointing to the B30.2 domain as the microtubule-targeting determinant.\",\n      \"evidence\": \"Direct subcellular imaging with domain-deletion analysis\",\n      \"pmids\": [\"10644436\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not define a molecular activity or substrate\", \"Single lab, localization only\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Resolved how MID2 is assembled at the cytoskeleton, showing it dimerises with MID1 and recruits the PP2A subunit Alpha4, with dimerisation required for microtubule association.\",\n      \"evidence\": \"Yeast two-hybrid and domain-specific deletion mapping\",\n      \"pmids\": [\"11806752\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Interactions not validated in a structural or reconstituted system\", \"Functional consequence of Alpha4 recruitment not tested here\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected MID2 to a developmental process, showing its loss disorganizes polarized microtubules during neural tube closure via cooperation with Mig12.\",\n      \"evidence\": \"Morpholino knockdown in Xenopus with live imaging and microtubule staining\",\n      \"pmids\": [\"20534674\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ubiquitin-ligase activity not implicated in this phenotype\", \"Molecular mechanism of MT stabilization undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the COS domain as required for cytoskeletal localization, linking a disease-associated missense mutation to mislocalization.\",\n      \"evidence\": \"GFP-tagged mutant expression and fluorescence microscopy in HEK293T\",\n      \"pmids\": [\"24115387\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single method (imaging) without biochemical validation\", \"Effect on ligase activity not assessed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the first MID2 ubiquitination substrate, establishing it as a RING E3 that degrades astrin at K409 to permit cytokinesis.\",\n      \"evidence\": \"Co-purification/MS, in vivo ubiquitination assay, K409A mutagenesis, and RNAi with cytokinesis phenotype\",\n      \"pmids\": [\"26748699\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Does not establish ubiquitin chain linkage type on astrin\", \"E2 partner not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed MID2 is functionally interchangeable with MID1 in a distinct cellular process, controlling lytic granule exocytosis in cytotoxic T cells.\",\n      \"evidence\": \"MID2 rescue in MID1-/- CTL, siRNA knockdown, exocytosis assay\",\n      \"pmids\": [\"25924778\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Substrate or molecular mechanism in exocytosis unknown\", \"Whether ligase activity is required not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed MID2 upstream of PP2A in Wnt/β-catenin signaling and EMT, linking it to phospho-regulation of PP2Ac and β-catenin stability.\",\n      \"evidence\": \"Overexpression/knockdown with TCF/LEF reporter, western blot, okadaic acid rescue, migration assay\",\n      \"pmids\": [\"32953821\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct ubiquitination substrate in this pathway not identified\", \"Mechanism linking MID2 to PP2Ac phosphorylation undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified an upstream transcriptional input, showing MORC4/STAT3 activates the MID2 promoter.\",\n      \"evidence\": \"Co-IP, ChIP-qPCR, dual-luciferase reporter\",\n      \"pmids\": [\"32764967\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Physiological context of MID2 induction not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a second physiological substrate and a regulatory switch, showing MID2 recruits LRRK2 (residues 911-919) to microtubules for degradation and restrains its kinase activity, with phospho-Ser910/935 partitioning LRRK2 between 14-3-3 and MID2.\",\n      \"evidence\": \"Quantitative MS interactome, co-IP, domain mapping, mutagenesis, proteasome inhibition, neurite outgrowth rescue\",\n      \"pmids\": [\"35266954\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ubiquitin chain type on LRRK2 not fully defined\", \"In vivo relevance to LRRK2-associated disease not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended MID2 activity to non-degradative signaling, showing K63-linked ubiquitination of HIF1α at K214 drives its nuclear translocation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, K214 mutagenesis, nuclear fractionation\",\n      \"pmids\": [\"38769340\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab\", \"Structural basis of K214 selection and chain assembly undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how MID2 substrate selection, chain-linkage choice (K48- vs K63-linked), and its PP2A/Alpha4-dependent functions are integrated into a unified regulatory logic at the microtubule cytoskeleton.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structural model of substrate engagement\", \"Determinants of degradative vs non-degradative ubiquitination unresolved\", \"Relationship between ligase activity and PP2A-dependent signaling not mechanistically connected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [5, 7, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 7, 10]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 7, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 8, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MID1\", \"Alpha4\", \"LRRK2\", \"HIF1A\", \"astrin\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}