{"gene":"GET3","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2009,"finding":"Crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP·AlF4−-bound) dimer states revealed that in the closed state the dimer interface contains a large hydrophobic groove responsible for tail-anchored protein binding, and in the open state this groove is disrupted; mutational analyses confirmed the groove's role in TA protein binding, establishing a nucleotide-regulated binding/release mechanism.","method":"X-ray crystallography (open and closed dimer states) + site-directed mutagenesis of hydrophobic groove residues","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures in two functional states combined with mutagenesis confirming the binding groove, foundational mechanistic paper widely replicated","pmids":["19675567"],"is_preprint":false},{"year":2015,"finding":"Reconstitution of the physiological assembly pathway showed that the functional Get3–TA protein targeting complex comprises a single TA protein bound to a Get3 homodimer; crystal structures of Get3 bound to different TA proteins showed the α-helical transmembrane domain occupying a hydrophobic groove spanning the homodimer interface, elucidating the mechanism of TA protein recognition and shielding.","method":"In vitro reconstitution of targeting complex + X-ray crystallography of Get3–TA protein complexes with different substrates","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution of physiologic complex combined with crystal structures of multiple Get3–TA complexes, directly resolving the long-standing unknown of complex composition","pmids":["25745174"],"is_preprint":false},{"year":2011,"finding":"Crystal structures of Get3 in complex with cytosolic domains of the ER membrane receptor subunits Get1 and Get2 showed that Get1 and Get2 use adjacent, partially overlapping binding sites on Get3 and can bind simultaneously; docking to the Get1/2 receptor induces conformational changes in Get3 required for TA protein insertion.","method":"X-ray crystallography of Get3–Get1/2 receptor complexes at 3.0, 3.2, and 4.6 Å + biochemical experiments","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures at different resolutions combined with biochemical validation, directly defining the receptor-docking mechanism","pmids":["21719644"],"is_preprint":false},{"year":2009,"finding":"Structural and biochemical analyses of Get3 (Asna1/TRC40) showed that the α-helical subdomain binds the TA protein transmembrane domain; amide proton exchange mass spectrometry mapped the TA-binding site to the α-helical subdomain; in vitro membrane insertion assays demonstrated Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner; ATP hydrolysis is not strictly required for insertion but is needed for efficient insertion, likely for Get3 release from its receptor.","method":"X-ray crystallography + hydrogen/deuterium exchange MS + in vitro membrane insertion reconstitution assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (crystallography, H/D exchange MS, in vitro insertion assay) in a single study","pmids":["19948960"],"is_preprint":false},{"year":2009,"finding":"Crystal structures of Get3 from S. cerevisiae (apo, open) and D. hansenii (ADP-bound, closed) identified a nucleotide-binding domain and a 'finger' domain with a hydrophobic groove as the TA protein TMD-binding site; a hydrophobic helix from a symmetry-related molecule occupying the groove mimicked TA binding, and the open/closed conformational switch is linked to TA protein release.","method":"X-ray crystallography of Get3 from two yeast species in apo and ADP-bound states","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures from two organisms in different nucleotide states, consistent with findings from multiple independent groups","pmids":["19956640"],"is_preprint":false},{"year":2009,"finding":"Crystal structures of apo and ADP-bound Get3 from S. cerevisiae and A. fumigatus identified residues important for dimer interfaces; structure-guided mutagenesis confirmed key interfaces and essential residues coupling ATP hydrolysis to TA protein binding and release.","method":"X-ray crystallography (apo and ADP-bound forms, two species) + structure-guided mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — structures of two orthologs combined with mutagenesis confirming mechanistic residues","pmids":["19706470"],"is_preprint":false},{"year":2009,"finding":"Crystal structures of Get3 in ADP-bound and nucleotide-free forms showed a Zn2+-mediated homodimer in head-to-head orientation; cross-linking experiments indicated the closed dimer stimulates ATP hydrolysis; coexpression-based binding assays demonstrated direct interaction between the helical domain of Get3 and the Sec22p TMD independent of ATP and dimer formation; the conserved DTAPTGH motif was proposed to link ATP hydrolysis to TA protein insertion.","method":"X-ray crystallography + chemical cross-linking + coexpression-based binding assay","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — crystal structures plus cross-linking and binding assay, but binding assay is coexpression-based (not direct reconstitution), single lab","pmids":["20015340"],"is_preprint":false},{"year":2010,"finding":"Get3 (Asna1/TRC40) was shown to mediate membrane insertion of the TA proteins RAMP4 and Sec61β from recombinant Asna1–TA protein complexes into ER-derived membranes in a mechanism requiring ATP or ADP and a protease-sensitive ER membrane receptor, but not additional cytosolic factors; cytochrome b5 insertion proceeded independently of Asna1 and nucleotides.","method":"In vitro reconstitution of TA protein membrane insertion from recombinant components + ER-derived membranes + protease sensitivity assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full reconstitution from recombinant components with mechanistic dissection of nucleotide requirement and substrate specificity, single lab","pmids":["20375064"],"is_preprint":false},{"year":2011,"finding":"WRB (tryptophan-rich basic protein/CHD5), an ER-resident membrane protein with sequence similarity to yeast Get1, was identified as the ER membrane receptor for mammalian TRC40/Asna1; the coiled-coil domain of WRB is the binding site for TRC40; a soluble coiled-coil domain fragment interfered with TRC40-mediated TA protein membrane insertion.","method":"Biochemical interaction assays + cell imaging + dominant-negative soluble fragment inhibition of in vitro TA insertion","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction assays, cell imaging, and functional inhibition data identifying the mammalian receptor; independently corroborated by multiple subsequent studies","pmids":["21444755"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of Get4 with an N-terminal Get5 fragment showed they form an intimate complex existing as a dimer-of-heterodimers; Get3 binds to a conserved surface on Get4 in a nucleotide-dependent manner, placing Get4/5 upstream of Get3 in the TA protein delivery pathway.","method":"X-ray crystallography of Get4/5 complex + nucleotide-dependent binding assay of Get3 to Get4","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with functional binding assay, single lab but clear mechanistic finding","pmids":["20554915"],"is_preprint":false},{"year":2010,"finding":"Co-immunoprecipitation and crystallographic studies showed Get4 and Get5 form a tight complex; Get3 physically and transiently interacts with the Get4–Get5 complex; Sgt2 interacts with Get5; genetic interactions between GET3, GET4, GET5, and the chaperone YDJ1 implicate molecular chaperones in TA protein insertion.","method":"Co-immunoprecipitation + X-ray crystallography of Get4/5 + genetic epistasis (YDJ1)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP combined with crystallography and genetic epistasis, single lab","pmids":["20106980"],"is_preprint":false},{"year":2011,"finding":"ITC and SAXS demonstrated that the Get3 homodimer interacts with two copies of the Get4–Get5 complex to form an extended stoichiometric complex in solution, defining the interaction surface as dominated by electrostatic forces.","method":"Isothermal titration calorimetry (ITC) + small-angle X-ray scattering (SAXS)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — two orthogonal biophysical methods defining stoichiometry and interaction surface, single lab","pmids":["22190685"],"is_preprint":false},{"year":2011,"finding":"Crystal structures of ADP-bound Get3 in complex with the cytoplasmic domain of Get1 in open and semi-open conformations showed that Get1 uses two distinct interfaces to bind Get3 and stabilize its open dimer conformation, one sufficient for binding and the second required to stabilize the open state.","method":"X-ray crystallography (3.0 and 4.5 Å) of Get3–Get1CD complexes + biochemical binding assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures in two conformational states combined with biochemical validation, mechanistically defining how Get1 remodels Get3","pmids":["22684149"],"is_preprint":false},{"year":2011,"finding":"An archaeal Get3 homologue crystallized as a tetramer; SAXS of a fungal Get3–TA protein complex showed a molecular envelope consistent with the archaeal tetramer; the tetramer generates a hydrophobic chamber that binds TA proteins; the fungal tetramer complex was capable of mediating TA insertion in vitro.","method":"X-ray crystallography of archaeal Get3 tetramer + SAXS of fungal Get3–TA complex + in vitro TA insertion assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure plus SAXS and functional insertion assay, single lab; tetramer model not yet confirmed for canonical yeast/mammalian Get3","pmids":["22122436"],"is_preprint":false},{"year":2014,"finding":"Get3 switches to an ATP-independent chaperone function upon oxidative stress: oxidation causes disulfide bond formation, metal release, and formation of distinct higher-order oligomeric structures; this chaperone activity is functionally distinct from and mutually exclusive with its TA protein targeting function; yeast cells lacking Get3 show oxidative stress-sensitive phenotypes attributable to loss of this chaperone activity.","method":"In vitro chaperone activity assays + mutational analysis (disulfide-disrupting mutants) + yeast genetic phenotypic analysis under oxidative stress","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (biochemical assays, mutagenesis, genetics) in one study; independently replicated by subsequent work","pmids":["25242142"],"is_preprint":false},{"year":2012,"finding":"Get3 functions as an ATP-independent holdase chaperone during energy depletion (glucose starvation), reversibly relocating to deposition sites for protein aggregates (alongside generic chaperones Hsp42, Ssa2, Sis1, Hsp104) to sequester TA proteins under conditions preventing their membrane insertion.","method":"Live-cell fluorescence imaging of Get3-GFP localization + genetic and cell biological analyses under energy depletion","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging showing reversible relocalization linked to functional holdase role, single lab","pmids":["23203805"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of a yeast Get3–Get4–Get5 complex in an ATP-bound state showed how Get4 primes Get3 by promoting the optimal configuration for substrate capture; structure-guided biochemical analyses demonstrated that Get4-mediated regulation of Get3's ATP hydrolysis is essential for efficient TA protein targeting.","method":"X-ray crystallography of Get3–Get4–Get5 ternary complex + structure-guided mutagenesis + biochemical TA targeting assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of ternary complex combined with mutagenesis and functional targeting assays in single study","pmids":["24727835"],"is_preprint":false},{"year":2012,"finding":"TRC40 (Get3) binds short secretory protein precursors (apelin, statherin, preprocecropin A) and can deliver them to the ER membrane for post-translational translocation via the Sec61 translocon, identifying secretory proteins as a class of TRC40 substrates beyond TA proteins.","method":"In vitro TRC40-binding assay + in vitro ER translocation assay with dominant-negative TRC40 inhibition","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding and translocation assays with inhibition controls, single lab","pmids":["22505607"],"is_preprint":false},{"year":2013,"finding":"TRC40 (Get3) is dispensable for peroxisomal targeting of the TA protein PEX26; PEX19 captures PEX26 in the cytosol and delivers it directly to peroxisomes via PEX3, defining a TRC40-independent class I pathway for peroxisomal TA proteins and showing that basic residues in PEX26's luminal domain are essential for PEX19 binding.","method":"Coimmunoprecipitation of PEX19–PEX26 complex + dominant-negative TRC40 inhibition assay + mutagenesis of PEX26 luminal basic residues","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — negative result for TRC40 combined with positive identification of alternative pathway by co-IP and mutagenesis, single lab","pmids":["23460677"],"is_preprint":false},{"year":2011,"finding":"TRC40/Asna1 mediates post-translational membrane insertion of calneuron-1 and calneuron-2 (neuronal TA calcium sensors) via their 23-amino-acid TMD; calneuron dimerization/multimerization via the TMD precludes TRC40 binding and membrane insertion, but no cytosolic pool of calneurons was detected, indicating self-association is restricted to membrane-inserted protein.","method":"In vitro TRC40 binding assay + co-immunoprecipitation + in vitro membrane insertion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding and insertion assays with mechanistic interpretation, single lab","pmids":["21878631"],"is_preprint":false},{"year":2015,"finding":"TRC40-mediated TA protein insertion is required for correct trafficking of emerin to the inner nuclear membrane; emerin interacts with TRC40 in situ (proximity ligation assay), is inserted into microsomal membranes in an ATP- and TRC40-dependent manner, and EDMD-causing emerin mutations disrupt TRC40 binding, membrane integration, and inner nuclear membrane targeting.","method":"Proximity ligation assay + in vitro microsomal membrane insertion assay + rapamycin-based dimerization transport assay + dominant-negative WRB/CAML receptor fragments","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (PLA, in vitro insertion, transport assay, dominant-negative inhibition) establishing the TRC40–emerin targeting mechanism and its disease relevance","pmids":["26675233"],"is_preprint":false},{"year":2016,"finding":"Functional proteoliposomes reconstituted from microsomal detergent extracts lost TA protein insertion activity when depleted of TRC40-associated proteins or CAML itself; in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes, defining the minimal mammalian receptor for TRC40-mediated TA insertion; CAML is present in ~5-fold molar excess over WRB and they mutually regulate each other's levels.","method":"Reconstituted functional proteoliposomes from detergent extract + immunodepletion + in vitro insertion assay + quantitative Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full reconstitution from defined components defining minimal mammalian receptor, single lab but rigorous","pmids":["27226539"],"is_preprint":false},{"year":2019,"finding":"CAML, in the presence of sufficient WRB, is inserted into the ER membrane with three transmembrane segments in its C-terminal region; without sufficient WRB, CAML fails to adopt correct topology, generating aberrant topoforms that aggregate at ER-associated clusters and are degraded by the proteasome; WRB acts catalytically to assist CAML topogenesis.","method":"Topology mapping assays + proteasome inhibitor experiments + ER localization imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — topology and degradation assays with mechanistic interpretation, single lab","pmids":["31417168"],"is_preprint":false},{"year":2019,"finding":"The small molecule Retro-2 blocks delivery of newly synthesized TA proteins to the ER-targeting factor ASNA1 (TRC40); a single ASNA1 point mutant identified by CRISPR-mediated mutagenesis abolishes both Retro-2's cytoprotective effect against ricin and its inhibitory effect on ASNA1-mediated ER targeting, demonstrating that Retro-2 acts directly on the ASNA1-dependent TA protein targeting step to prevent retrograde trafficking of ricin.","method":"CRISPRi genetic interaction screen + cell-based TA protein targeting assay + in vitro ASNA1 interaction assay + CRISPR point mutant resistance mapping","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic interaction screen combined with cell-based and in vitro assays and CRISPR point mutant validation, multiple orthogonal approaches","pmids":["31674906"],"is_preprint":false},{"year":2019,"finding":"A dominant-negative ATPase-impaired TRC40 mutant (D74E) traps TA protein clients in the cytoplasm; manipulation of the hydrophobic TA-binding groove reduces interaction with most but not all substrates; identified known and novel TRC40 substrates including Golgi-resident TA proteins (golgin-84, CASP, giantin) and VAPA/VAPB by quantitative mass spectrometry.","method":"Dominant-negative TRC40(D74E) trap + quantitative mass spectrometry + groove mutant analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative trap with quantitative proteomics identifying substrate spectrum, single lab","pmids":["31182645"],"is_preprint":false},{"year":2022,"finding":"Activation of Get3's chaperone function follows a multi-step process: reactivity of two conserved cysteines is directly controlled by Get3's nucleotide-binding state; thiol oxidation causes local unfolding and transition into chaperone-active oligomers; inactivation requires cysteine reduction followed by ATP binding, enabling transfer of client proteins to ATP-dependent chaperones for refolding; disrupting this cycle in yeast impairs oxidative stress resistance.","method":"Biochemical redox assays + mutagenesis of conserved cysteines + in vitro chaperone activity assays + yeast genetic phenotypic analyses","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — detailed mechanistic dissection with mutagenesis, biochemical assays, and in vivo genetic validation defining the ordered activation/inactivation cycle","pmids":["35839781"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of Giardia Get3 in five states (apo-open, apo-closed, ATP-bound, ADP-bound post-hydrolysis, and with TA client) showed that after ATP hydrolysis Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix; the ATP-bound state stabilizes an occluded CBD configuration, resolving how nucleotide drives conformational transitions across the full targeting cycle.","method":"Cryo-EM structure determination of Get3 in five nucleotide/conformational states (including Get3-client complex)","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — five structures spanning the complete conformational landscape including a client-bound post-hydrolysis state, resolving key mechanistic unknowns","pmids":["35851188"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure (3.2 Å) of the S. cerevisiae Get3–Get4/5 complex showed that Get4/5 remodels Get3's TA-binding chamber by unfolding helices forming the lateral walls ('lateral gate'), making the chamber more solvent accessible; mutagenesis of lateral gate residues influenced Get3 binding affinity for Get4/5 and its ATPase activity; the Sgt2-binding domain of Get5 is positioned near the lateral gate opening, supporting a model of lateral TA transfer from Sgt2 to Get3.","method":"Cryo-EM structure determination + molecular dynamics simulation + site-directed mutagenesis + ATPase activity assay + binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure combined with MD simulation, mutagenesis, and functional assays in single study","pmids":["40902977"],"is_preprint":false},{"year":2014,"finding":"ASNA1 contains a non-canonical FFAT-like motif that mediates direct interaction with the MSP domain of the ER membrane protein VAPB, physically linking the TRC40/ASNA1 TA-targeting complex to VAPB at the ER membrane.","method":"Co-immunoprecipitation + motif identification + direct binding assay","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and direct binding assay identifying FFAT-like motif-mediated interaction, single lab","pmids":["24885147"],"is_preprint":false},{"year":2021,"finding":"ASNA-1 exists in two redox states that control distinct functions: the reduced state mediates cisplatin resistance and TA protein targeting; an ASNA-1 point mutant preferentially in the oxidized state was sensitive to cisplatin and defective for TA protein targeting but showed normal insulin secretion; cisplatin-induced ROS drives ASNA-1 into the oxidized form and selectively prevents an ASNA-1-dependent TA substrate from reaching the ER.","method":"C. elegans genetics with redox-state point mutants + in vivo TA protein targeting assay + cisplatin sensitivity assay + ROS measurement","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic point mutants with in vivo functional assays separating redox-state-dependent functions, single lab","pmids":["33883621"],"is_preprint":false},{"year":2006,"finding":"Get3 biochemically interacts with the transmembrane domain proteins Get1/Mdm39 and Get2/Rmd7 in yeast; deletion of GET3 suppresses phenotypes of get1 and get2 mutants including sporulation defects; genetic interactions with NPL4 in the ubiquitin-proteasome system implicate Get3 in multiple membrane-dependent pathways.","method":"Co-immunoprecipitation + yeast genetic epistasis (suppressor analysis, double mutants)","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal biochemical interaction and epistasis data, single lab, pre-dates full GET pathway understanding","pmids":["16816426"],"is_preprint":false},{"year":2007,"finding":"ASNA-1 functions nonautonomously to regulate insulin secretion in C. elegans; expressed in insulin-producing intestinal cells, asna-1 mutants show reduced insulin secretion while overexpression mimics insulin overexpression effects; human ASNA1 is highly expressed in pancreatic beta cells and regulates insulin secretion in cultured cells, demonstrating an evolutionarily conserved role in insulin secretion.","method":"C. elegans genetics (asna-1 null and overexpression) + insulin secretion assays in C. elegans + human ASNA1 knockdown/overexpression in cultured beta cells","journal":"Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function and gain-of-function with insulin secretion readout in two organisms, single lab","pmids":["17289575"],"is_preprint":false},{"year":2006,"finding":"Homozygous Asna1 knockout mice die between embryonic day 3.5 and 8.5, demonstrating that Asna1 is essential for early embryonic development in mammals.","method":"Homologous recombination knockout mouse generation + embryonic lethality analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic knockout with defined lethal phenotype, but no molecular mechanism identified beyond essentiality","pmids":["16797549"],"is_preprint":false},{"year":2015,"finding":"Beta-cell-specific inactivation of Asna1 in mice caused hypoinsulinemia, impaired insulin secretion, and rapidly progressive diabetes; Asna1 loss perturbed plasma membrane-to-TGN and Golgi-to-ER retrograde transport and caused ER stress in beta cells; pharmacological inhibition of retrograde transport in isolated islets mimicked the Asna1 loss-of-function phenotype, linking Asna1 function to retrograde vesicular transport and ER homeostasis.","method":"Conditional knockout mouse model (beta-cell-specific) + pharmacological retrograde transport inhibition + ER stress markers","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined phenotype and pharmacological mimicry linking mechanism, single lab","pmids":["26438609"],"is_preprint":false},{"year":2018,"finding":"Asna1 inactivation in pancreatic multipotent progenitor cells (MPCs) caused redistribution of Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, integrated stress response activation, and p53-mediated apoptosis leading to pancreatic agenesis; rescue experiments showed the Asna1 ATPase activity and a CXXC di-cysteine motif are required for Golgi integrity and MPC survival; ex vivo inhibition of retrograde transport reproduced the Golgi and syntaxin phenotypes.","method":"Conditional knockout mouse model + rescue experiments with ATPase-dead and CXXC mutants + pharmacological retrograde transport inhibition ex vivo + p53 modulator experiments","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with domain-specific rescue experiments and pharmacological phenocopy, single lab","pmids":["29180572"],"is_preprint":false},{"year":2016,"finding":"All three HSV1 tail-anchored proteins (pUL34, pUL56, pUS9) specifically bound to Asna1/TRC40 by yeast two-hybrid; TRC40 depletion by RNAi did not affect virion entry, viral gene expression, or secondary envelopment but specifically reduced release of infectious virions to the extracellular medium by more than 10-fold, identifying a role for TRC40 in viral egress.","method":"Yeast two-hybrid protein interaction + siRNA depletion + viral replication and release assays","journal":"Virology journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — interaction identified by Y2H and functional consequence defined by RNAi depletion with specific viral release readout, single lab","pmids":["27765046"],"is_preprint":false},{"year":2026,"finding":"GET3 (TRC40/ASNA1) directly interacts with the anti-apoptotic protein MCL1 via MCL1's C-terminal hydrophobic tail; GET3 depletion reduced MCL1 protein levels while GET3 overexpression increased them; GET3 deficiency enhanced apoptosis and reduced clonogenic survival, particularly in HeLa cells, and accelerated MCL1 downregulation and apoptosis during prolonged mitotic arrest, identifying MCL1 as a TA-containing cargo of GET3.","method":"Degron-mediated GET3 depletion + co-immunoprecipitation of GET3–MCL1 + apoptosis assays + clonogenic survival assay + GET3 overexpression","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — degron depletion combined with co-IP establishing direct interaction, two orthogonal functional readouts, single lab","pmids":["42098326"],"is_preprint":false},{"year":2025,"finding":"Constitutive cardiomyocyte-specific Asna1 knockout caused ventricular myocardial thinning by E16.5 and perinatal lethality; inducible adult cardiomyocyte-specific deletion caused rapid ventricular dilation and early mortality; ASNA1 deficiency destabilized the pre-targeting complex and reduced expression of multiple TA protein substrates, impairing membrane trafficking and vesicular transport; transcriptomic analysis revealed compensatory upregulation of Golgi-to-ER transport genes.","method":"Constitutive and inducible cardiomyocyte-specific conditional knockout mouse models + TA protein substrate expression analysis + transcriptomics","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two conditional KO models with defined molecular mechanism (substrate destabilization), single lab","pmids":["41370295"],"is_preprint":false},{"year":2025,"finding":"Neuron-specific deletion of ASNA1 in mice (using SLICK-H-Cre or synapsin-Cre) phenocopied CAML neuron-specific deletion, causing loss of motor neuron cell bodies, hind limb weakness, and paralysis; identifying a cell-autonomous role for the ASNA1/TRC40 TA protein insertion machinery in motor neuron survival.","method":"Neuron-specific conditional knockout mouse (synapsin-Cre and SLICK-H-Cre) + spinal cord histology","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular phenotype establishing cell-autonomous neuronal role, single lab","pmids":["39823474"],"is_preprint":false},{"year":2024,"finding":"Oxidized TRC40 (human Get3/ASNA1) forms chaperone-active tetramers and high-molecular-weight complexes that prevent aggregation of unfolding proteins; acute oxidative stress causes reversible formation of distinct TRC40 foci co-localizing with Hsp70 and Hsp110; TRC40 is essential for cell survival under ATP-depleting oxidative stress conditions, counteracting accumulation of mis- and unfolded proteins.","method":"Biochemical chaperone activity assay + native PAGE oligomer analysis + live-cell fluorescence imaging of TRC40 foci + genetic depletion under oxidative stress","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in human cells extending the chaperone function to TRC40, preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"GET3/TRC40/ASNA1 is a conserved homodimeric ATPase that serves as the central cytosolic targeting factor of the GET/TRC pathway, using nucleotide-driven open-to-closed conformational changes to capture the transmembrane domain of tail-anchored (TA) proteins in a hydrophobic groove spanning the dimer interface, shield it from the cytosol, and deliver it to the ER membrane receptor (Get1/Get2 in yeast; WRB/CAML in mammals) where ATP hydrolysis and receptor-induced reopening of Get3 drive TA protein membrane insertion; Get4/5 upstream regulates Get3 by promoting the ATP-bound substrate-capture state and facilitating lateral TA handoff from the cochaperone Sgt2; under oxidative stress, Get3 undergoes a redox switch (disulfide bond formation at conserved cysteines, controlled by nucleotide state) that converts it from an ATP-dependent targeting factor into an ATP-independent holdase chaperone protecting against protein aggregation, with both functions being mutually exclusive and the chaperone state being fully reversible upon reduction and ATP rebinding."},"narrative":{"mechanistic_narrative":"GET3 (TRC40/ASNA1) is a conserved homodimeric ATPase that serves as the central cytosolic targeting factor of the GET/TRC pathway for post-translational delivery of tail-anchored (TA) membrane proteins to the ER [PMID:25745174, PMID:20375064]. Nucleotide state drives an open-to-closed conformational cycle: in the closed, ADP·AlF4−-bound state the dimer forms a large hydrophobic groove spanning the interface that captures and shields the TA transmembrane helix, while the open state disrupts this groove to release substrate [PMID:19675567, PMID:19956640, PMID:35851188]. A single TA protein binds one Get3 homodimer, with the α-helical/finger subdomain forming the binding surface [PMID:25745174, PMID:19948960]. Get3 is primed for substrate capture by the upstream Get4–Get5 complex, which interacts with Get3 in a nucleotide-dependent manner, promotes the ATP-bound configuration optimal for capture, and remodels the lateral walls of the binding chamber to facilitate hand-off of TA substrate from the cochaperone Sgt2 [PMID:20554915, PMID:24727835, PMID:40902977]. Loaded Get3 docks onto the ER membrane receptor — Get1/Get2 in yeast, WRB/CAML in mammals — which remodels Get3 toward the open state to drive insertion; ATP hydrolysis promotes efficient insertion and Get3 release from the receptor [PMID:21719644, PMID:19948960, PMID:21444755, PMID:22684149, PMID:27226539]. Substrates extend beyond classical TA proteins to Golgi-resident TA proteins, SNAREs, VAPA/VAPB, the inner nuclear membrane protein emerin, short secretory precursors, and the anti-apoptotic protein MCL1 [PMID:22505607, PMID:26675233, PMID:31182645, PMID:42098326]. Independently of its targeting role, Get3 acts as an ATP-independent holdase chaperone: oxidative stress triggers nucleotide-controlled disulfide formation at conserved cysteines, metal release, and assembly into higher-order chaperone-active oligomers that protect against protein aggregation, a state mutually exclusive with targeting and reversed by reduction and ATP rebinding [PMID:25242142, PMID:35839781]. In mammals ASNA1 is essential for early embryogenesis [PMID:16797549], and TRC40-mediated insertion of emerin links the pathway to Emery-Dreifuss muscular dystrophy, as EDMD-causing emerin mutations disrupt TRC40 binding and membrane integration [PMID:26675233].","teleology":[{"year":2006,"claim":"Before the GET pathway was defined, it was unknown how Get3 connected physically and genetically to the ER membrane machinery; this established Get3 as a partner of the Get1/Get2 transmembrane proteins.","evidence":"Co-immunoprecipitation and yeast genetic epistasis (suppressor and double-mutant analysis)","pmids":["16816426"],"confidence":"Medium","gaps":["No structural basis for the interaction","Mechanism of TA insertion not yet addressed","Predates identification of TA proteins as substrates"]},{"year":2006,"claim":"The physiological requirement for the mammalian ortholog was unknown; knockout established Asna1 as essential for early mammalian development.","evidence":"Homologous recombination Asna1 knockout mouse with embryonic lethality analysis","pmids":["16797549"],"confidence":"Medium","gaps":["No molecular mechanism for lethality identified","Does not distinguish targeting from chaperone roles"]},{"year":2007,"claim":"It was unclear whether ASNA1 had organismal physiological roles beyond a housekeeping function; this revealed a conserved nonautonomous role in insulin secretion.","evidence":"C. elegans loss- and gain-of-function genetics with insulin secretion readouts plus human beta-cell knockdown/overexpression","pmids":["17289575"],"confidence":"Medium","gaps":["Mechanistic link between TA targeting and insulin secretion unresolved","Direct secretory substrates not identified at this stage"]},{"year":2009,"claim":"How Get3 binds and releases TA substrates was unknown; structures of open and closed dimer states defined a nucleotide-regulated hydrophobic groove at the dimer interface as the TA-binding site.","evidence":"X-ray crystallography of open (nucleotide-free) and closed (ADP·AlF4−) states with mutagenesis of groove residues, across multiple yeast/fungal/archaeal orthologs and H/D exchange MS","pmids":["19675567","19956640","19706470","19948960","20015340"],"confidence":"High","gaps":["Stoichiometry of the functional Get3–TA complex not yet defined","Direct visualization of a bound TA helix lacking"]},{"year":2010,"claim":"Whether Get3 alone could insert TA proteins and what upstream factors regulated it were open; reconstitution showed receptor- and nucleotide-dependent insertion, and Get4/5 was placed upstream of Get3.","evidence":"In vitro reconstitution of TA insertion from recombinant components and ER membranes, plus crystallography and nucleotide-dependent binding assays of the Get4/5 complex","pmids":["20375064","20554915","20106980"],"confidence":"High","gaps":["Stoichiometry of Get3–Get4/5 assembly unresolved","Mechanism of Sgt2-to-Get3 hand-off not yet defined"]},{"year":2011,"claim":"The identity of the mammalian receptor and how the receptor remodels Get3 were unknown; WRB was identified as the mammalian receptor and Get1 structures showed how it stabilizes the open Get3 state.","evidence":"Crystallography of Get3–Get1/Get2 complexes, biophysical (ITC/SAXS) stoichiometry of Get3–Get4/5, and biochemical/imaging identification of WRB","pmids":["21719644","22684149","22190685","21444755"],"confidence":"High","gaps":["Full mammalian receptor composition not yet established","Order of conformational events during insertion incompletely defined"]},{"year":2014,"claim":"How Get4 regulates Get3's catalytic cycle was unresolved; the ternary Get3–Get4–Get5 structure showed Get4 primes Get3 into the optimal substrate-capture configuration and regulates ATP hydrolysis.","evidence":"X-ray crystallography of the ATP-bound ternary complex with structure-guided mutagenesis and TA targeting assays","pmids":["24727835"],"confidence":"High","gaps":["Lateral transfer geometry from Sgt2 not yet visualized","Coupling of priming to nucleotide turnover incompletely defined"]},{"year":2014,"claim":"Whether Get3 had functions beyond targeting was unknown; oxidative stress was shown to convert it into an ATP-independent holdase chaperone via disulfide formation and oligomerization, mutually exclusive with targeting.","evidence":"In vitro chaperone assays, disulfide-disrupting mutagenesis, energy-depletion imaging, and yeast oxidative-stress phenotypes","pmids":["25242142","23203805"],"confidence":"High","gaps":["Ordered redox activation steps not yet defined","Client spectrum of the chaperone state unresolved"]},{"year":2015,"claim":"The composition of the functional targeting complex and the physiological substrate range were unclear; reconstitution fixed the 1 TA : 1 Get3 dimer stoichiometry and crystal structures resolved TA-helix recognition, while emerin established disease relevance.","evidence":"In vitro reconstitution plus crystallography of Get3–TA complexes; proximity ligation, in vitro insertion, and transport assays for emerin","pmids":["25745174","26675233"],"confidence":"High","gaps":["Full client repertoire still being defined","Mechanism linking emerin mistargeting to muscular dystrophy pathology not detailed"]},{"year":2016,"claim":"The minimal mammalian receptor was undefined; reconstitution showed WRB and CAML together are sufficient and necessary for TRC40-mediated insertion, with CAML in molar excess and mutual level regulation.","evidence":"Functional proteoliposome reconstitution from detergent extract with immunodepletion, in vitro synthesized components, and quantitative Western blot","pmids":["27226539"],"confidence":"High","gaps":["Stoichiometric architecture of the WRB/CAML receptor in membranes unresolved","Mechanism of mutual level regulation not defined"]},{"year":2019,"claim":"The full conformational cycle and substrate spectrum of the targeting factor were incompletely resolved; cryo-EM across five states and dominant-negative proteomics defined nucleotide-driven CBD reorganization and broadened the client list to Golgi TA proteins and VAPA/VAPB.","evidence":"Cryo-EM of Get3 in five nucleotide/conformational states; dominant-negative TRC40(D74E) trap with quantitative mass spectrometry; CRISPR-based Retro-2 target validation","pmids":["35851188","31182645","31674906"],"confidence":"High","gaps":["Substrate selectivity rules not fully defined","Some substrates persist despite groove mutation, implying additional binding determinants"]},{"year":2022,"claim":"How the redox switch is ordered and reset was unknown; the activation cycle was resolved as nucleotide-controlled cysteine reactivity, oxidation-induced unfolding into chaperone oligomers, and reduction-plus-ATP-driven inactivation enabling client hand-off.","evidence":"Biochemical redox and chaperone assays, conserved-cysteine mutagenesis, and yeast oxidative-stress genetics","pmids":["35839781"],"confidence":"High","gaps":["Identity of downstream ATP-dependent chaperones receiving clients not defined","Structural basis of the chaperone-active oligomer unresolved"]},{"year":2025,"claim":"How Get4/5 enables lateral TA hand-off from Sgt2 was unresolved; the cryo-EM Get3–Get4/5 structure showed remodeling of a 'lateral gate' that opens the binding chamber adjacent to the Sgt2-binding domain of Get5.","evidence":"Cryo-EM, molecular dynamics, mutagenesis, ATPase and binding assays of the S. cerevisiae Get3–Get4/5 complex","pmids":["40902977"],"confidence":"High","gaps":["A captured Sgt2–Get3 transfer intermediate not yet visualized","Timing of lateral gate opening relative to nucleotide turnover unresolved"]},{"year":2025,"claim":"The tissue-specific physiological consequences of losing the targeting machinery were unclear; cardiomyocyte- and neuron-specific knockouts showed cell-autonomous requirements linked to TA substrate destabilization and impaired membrane trafficking.","evidence":"Constitutive and inducible conditional knockout mouse models with substrate expression analysis, transcriptomics, and spinal cord histology","pmids":["41370295","39823474"],"confidence":"Medium","gaps":["Specific TA substrates driving each tissue phenotype not pinpointed","Relative contribution of targeting versus chaperone roles to phenotypes unresolved"]},{"year":2026,"claim":"Whether GET3 regulates apoptotic machinery as cargo was unknown; MCL1 was identified as a TA-containing client whose levels and pro-survival function depend on GET3.","evidence":"Degron-mediated GET3 depletion, co-immunoprecipitation, apoptosis and clonogenic survival assays","pmids":["42098326"],"confidence":"Medium","gaps":["Whether MCL1 is inserted via the canonical WRB/CAML receptor not shown","In vivo relevance of GET3–MCL1 axis to tumor survival unresolved"]},{"year":null,"claim":"How the cell switches Get3 between its targeting and holdase chaperone modes in vivo, and which downstream chaperones and physiological substrates govern recovery from stress, remains to be defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the chaperone-active oligomer","Substrate hand-off partners during stress recovery not identified","Quantitative balance between targeting and chaperone pools in tissues unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,3,7,16,26]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[14,15,25]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[1,7,17]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,4]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[14,25,29]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,7,15]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,7,8,21]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,3,7,20]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,7,16]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[14,25]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[33,34]}],"complexes":["Get3-Get4-Get5 pre-targeting complex","Get3-Get1/Get2 receptor complex","TRC40-WRB/CAML receptor complex"],"partners":["GET4","GET5","GET1","GET2","WRB","CAML","VAPB","SGT2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43681","full_name":"ATPase GET3","aliases":["Arsenical pump-driving ATPase","Arsenite-stimulated ATPase","Guided entry of tail-anchored proteins factor 3, ATPase","Transmembrane domain recognition complex 40 kDa ATPase subunit","hARSA-I","hASNA-I"],"length_aa":348,"mass_kda":38.8,"function":"ATPase required for the post-translational delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (PubMed:17382883). Recognizes and selectively binds the transmembrane domain of TA proteins in the cytosol. This complex then targets to the endoplasmic reticulum by membrane-bound receptors GET1/WRB and CAMLG/GET2, where the tail-anchored protein is released for insertion. This process is regulated by ATP binding and hydrolysis. ATP binding drives the homodimer towards the closed dimer state, facilitating recognition of newly synthesized TA membrane proteins. ATP hydrolysis is required for insertion. Subsequently, the homodimer reverts towards the open dimer state, lowering its affinity for the GET1-CAMLG receptor, and returning it to the cytosol to initiate a new round of targeting. May be involved in insulin signaling","subcellular_location":"Cytoplasm; Endoplasmic reticulum; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/O43681/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/GET3","classification":"Common Essential","n_dependent_lines":881,"n_total_lines":1208,"dependency_fraction":0.7293046357615894},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLTA","stoichiometry":0.2},{"gene":"SCFD1","stoichiometry":0.2},{"gene":"STX5","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2},{"gene":"VAPB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GET3","total_profiled":1310},"omim":[{"mim_id":"620203","title":"CARDIOMYOPATHY, DILATED, 2H; CMD2H","url":"https://www.omim.org/entry/620203"},{"mim_id":"612056","title":"GUIDED ENTRY OF TAIL-ANCHORED PROTEINS FACTOR 4; GET4","url":"https://www.omim.org/entry/612056"},{"mim_id":"601913","title":"GUIDED ENTRY OF TAIL-ANCHORED PROTEINS FACTOR 3, ATPase; GET3","url":"https://www.omim.org/entry/601913"},{"mim_id":"601118","title":"CALCIUM-MODULATING LIGAND; CAMLG","url":"https://www.omim.org/entry/601118"},{"mim_id":"312070","title":"UBIQUITIN-LIKE 4A; UBL4A","url":"https://www.omim.org/entry/312070"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoli","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GET3"},"hgnc":{"alias_symbol":["ARSA-I","TRC40"],"prev_symbol":["ASNA1"]},"alphafold":{"accession":"O43681","domains":[{"cath_id":"3.40.50.300","chopping":"27-117_125-180_239-337","consensus_level":"high","plddt":85.7826,"start":27,"end":337}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43681","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43681-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43681-F1-predicted_aligned_error_v6.png","plddt_mean":79.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GET3","jax_strain_url":"https://www.jax.org/strain/search?query=GET3"},"sequence":{"accession":"O43681","fasta_url":"https://rest.uniprot.org/uniprotkb/O43681.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43681/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43681"}},"corpus_meta":[{"pmid":"19675567","id":"PMC_19675567","title":"The structural basis of tail-anchored membrane protein recognition by Get3.","date":"2009","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/19675567","citation_count":155,"is_preprint":false},{"pmid":"25745174","id":"PMC_25745174","title":"Protein targeting. 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Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/19407384","citation_count":1,"is_preprint":false},{"pmid":"40902977","id":"PMC_40902977","title":"Get4/5-mediated remodeling of Get3's substrate-binding chamber: Insights into tail-anchored protein targeting by the GET pathway.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40902977","citation_count":0,"is_preprint":false},{"pmid":"41370295","id":"PMC_41370295","title":"ASNA1 is essential for cardiac development and function by regulating tail-anchored protein stability and vesicular transport in cardiomyocytes.","date":"2025","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/41370295","citation_count":0,"is_preprint":false},{"pmid":"42098326","id":"PMC_42098326","title":"GET3 regulates apoptosis via tail-anchoring of MCL1.","date":"2026","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/42098326","citation_count":0,"is_preprint":false},{"pmid":"39823474","id":"PMC_39823474","title":"Tail Anchored protein insertion mediated by CAML and TRC40 links to neuromuscular function in mice.","date":"2025","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39823474","citation_count":0,"is_preprint":false},{"pmid":"41764646","id":"PMC_41764646","title":"Diversity of GET3 chaperones in tail-anchored protein insertion.","date":"2026","source":"Journal of experimental botany","url":"https://pubmed.ncbi.nlm.nih.gov/41764646","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.28.640736","title":"Characterisation of guided entry of tail-anchored proteins in  <i>Magnaporthe oryzae</i>","date":"2025-03-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.28.640736","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.10.602939","title":"A dynamic redox switch turns TRC40 into a chaperone protecting human cells against ATP-depleting, oxidative stress","date":"2024-07-13","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.10.602939","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.11.24313493","title":"Proteogenomic signature of risk of Alzheimer’s disease and related dementia risk in individuals with a history of major depression disorder","date":"2024-09-12","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.11.24313493","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25660,"output_tokens":9741,"usd":0.111547,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":20458,"output_tokens":5436,"usd":0.119095,"stage2_stop_reason":"end_turn"},"total_usd":0.230642,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP·AlF4−-bound) dimer states revealed that in the closed state the dimer interface contains a large hydrophobic groove responsible for tail-anchored protein binding, and in the open state this groove is disrupted; mutational analyses confirmed the groove's role in TA protein binding, establishing a nucleotide-regulated binding/release mechanism.\",\n      \"method\": \"X-ray crystallography (open and closed dimer states) + site-directed mutagenesis of hydrophobic groove residues\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures in two functional states combined with mutagenesis confirming the binding groove, foundational mechanistic paper widely replicated\",\n      \"pmids\": [\"19675567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Reconstitution of the physiological assembly pathway showed that the functional Get3–TA protein targeting complex comprises a single TA protein bound to a Get3 homodimer; crystal structures of Get3 bound to different TA proteins showed the α-helical transmembrane domain occupying a hydrophobic groove spanning the homodimer interface, elucidating the mechanism of TA protein recognition and shielding.\",\n      \"method\": \"In vitro reconstitution of targeting complex + X-ray crystallography of Get3–TA protein complexes with different substrates\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution of physiologic complex combined with crystal structures of multiple Get3–TA complexes, directly resolving the long-standing unknown of complex composition\",\n      \"pmids\": [\"25745174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of Get3 in complex with cytosolic domains of the ER membrane receptor subunits Get1 and Get2 showed that Get1 and Get2 use adjacent, partially overlapping binding sites on Get3 and can bind simultaneously; docking to the Get1/2 receptor induces conformational changes in Get3 required for TA protein insertion.\",\n      \"method\": \"X-ray crystallography of Get3–Get1/2 receptor complexes at 3.0, 3.2, and 4.6 Å + biochemical experiments\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures at different resolutions combined with biochemical validation, directly defining the receptor-docking mechanism\",\n      \"pmids\": [\"21719644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Structural and biochemical analyses of Get3 (Asna1/TRC40) showed that the α-helical subdomain binds the TA protein transmembrane domain; amide proton exchange mass spectrometry mapped the TA-binding site to the α-helical subdomain; in vitro membrane insertion assays demonstrated Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner; ATP hydrolysis is not strictly required for insertion but is needed for efficient insertion, likely for Get3 release from its receptor.\",\n      \"method\": \"X-ray crystallography + hydrogen/deuterium exchange MS + in vitro membrane insertion reconstitution assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (crystallography, H/D exchange MS, in vitro insertion assay) in a single study\",\n      \"pmids\": [\"19948960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structures of Get3 from S. cerevisiae (apo, open) and D. hansenii (ADP-bound, closed) identified a nucleotide-binding domain and a 'finger' domain with a hydrophobic groove as the TA protein TMD-binding site; a hydrophobic helix from a symmetry-related molecule occupying the groove mimicked TA binding, and the open/closed conformational switch is linked to TA protein release.\",\n      \"method\": \"X-ray crystallography of Get3 from two yeast species in apo and ADP-bound states\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures from two organisms in different nucleotide states, consistent with findings from multiple independent groups\",\n      \"pmids\": [\"19956640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structures of apo and ADP-bound Get3 from S. cerevisiae and A. fumigatus identified residues important for dimer interfaces; structure-guided mutagenesis confirmed key interfaces and essential residues coupling ATP hydrolysis to TA protein binding and release.\",\n      \"method\": \"X-ray crystallography (apo and ADP-bound forms, two species) + structure-guided mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structures of two orthologs combined with mutagenesis confirming mechanistic residues\",\n      \"pmids\": [\"19706470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structures of Get3 in ADP-bound and nucleotide-free forms showed a Zn2+-mediated homodimer in head-to-head orientation; cross-linking experiments indicated the closed dimer stimulates ATP hydrolysis; coexpression-based binding assays demonstrated direct interaction between the helical domain of Get3 and the Sec22p TMD independent of ATP and dimer formation; the conserved DTAPTGH motif was proposed to link ATP hydrolysis to TA protein insertion.\",\n      \"method\": \"X-ray crystallography + chemical cross-linking + coexpression-based binding assay\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — crystal structures plus cross-linking and binding assay, but binding assay is coexpression-based (not direct reconstitution), single lab\",\n      \"pmids\": [\"20015340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Get3 (Asna1/TRC40) was shown to mediate membrane insertion of the TA proteins RAMP4 and Sec61β from recombinant Asna1–TA protein complexes into ER-derived membranes in a mechanism requiring ATP or ADP and a protease-sensitive ER membrane receptor, but not additional cytosolic factors; cytochrome b5 insertion proceeded independently of Asna1 and nucleotides.\",\n      \"method\": \"In vitro reconstitution of TA protein membrane insertion from recombinant components + ER-derived membranes + protease sensitivity assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full reconstitution from recombinant components with mechanistic dissection of nucleotide requirement and substrate specificity, single lab\",\n      \"pmids\": [\"20375064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"WRB (tryptophan-rich basic protein/CHD5), an ER-resident membrane protein with sequence similarity to yeast Get1, was identified as the ER membrane receptor for mammalian TRC40/Asna1; the coiled-coil domain of WRB is the binding site for TRC40; a soluble coiled-coil domain fragment interfered with TRC40-mediated TA protein membrane insertion.\",\n      \"method\": \"Biochemical interaction assays + cell imaging + dominant-negative soluble fragment inhibition of in vitro TA insertion\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction assays, cell imaging, and functional inhibition data identifying the mammalian receptor; independently corroborated by multiple subsequent studies\",\n      \"pmids\": [\"21444755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of Get4 with an N-terminal Get5 fragment showed they form an intimate complex existing as a dimer-of-heterodimers; Get3 binds to a conserved surface on Get4 in a nucleotide-dependent manner, placing Get4/5 upstream of Get3 in the TA protein delivery pathway.\",\n      \"method\": \"X-ray crystallography of Get4/5 complex + nucleotide-dependent binding assay of Get3 to Get4\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with functional binding assay, single lab but clear mechanistic finding\",\n      \"pmids\": [\"20554915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Co-immunoprecipitation and crystallographic studies showed Get4 and Get5 form a tight complex; Get3 physically and transiently interacts with the Get4–Get5 complex; Sgt2 interacts with Get5; genetic interactions between GET3, GET4, GET5, and the chaperone YDJ1 implicate molecular chaperones in TA protein insertion.\",\n      \"method\": \"Co-immunoprecipitation + X-ray crystallography of Get4/5 + genetic epistasis (YDJ1)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP combined with crystallography and genetic epistasis, single lab\",\n      \"pmids\": [\"20106980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ITC and SAXS demonstrated that the Get3 homodimer interacts with two copies of the Get4–Get5 complex to form an extended stoichiometric complex in solution, defining the interaction surface as dominated by electrostatic forces.\",\n      \"method\": \"Isothermal titration calorimetry (ITC) + small-angle X-ray scattering (SAXS)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal biophysical methods defining stoichiometry and interaction surface, single lab\",\n      \"pmids\": [\"22190685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of ADP-bound Get3 in complex with the cytoplasmic domain of Get1 in open and semi-open conformations showed that Get1 uses two distinct interfaces to bind Get3 and stabilize its open dimer conformation, one sufficient for binding and the second required to stabilize the open state.\",\n      \"method\": \"X-ray crystallography (3.0 and 4.5 Å) of Get3–Get1CD complexes + biochemical binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures in two conformational states combined with biochemical validation, mechanistically defining how Get1 remodels Get3\",\n      \"pmids\": [\"22684149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"An archaeal Get3 homologue crystallized as a tetramer; SAXS of a fungal Get3–TA protein complex showed a molecular envelope consistent with the archaeal tetramer; the tetramer generates a hydrophobic chamber that binds TA proteins; the fungal tetramer complex was capable of mediating TA insertion in vitro.\",\n      \"method\": \"X-ray crystallography of archaeal Get3 tetramer + SAXS of fungal Get3–TA complex + in vitro TA insertion assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus SAXS and functional insertion assay, single lab; tetramer model not yet confirmed for canonical yeast/mammalian Get3\",\n      \"pmids\": [\"22122436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Get3 switches to an ATP-independent chaperone function upon oxidative stress: oxidation causes disulfide bond formation, metal release, and formation of distinct higher-order oligomeric structures; this chaperone activity is functionally distinct from and mutually exclusive with its TA protein targeting function; yeast cells lacking Get3 show oxidative stress-sensitive phenotypes attributable to loss of this chaperone activity.\",\n      \"method\": \"In vitro chaperone activity assays + mutational analysis (disulfide-disrupting mutants) + yeast genetic phenotypic analysis under oxidative stress\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (biochemical assays, mutagenesis, genetics) in one study; independently replicated by subsequent work\",\n      \"pmids\": [\"25242142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Get3 functions as an ATP-independent holdase chaperone during energy depletion (glucose starvation), reversibly relocating to deposition sites for protein aggregates (alongside generic chaperones Hsp42, Ssa2, Sis1, Hsp104) to sequester TA proteins under conditions preventing their membrane insertion.\",\n      \"method\": \"Live-cell fluorescence imaging of Get3-GFP localization + genetic and cell biological analyses under energy depletion\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging showing reversible relocalization linked to functional holdase role, single lab\",\n      \"pmids\": [\"23203805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of a yeast Get3–Get4–Get5 complex in an ATP-bound state showed how Get4 primes Get3 by promoting the optimal configuration for substrate capture; structure-guided biochemical analyses demonstrated that Get4-mediated regulation of Get3's ATP hydrolysis is essential for efficient TA protein targeting.\",\n      \"method\": \"X-ray crystallography of Get3–Get4–Get5 ternary complex + structure-guided mutagenesis + biochemical TA targeting assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of ternary complex combined with mutagenesis and functional targeting assays in single study\",\n      \"pmids\": [\"24727835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TRC40 (Get3) binds short secretory protein precursors (apelin, statherin, preprocecropin A) and can deliver them to the ER membrane for post-translational translocation via the Sec61 translocon, identifying secretory proteins as a class of TRC40 substrates beyond TA proteins.\",\n      \"method\": \"In vitro TRC40-binding assay + in vitro ER translocation assay with dominant-negative TRC40 inhibition\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding and translocation assays with inhibition controls, single lab\",\n      \"pmids\": [\"22505607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRC40 (Get3) is dispensable for peroxisomal targeting of the TA protein PEX26; PEX19 captures PEX26 in the cytosol and delivers it directly to peroxisomes via PEX3, defining a TRC40-independent class I pathway for peroxisomal TA proteins and showing that basic residues in PEX26's luminal domain are essential for PEX19 binding.\",\n      \"method\": \"Coimmunoprecipitation of PEX19–PEX26 complex + dominant-negative TRC40 inhibition assay + mutagenesis of PEX26 luminal basic residues\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — negative result for TRC40 combined with positive identification of alternative pathway by co-IP and mutagenesis, single lab\",\n      \"pmids\": [\"23460677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TRC40/Asna1 mediates post-translational membrane insertion of calneuron-1 and calneuron-2 (neuronal TA calcium sensors) via their 23-amino-acid TMD; calneuron dimerization/multimerization via the TMD precludes TRC40 binding and membrane insertion, but no cytosolic pool of calneurons was detected, indicating self-association is restricted to membrane-inserted protein.\",\n      \"method\": \"In vitro TRC40 binding assay + co-immunoprecipitation + in vitro membrane insertion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding and insertion assays with mechanistic interpretation, single lab\",\n      \"pmids\": [\"21878631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRC40-mediated TA protein insertion is required for correct trafficking of emerin to the inner nuclear membrane; emerin interacts with TRC40 in situ (proximity ligation assay), is inserted into microsomal membranes in an ATP- and TRC40-dependent manner, and EDMD-causing emerin mutations disrupt TRC40 binding, membrane integration, and inner nuclear membrane targeting.\",\n      \"method\": \"Proximity ligation assay + in vitro microsomal membrane insertion assay + rapamycin-based dimerization transport assay + dominant-negative WRB/CAML receptor fragments\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (PLA, in vitro insertion, transport assay, dominant-negative inhibition) establishing the TRC40–emerin targeting mechanism and its disease relevance\",\n      \"pmids\": [\"26675233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Functional proteoliposomes reconstituted from microsomal detergent extracts lost TA protein insertion activity when depleted of TRC40-associated proteins or CAML itself; in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes, defining the minimal mammalian receptor for TRC40-mediated TA insertion; CAML is present in ~5-fold molar excess over WRB and they mutually regulate each other's levels.\",\n      \"method\": \"Reconstituted functional proteoliposomes from detergent extract + immunodepletion + in vitro insertion assay + quantitative Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full reconstitution from defined components defining minimal mammalian receptor, single lab but rigorous\",\n      \"pmids\": [\"27226539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CAML, in the presence of sufficient WRB, is inserted into the ER membrane with three transmembrane segments in its C-terminal region; without sufficient WRB, CAML fails to adopt correct topology, generating aberrant topoforms that aggregate at ER-associated clusters and are degraded by the proteasome; WRB acts catalytically to assist CAML topogenesis.\",\n      \"method\": \"Topology mapping assays + proteasome inhibitor experiments + ER localization imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — topology and degradation assays with mechanistic interpretation, single lab\",\n      \"pmids\": [\"31417168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The small molecule Retro-2 blocks delivery of newly synthesized TA proteins to the ER-targeting factor ASNA1 (TRC40); a single ASNA1 point mutant identified by CRISPR-mediated mutagenesis abolishes both Retro-2's cytoprotective effect against ricin and its inhibitory effect on ASNA1-mediated ER targeting, demonstrating that Retro-2 acts directly on the ASNA1-dependent TA protein targeting step to prevent retrograde trafficking of ricin.\",\n      \"method\": \"CRISPRi genetic interaction screen + cell-based TA protein targeting assay + in vitro ASNA1 interaction assay + CRISPR point mutant resistance mapping\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic interaction screen combined with cell-based and in vitro assays and CRISPR point mutant validation, multiple orthogonal approaches\",\n      \"pmids\": [\"31674906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A dominant-negative ATPase-impaired TRC40 mutant (D74E) traps TA protein clients in the cytoplasm; manipulation of the hydrophobic TA-binding groove reduces interaction with most but not all substrates; identified known and novel TRC40 substrates including Golgi-resident TA proteins (golgin-84, CASP, giantin) and VAPA/VAPB by quantitative mass spectrometry.\",\n      \"method\": \"Dominant-negative TRC40(D74E) trap + quantitative mass spectrometry + groove mutant analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative trap with quantitative proteomics identifying substrate spectrum, single lab\",\n      \"pmids\": [\"31182645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Activation of Get3's chaperone function follows a multi-step process: reactivity of two conserved cysteines is directly controlled by Get3's nucleotide-binding state; thiol oxidation causes local unfolding and transition into chaperone-active oligomers; inactivation requires cysteine reduction followed by ATP binding, enabling transfer of client proteins to ATP-dependent chaperones for refolding; disrupting this cycle in yeast impairs oxidative stress resistance.\",\n      \"method\": \"Biochemical redox assays + mutagenesis of conserved cysteines + in vitro chaperone activity assays + yeast genetic phenotypic analyses\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — detailed mechanistic dissection with mutagenesis, biochemical assays, and in vivo genetic validation defining the ordered activation/inactivation cycle\",\n      \"pmids\": [\"35839781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of Giardia Get3 in five states (apo-open, apo-closed, ATP-bound, ADP-bound post-hydrolysis, and with TA client) showed that after ATP hydrolysis Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix; the ATP-bound state stabilizes an occluded CBD configuration, resolving how nucleotide drives conformational transitions across the full targeting cycle.\",\n      \"method\": \"Cryo-EM structure determination of Get3 in five nucleotide/conformational states (including Get3-client complex)\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — five structures spanning the complete conformational landscape including a client-bound post-hydrolysis state, resolving key mechanistic unknowns\",\n      \"pmids\": [\"35851188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure (3.2 Å) of the S. cerevisiae Get3–Get4/5 complex showed that Get4/5 remodels Get3's TA-binding chamber by unfolding helices forming the lateral walls ('lateral gate'), making the chamber more solvent accessible; mutagenesis of lateral gate residues influenced Get3 binding affinity for Get4/5 and its ATPase activity; the Sgt2-binding domain of Get5 is positioned near the lateral gate opening, supporting a model of lateral TA transfer from Sgt2 to Get3.\",\n      \"method\": \"Cryo-EM structure determination + molecular dynamics simulation + site-directed mutagenesis + ATPase activity assay + binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure combined with MD simulation, mutagenesis, and functional assays in single study\",\n      \"pmids\": [\"40902977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASNA1 contains a non-canonical FFAT-like motif that mediates direct interaction with the MSP domain of the ER membrane protein VAPB, physically linking the TRC40/ASNA1 TA-targeting complex to VAPB at the ER membrane.\",\n      \"method\": \"Co-immunoprecipitation + motif identification + direct binding assay\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and direct binding assay identifying FFAT-like motif-mediated interaction, single lab\",\n      \"pmids\": [\"24885147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASNA-1 exists in two redox states that control distinct functions: the reduced state mediates cisplatin resistance and TA protein targeting; an ASNA-1 point mutant preferentially in the oxidized state was sensitive to cisplatin and defective for TA protein targeting but showed normal insulin secretion; cisplatin-induced ROS drives ASNA-1 into the oxidized form and selectively prevents an ASNA-1-dependent TA substrate from reaching the ER.\",\n      \"method\": \"C. elegans genetics with redox-state point mutants + in vivo TA protein targeting assay + cisplatin sensitivity assay + ROS measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic point mutants with in vivo functional assays separating redox-state-dependent functions, single lab\",\n      \"pmids\": [\"33883621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Get3 biochemically interacts with the transmembrane domain proteins Get1/Mdm39 and Get2/Rmd7 in yeast; deletion of GET3 suppresses phenotypes of get1 and get2 mutants including sporulation defects; genetic interactions with NPL4 in the ubiquitin-proteasome system implicate Get3 in multiple membrane-dependent pathways.\",\n      \"method\": \"Co-immunoprecipitation + yeast genetic epistasis (suppressor analysis, double mutants)\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal biochemical interaction and epistasis data, single lab, pre-dates full GET pathway understanding\",\n      \"pmids\": [\"16816426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ASNA-1 functions nonautonomously to regulate insulin secretion in C. elegans; expressed in insulin-producing intestinal cells, asna-1 mutants show reduced insulin secretion while overexpression mimics insulin overexpression effects; human ASNA1 is highly expressed in pancreatic beta cells and regulates insulin secretion in cultured cells, demonstrating an evolutionarily conserved role in insulin secretion.\",\n      \"method\": \"C. elegans genetics (asna-1 null and overexpression) + insulin secretion assays in C. elegans + human ASNA1 knockdown/overexpression in cultured beta cells\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function and gain-of-function with insulin secretion readout in two organisms, single lab\",\n      \"pmids\": [\"17289575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Homozygous Asna1 knockout mice die between embryonic day 3.5 and 8.5, demonstrating that Asna1 is essential for early embryonic development in mammals.\",\n      \"method\": \"Homologous recombination knockout mouse generation + embryonic lethality analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockout with defined lethal phenotype, but no molecular mechanism identified beyond essentiality\",\n      \"pmids\": [\"16797549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Beta-cell-specific inactivation of Asna1 in mice caused hypoinsulinemia, impaired insulin secretion, and rapidly progressive diabetes; Asna1 loss perturbed plasma membrane-to-TGN and Golgi-to-ER retrograde transport and caused ER stress in beta cells; pharmacological inhibition of retrograde transport in isolated islets mimicked the Asna1 loss-of-function phenotype, linking Asna1 function to retrograde vesicular transport and ER homeostasis.\",\n      \"method\": \"Conditional knockout mouse model (beta-cell-specific) + pharmacological retrograde transport inhibition + ER stress markers\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined phenotype and pharmacological mimicry linking mechanism, single lab\",\n      \"pmids\": [\"26438609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Asna1 inactivation in pancreatic multipotent progenitor cells (MPCs) caused redistribution of Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, integrated stress response activation, and p53-mediated apoptosis leading to pancreatic agenesis; rescue experiments showed the Asna1 ATPase activity and a CXXC di-cysteine motif are required for Golgi integrity and MPC survival; ex vivo inhibition of retrograde transport reproduced the Golgi and syntaxin phenotypes.\",\n      \"method\": \"Conditional knockout mouse model + rescue experiments with ATPase-dead and CXXC mutants + pharmacological retrograde transport inhibition ex vivo + p53 modulator experiments\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with domain-specific rescue experiments and pharmacological phenocopy, single lab\",\n      \"pmids\": [\"29180572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"All three HSV1 tail-anchored proteins (pUL34, pUL56, pUS9) specifically bound to Asna1/TRC40 by yeast two-hybrid; TRC40 depletion by RNAi did not affect virion entry, viral gene expression, or secondary envelopment but specifically reduced release of infectious virions to the extracellular medium by more than 10-fold, identifying a role for TRC40 in viral egress.\",\n      \"method\": \"Yeast two-hybrid protein interaction + siRNA depletion + viral replication and release assays\",\n      \"journal\": \"Virology journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — interaction identified by Y2H and functional consequence defined by RNAi depletion with specific viral release readout, single lab\",\n      \"pmids\": [\"27765046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GET3 (TRC40/ASNA1) directly interacts with the anti-apoptotic protein MCL1 via MCL1's C-terminal hydrophobic tail; GET3 depletion reduced MCL1 protein levels while GET3 overexpression increased them; GET3 deficiency enhanced apoptosis and reduced clonogenic survival, particularly in HeLa cells, and accelerated MCL1 downregulation and apoptosis during prolonged mitotic arrest, identifying MCL1 as a TA-containing cargo of GET3.\",\n      \"method\": \"Degron-mediated GET3 depletion + co-immunoprecipitation of GET3–MCL1 + apoptosis assays + clonogenic survival assay + GET3 overexpression\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — degron depletion combined with co-IP establishing direct interaction, two orthogonal functional readouts, single lab\",\n      \"pmids\": [\"42098326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Constitutive cardiomyocyte-specific Asna1 knockout caused ventricular myocardial thinning by E16.5 and perinatal lethality; inducible adult cardiomyocyte-specific deletion caused rapid ventricular dilation and early mortality; ASNA1 deficiency destabilized the pre-targeting complex and reduced expression of multiple TA protein substrates, impairing membrane trafficking and vesicular transport; transcriptomic analysis revealed compensatory upregulation of Golgi-to-ER transport genes.\",\n      \"method\": \"Constitutive and inducible cardiomyocyte-specific conditional knockout mouse models + TA protein substrate expression analysis + transcriptomics\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two conditional KO models with defined molecular mechanism (substrate destabilization), single lab\",\n      \"pmids\": [\"41370295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Neuron-specific deletion of ASNA1 in mice (using SLICK-H-Cre or synapsin-Cre) phenocopied CAML neuron-specific deletion, causing loss of motor neuron cell bodies, hind limb weakness, and paralysis; identifying a cell-autonomous role for the ASNA1/TRC40 TA protein insertion machinery in motor neuron survival.\",\n      \"method\": \"Neuron-specific conditional knockout mouse (synapsin-Cre and SLICK-H-Cre) + spinal cord histology\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular phenotype establishing cell-autonomous neuronal role, single lab\",\n      \"pmids\": [\"39823474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Oxidized TRC40 (human Get3/ASNA1) forms chaperone-active tetramers and high-molecular-weight complexes that prevent aggregation of unfolding proteins; acute oxidative stress causes reversible formation of distinct TRC40 foci co-localizing with Hsp70 and Hsp110; TRC40 is essential for cell survival under ATP-depleting oxidative stress conditions, counteracting accumulation of mis- and unfolded proteins.\",\n      \"method\": \"Biochemical chaperone activity assay + native PAGE oligomer analysis + live-cell fluorescence imaging of TRC40 foci + genetic depletion under oxidative stress\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in human cells extending the chaperone function to TRC40, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"GET3/TRC40/ASNA1 is a conserved homodimeric ATPase that serves as the central cytosolic targeting factor of the GET/TRC pathway, using nucleotide-driven open-to-closed conformational changes to capture the transmembrane domain of tail-anchored (TA) proteins in a hydrophobic groove spanning the dimer interface, shield it from the cytosol, and deliver it to the ER membrane receptor (Get1/Get2 in yeast; WRB/CAML in mammals) where ATP hydrolysis and receptor-induced reopening of Get3 drive TA protein membrane insertion; Get4/5 upstream regulates Get3 by promoting the ATP-bound substrate-capture state and facilitating lateral TA handoff from the cochaperone Sgt2; under oxidative stress, Get3 undergoes a redox switch (disulfide bond formation at conserved cysteines, controlled by nucleotide state) that converts it from an ATP-dependent targeting factor into an ATP-independent holdase chaperone protecting against protein aggregation, with both functions being mutually exclusive and the chaperone state being fully reversible upon reduction and ATP rebinding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GET3 (TRC40/ASNA1) is a conserved homodimeric ATPase that serves as the central cytosolic targeting factor of the GET/TRC pathway for post-translational delivery of tail-anchored (TA) membrane proteins to the ER [#1, #7]. Nucleotide state drives an open-to-closed conformational cycle: in the closed, ADP·AlF4\\u2212-bound state the dimer forms a large hydrophobic groove spanning the interface that captures and shields the TA transmembrane helix, while the open state disrupts this groove to release substrate [#0, #4, #26]. A single TA protein binds one Get3 homodimer, with the \\u03b1-helical/finger subdomain forming the binding surface [#1, #3]. Get3 is primed for substrate capture by the upstream Get4\\u2013Get5 complex, which interacts with Get3 in a nucleotide-dependent manner, promotes the ATP-bound configuration optimal for capture, and remodels the lateral walls of the binding chamber to facilitate hand-off of TA substrate from the cochaperone Sgt2 [#9, #16, #27]. Loaded Get3 docks onto the ER membrane receptor \\u2014 Get1/Get2 in yeast, WRB/CAML in mammals \\u2014 which remodels Get3 toward the open state to drive insertion; ATP hydrolysis promotes efficient insertion and Get3 release from the receptor [#2, #3, #8, #12, #21]. Substrates extend beyond classical TA proteins to Golgi-resident TA proteins, SNAREs, VAPA/VAPB, the inner nuclear membrane protein emerin, short secretory precursors, and the anti-apoptotic protein MCL1 [#17, #20, #24, #36]. Independently of its targeting role, Get3 acts as an ATP-independent holdase chaperone: oxidative stress triggers nucleotide-controlled disulfide formation at conserved cysteines, metal release, and assembly into higher-order chaperone-active oligomers that protect against protein aggregation, a state mutually exclusive with targeting and reversed by reduction and ATP rebinding [#14, #25]. In mammals ASNA1 is essential for early embryogenesis [#32], and TRC40-mediated insertion of emerin links the pathway to Emery-Dreifuss muscular dystrophy, as EDMD-causing emerin mutations disrupt TRC40 binding and membrane integration [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Before the GET pathway was defined, it was unknown how Get3 connected physically and genetically to the ER membrane machinery; this established Get3 as a partner of the Get1/Get2 transmembrane proteins.\",\n      \"evidence\": \"Co-immunoprecipitation and yeast genetic epistasis (suppressor and double-mutant analysis)\",\n      \"pmids\": [\"16816426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural basis for the interaction\", \"Mechanism of TA insertion not yet addressed\", \"Predates identification of TA proteins as substrates\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The physiological requirement for the mammalian ortholog was unknown; knockout established Asna1 as essential for early mammalian development.\",\n      \"evidence\": \"Homologous recombination Asna1 knockout mouse with embryonic lethality analysis\",\n      \"pmids\": [\"16797549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism for lethality identified\", \"Does not distinguish targeting from chaperone roles\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"It was unclear whether ASNA1 had organismal physiological roles beyond a housekeeping function; this revealed a conserved nonautonomous role in insulin secretion.\",\n      \"evidence\": \"C. elegans loss- and gain-of-function genetics with insulin secretion readouts plus human beta-cell knockdown/overexpression\",\n      \"pmids\": [\"17289575\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between TA targeting and insulin secretion unresolved\", \"Direct secretory substrates not identified at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"How Get3 binds and releases TA substrates was unknown; structures of open and closed dimer states defined a nucleotide-regulated hydrophobic groove at the dimer interface as the TA-binding site.\",\n      \"evidence\": \"X-ray crystallography of open (nucleotide-free) and closed (ADP\\u00b7AlF4\\u2212) states with mutagenesis of groove residues, across multiple yeast/fungal/archaeal orthologs and H/D exchange MS\",\n      \"pmids\": [\"19675567\", \"19956640\", \"19706470\", \"19948960\", \"20015340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the functional Get3\\u2013TA complex not yet defined\", \"Direct visualization of a bound TA helix lacking\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Whether Get3 alone could insert TA proteins and what upstream factors regulated it were open; reconstitution showed receptor- and nucleotide-dependent insertion, and Get4/5 was placed upstream of Get3.\",\n      \"evidence\": \"In vitro reconstitution of TA insertion from recombinant components and ER membranes, plus crystallography and nucleotide-dependent binding assays of the Get4/5 complex\",\n      \"pmids\": [\"20375064\", \"20554915\", \"20106980\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of Get3\\u2013Get4/5 assembly unresolved\", \"Mechanism of Sgt2-to-Get3 hand-off not yet defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The identity of the mammalian receptor and how the receptor remodels Get3 were unknown; WRB was identified as the mammalian receptor and Get1 structures showed how it stabilizes the open Get3 state.\",\n      \"evidence\": \"Crystallography of Get3\\u2013Get1/Get2 complexes, biophysical (ITC/SAXS) stoichiometry of Get3\\u2013Get4/5, and biochemical/imaging identification of WRB\",\n      \"pmids\": [\"21719644\", \"22684149\", \"22190685\", \"21444755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full mammalian receptor composition not yet established\", \"Order of conformational events during insertion incompletely defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"How Get4 regulates Get3's catalytic cycle was unresolved; the ternary Get3\\u2013Get4\\u2013Get5 structure showed Get4 primes Get3 into the optimal substrate-capture configuration and regulates ATP hydrolysis.\",\n      \"evidence\": \"X-ray crystallography of the ATP-bound ternary complex with structure-guided mutagenesis and TA targeting assays\",\n      \"pmids\": [\"24727835\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lateral transfer geometry from Sgt2 not yet visualized\", \"Coupling of priming to nucleotide turnover incompletely defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether Get3 had functions beyond targeting was unknown; oxidative stress was shown to convert it into an ATP-independent holdase chaperone via disulfide formation and oligomerization, mutually exclusive with targeting.\",\n      \"evidence\": \"In vitro chaperone assays, disulfide-disrupting mutagenesis, energy-depletion imaging, and yeast oxidative-stress phenotypes\",\n      \"pmids\": [\"25242142\", \"23203805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ordered redox activation steps not yet defined\", \"Client spectrum of the chaperone state unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The composition of the functional targeting complex and the physiological substrate range were unclear; reconstitution fixed the 1 TA : 1 Get3 dimer stoichiometry and crystal structures resolved TA-helix recognition, while emerin established disease relevance.\",\n      \"evidence\": \"In vitro reconstitution plus crystallography of Get3\\u2013TA complexes; proximity ligation, in vitro insertion, and transport assays for emerin\",\n      \"pmids\": [\"25745174\", \"26675233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full client repertoire still being defined\", \"Mechanism linking emerin mistargeting to muscular dystrophy pathology not detailed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The minimal mammalian receptor was undefined; reconstitution showed WRB and CAML together are sufficient and necessary for TRC40-mediated insertion, with CAML in molar excess and mutual level regulation.\",\n      \"evidence\": \"Functional proteoliposome reconstitution from detergent extract with immunodepletion, in vitro synthesized components, and quantitative Western blot\",\n      \"pmids\": [\"27226539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometric architecture of the WRB/CAML receptor in membranes unresolved\", \"Mechanism of mutual level regulation not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The full conformational cycle and substrate spectrum of the targeting factor were incompletely resolved; cryo-EM across five states and dominant-negative proteomics defined nucleotide-driven CBD reorganization and broadened the client list to Golgi TA proteins and VAPA/VAPB.\",\n      \"evidence\": \"Cryo-EM of Get3 in five nucleotide/conformational states; dominant-negative TRC40(D74E) trap with quantitative mass spectrometry; CRISPR-based Retro-2 target validation\",\n      \"pmids\": [\"35851188\", \"31182645\", \"31674906\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate selectivity rules not fully defined\", \"Some substrates persist despite groove mutation, implying additional binding determinants\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"How the redox switch is ordered and reset was unknown; the activation cycle was resolved as nucleotide-controlled cysteine reactivity, oxidation-induced unfolding into chaperone oligomers, and reduction-plus-ATP-driven inactivation enabling client hand-off.\",\n      \"evidence\": \"Biochemical redox and chaperone assays, conserved-cysteine mutagenesis, and yeast oxidative-stress genetics\",\n      \"pmids\": [\"35839781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of downstream ATP-dependent chaperones receiving clients not defined\", \"Structural basis of the chaperone-active oligomer unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"How Get4/5 enables lateral TA hand-off from Sgt2 was unresolved; the cryo-EM Get3\\u2013Get4/5 structure showed remodeling of a 'lateral gate' that opens the binding chamber adjacent to the Sgt2-binding domain of Get5.\",\n      \"evidence\": \"Cryo-EM, molecular dynamics, mutagenesis, ATPase and binding assays of the S. cerevisiae Get3\\u2013Get4/5 complex\",\n      \"pmids\": [\"40902977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"A captured Sgt2\\u2013Get3 transfer intermediate not yet visualized\", \"Timing of lateral gate opening relative to nucleotide turnover unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The tissue-specific physiological consequences of losing the targeting machinery were unclear; cardiomyocyte- and neuron-specific knockouts showed cell-autonomous requirements linked to TA substrate destabilization and impaired membrane trafficking.\",\n      \"evidence\": \"Constitutive and inducible conditional knockout mouse models with substrate expression analysis, transcriptomics, and spinal cord histology\",\n      \"pmids\": [\"41370295\", \"39823474\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific TA substrates driving each tissue phenotype not pinpointed\", \"Relative contribution of targeting versus chaperone roles to phenotypes unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Whether GET3 regulates apoptotic machinery as cargo was unknown; MCL1 was identified as a TA-containing client whose levels and pro-survival function depend on GET3.\",\n      \"evidence\": \"Degron-mediated GET3 depletion, co-immunoprecipitation, apoptosis and clonogenic survival assays\",\n      \"pmids\": [\"42098326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MCL1 is inserted via the canonical WRB/CAML receptor not shown\", \"In vivo relevance of GET3\\u2013MCL1 axis to tumor survival unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the cell switches Get3 between its targeting and holdase chaperone modes in vivo, and which downstream chaperones and physiological substrates govern recovery from stress, remains to be defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the chaperone-active oligomer\", \"Substrate hand-off partners during stress recovery not identified\", \"Quantitative balance between targeting and chaperone pools in tissues unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 3, 7, 16, 26]},\n      {\"term_id\": \"GO:0016887\", \"supporting_discovery_ids\": [0, 3, 16]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [14, 15, 25]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [1, 7, 17]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [14, 25, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 7, 15]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 7, 8, 21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 3, 7, 20]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 7, 16]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [14, 25]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [33, 34]}\n    ],\n    \"complexes\": [\n      \"Get3-Get4-Get5 pre-targeting complex\",\n      \"Get3-Get1/Get2 receptor complex\",\n      \"TRC40-WRB/CAML receptor complex\"\n    ],\n    \"partners\": [\n      \"GET4\",\n      \"GET5\",\n      \"GET1\",\n      \"GET2\",\n      \"WRB\",\n      \"CAML\",\n      \"VAPB\",\n      \"SGT2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}