{"gene":"ALG2","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1996,"finding":"ALG-2 is a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death; depletion of ALG-2 protects T cell hybridoma from apoptosis, placing ALG-2 as a required mediator of Ca2+-regulated death signals.","method":"Functional selection/death trap assay, antisense depletion with apoptosis readout","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — foundational loss-of-function study with defined cellular phenotype, highly cited, replicated","pmids":["8560270"],"is_preprint":false},{"year":1997,"finding":"ALG-2 functions downstream of ICE/Ced-3 caspase activation in apoptosis; ALG-2-depleted cells still activate caspases normally (cleave PARP) yet do not die, placing ALG-2 downstream of or independent of caspases.","method":"ALG-2 antisense depletion combined with fluorogenic and PARP substrate caspase activity assays","journal":"Journal of Immunology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with defined biochemical readouts, replicated in original lab","pmids":["9164928"],"is_preprint":false},{"year":1999,"finding":"ALG-2 directly interacts with AIP1 (Alix) in a strictly Ca2+-dependent manner; both proteins co-localize in the cytosol, and overexpression of a truncated AIP1 protects cells from trophic-factor withdrawal death.","method":"Yeast two-hybrid screen, co-immunoprecipitation, subcellular co-localization","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction validated by multiple methods, Ca2+ dependence established, replicated in subsequent papers","pmids":["9880530"],"is_preprint":false},{"year":1999,"finding":"ALG-2 binds to itself (Ca2+-independent homodimerization) and to Alix (Ca2+-dependent); the fifth EF-hand participates in dimer formation as shown by biochemical and gel-filtration analysis.","method":"Yeast two-hybrid, co-immunoprecipitation, gel filtration","journal":"Cell Death and Differentiation","confidence":"High","confidence_rationale":"Tier 2 — two orthogonal methods, confirmed by subsequent structural studies","pmids":["10200558"],"is_preprint":false},{"year":1998,"finding":"Ca2+ binding induces exposure of a hydrophobic surface on ALG-2 in a concentration-dependent manner (~6 µM half-maximal), a conformational change that is Mg2+-insensitive and drives association with macromolecules in cells.","method":"Fluorescent hydrophobicity probe (TNS) spectroscopy, recombinant protein characterization","journal":"Journal of Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro biochemical reconstitution with recombinant protein","pmids":["9832622"],"is_preprint":false},{"year":1999,"finding":"ALG-2 possesses two high-affinity Ca2+-binding sites and one low-affinity site; Ca2+ binding to both strong sites is required for Ca2+-induced aggregation; the fifth EF-hand mediates homodimerization.","method":"Gel filtration, chemical cross-linking, mutagenesis (E47A/E114A), fluorescence spectroscopy, circular dichroism","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro assay with mutagenesis and multiple spectroscopic methods","pmids":["10360947"],"is_preprint":false},{"year":2000,"finding":"Two alternatively spliced isoforms of ALG-2 exist (ALG-2,5 and ALG-2,1, differing by Gly121-Phe122); ALG-2,1 lacks the ability to interact with AIP1 and has a higher Ca2+-binding affinity, demonstrating that two residues govern target specificity.","method":"Yeast two-hybrid, Ca2+ binding assays, sequence analysis of isoforms","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical assays plus mutagenic isoform comparison, confirmed by structural studies","pmids":["10744743"],"is_preprint":false},{"year":2001,"finding":"X-ray crystal structure of Ca2+-loaded ALG-2 (des1-20, 2.3 Å) reveals five EF-hand fold with Ca2+ bound to EF1, EF3, and EF5; Ca2+ induces a rigid-body rotation of EF1-2 relative to EF4-5, exposing a hydrophobic cleft that accepts a Gly/Pro-rich peptide.","method":"X-ray crystallography at 2.3 Å resolution","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure","pmids":["11525164"],"is_preprint":false},{"year":2001,"finding":"Peflin forms a heterodimer with ALG-2 in Jurkat cells; the heterodimer dissociates upon Ca2+ addition, and peflin translocates to membranes/cytoskeleton in the presence of Ca2+, while ALG-2 persists in both cytoplasm and nucleus.","method":"Co-immunoprecipitation, gel filtration, immunofluorescence, subcellular fractionation","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, endogenous protein interaction","pmids":["11278427"],"is_preprint":false},{"year":2002,"finding":"ALG-2 directly interacts with the N-terminal domain of annexin XI (and similarly annexin VII) in a Ca2+-dependent manner; dissociation constants ~70 nM (high affinity) and ~500–700 nM (low affinity) were measured.","method":"Yeast two-hybrid, biotin-ALG-2 overlay assay, surface plasmon resonance (SPR)","journal":"Biochemical and Biophysical Research Communications / Biochimica et Biophysica Acta","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding demonstrated by SPR with kinetics and mutagenesis","pmids":["11883939","12445460"],"is_preprint":false},{"year":2002,"finding":"ALG-2 interacts with the C-terminus of ASK1 in a Ca2+-dependent and isoform-specific manner (ALG-2,1 does not bind); co-transfection of ALG-2 causes nuclear redistribution of ASK1 and suppresses ASK1-mediated JNK activation.","method":"Co-immunoprecipitation, in vitro binding, cotransfection/subcellular localization, JNK reporter assay","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 — direct interaction plus functional readout, single lab","pmids":["12372597"],"is_preprint":false},{"year":2002,"finding":"The fifth EF-hand of ALG-2 (and peflin) is essential for dimerization and protein stability; deletion of EF5 leads to rapid proteasomal degradation of the monomer.","method":"Exogenous expression of deletion mutants, pulse-chase, proteasome inhibitor (MG132) rescue, Western blot","journal":"Archives of Biochemistry and Biophysics","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with defined degradation phenotype, single lab","pmids":["11883899"],"is_preprint":false},{"year":2003,"finding":"Human ALG2 (hALG2) is an α1,3-mannosyltransferase that elongates Man1GlcNAc2-PP-dolichol; deficiency causes CDG-Ii with accumulation of Man1 and Man2 LLO intermediates. Expression of wild-type but not mutant hALG2 rescues both patient fibroblasts and yeast alg2-1 cells.","method":"Mannosyltransferase activity assay in patient fibroblast extracts, complementation of yeast alg2-1, genetic analysis of patient mutations","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — enzymatic assay with patient mutations and genetic complementation in two systems","pmids":["12684507"],"is_preprint":false},{"year":2004,"finding":"ALG-2 interacts with the ESCRT-I component TSG101 directly via its Pro-rich region in a Ca2+-dependent manner; indirect Ca2+-dependent association with hVps28 and hVps37A occurs through TSG101. ALG-2 co-localizes with aberrant endosomes (SKD1 dominant-negative) in a Ca2+-dependent fashion.","method":"GST pulldown, yeast two-hybrid, biotin-ALG-2 overlay assay, immunofluorescence with Ca2+ chelator (BAPTA)","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding demonstrated by overlay assay plus multiple interaction methods, subcellular localization linked to Ca2+","pmids":["16004603"],"is_preprint":false},{"year":2004,"finding":"ALG-2 interacts directly with the Alix C-terminal Pro-rich region (residues 794–827) via four tandem PxY repeats; alanine substitution of critical Pro and Tyr residues ablates binding. ALG-2 is required for the punctate subcellular distribution of Alix.","method":"Yeast two-hybrid, biotin-ALG-2 overlay, co-immunoprecipitation, Ca2+-binding-deficient mutant, fluorescence microscopy","journal":"Journal of Biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding motif defined by mutagenesis, confirmed by multiple methods","pmids":["14999017"],"is_preprint":false},{"year":2004,"finding":"Yeast Alg1, Alg2, and Alg11 mannosyltransferases form distinct physical complexes in the ER membrane; Alg1 self-assembles via a C-terminal domain distinct from its Alg2/Alg11 interaction domain; catalytically inactive Alg1 alleles cause dominant-negative phenotypes consistent with functional complex assembly.","method":"Co-immunoprecipitation, genetic complementation, dominant-negative analysis in S. cerevisiae","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 2 — biochemical and genetic epistasis in multiple assays, yeast ortholog of human ALG2","pmids":["15044395"],"is_preprint":false},{"year":2006,"finding":"ALG-2 is recruited to ER exit sites (ERES) by Ca2+-dependent binding to Sec31A (a COPII outer coat component); ALG-2 in turn stabilizes Sec31A at ERES. Ca2+-binding-deficient ALG-2 (E47A/E114A) fails to localize to ERES and fails to stabilize Sec31A.","method":"Co-immunoprecipitation, RNAi, Ca2+ ionophore (A23187) and chelator (BAPTA-AM) treatment, immunofluorescence confocal microscopy, GST pulldown","journal":"Molecular Biology of the Cell / Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, functional consequence of localization established, replicated across two labs","pmids":["16957052","17196169"],"is_preprint":false},{"year":2006,"finding":"In vitro reconstitution demonstrates that S. cerevisiae Alg2 is a bifunctional enzyme catalyzing sequential α1,3- then α1,6-mannosylation to form the first branched pentasaccharide (Man3GlcNAc2-PP-Dol) intermediate in the dolichol pathway.","method":"In vitro mannosyltransferase assay with recombinant Alg2 and defined dolichylpyrophosphate-linked substrates from E. coli overexpression","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified enzyme and defined substrates","pmids":["16878994"],"is_preprint":false},{"year":2006,"finding":"ALG-2 subcellular localization oscillates in synchrony with intracellular Ca2+ oscillations induced by physiological stimuli (ATP, EGF, prostaglandin, histamine); Ca2+-binding-deficient ALG-2 does not redistribute, linking ALG-2 dynamics to Ca2+ signaling.","method":"Live-cell imaging of tagged ALG-2 with simultaneous Ca2+ indicator, Ca2+-binding mutant comparison","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 2 — live-cell imaging with functional mutant control, directly links localization to Ca2+ signaling","pmids":["17214967"],"is_preprint":false},{"year":2006,"finding":"Nuclear translocation of ALG-2 is induced by the RNA-binding protein RBM22; when co-expressed, ALG-2 translocates to the nucleus where RBM22 is located, indicating RBM22 drives ALG-2 nuclear import.","method":"Fluorescent fusion protein co-expression, confocal microscopy in NIH 3T3 cells and zebrafish embryos","journal":"Biochimica et Biophysica Acta","confidence":"Medium","confidence_rationale":"Tier 2 — live-cell imaging with two model systems, single lab","pmids":["17045351"],"is_preprint":false},{"year":2008,"finding":"Alix and ALG-2 interact with pro-caspase-8 in a Ca2+-dependent manner and form a complex with TNF-R1 on endosomes; deletion of the ALG-2-binding site in Alix significantly reduces TNF-R1-induced cell death without affecting receptor endocytosis.","method":"Mass spectrometry of co-immunoprecipitates, co-immunoprecipitation, deletion mutant Alix (ΔAlg-2 binding site), cell death assay","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — complex formation identified by MS and confirmed by co-IP, functional consequence established by deletion mutant","pmids":["18936101"],"is_preprint":false},{"year":2009,"finding":"ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101: Alix requires its ALG-2-binding site (not its PSAP-TSG101 binding motif) for Ca2+-dependent pulldown of TSG101; ALG-2 knockdown abolishes the association; the ALG-2 dimer bridges the two binding partners.","method":"Strep-tag pulldown assays, ALG-2 knockdown, recombinant ALG-2 add-back, dimerization-defective mutant","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 1–2 — reconstitution with purified protein, knockdown rescue, mutant analysis","pmids":["19520058"],"is_preprint":false},{"year":2009,"finding":"ALG-2 directly binds the NH2-terminal cytosolic tail of mucolipin-1 (MCOLN1) in a strictly Ca2+-dependent manner via residues 37–49 of MCOLN1; ALG-2 regulates MCOLN1 function as mutation of the ALG-2-binding domain in MCOLN1 reduces MCOLN1-induced aberrant endosome aggregation.","method":"In vitro binding assay, co-localization, mutagenesis of MCOLN1 ALG-2-binding domain","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding demonstrated, functional mutagenesis with endosome phenotype readout","pmids":["19864416"],"is_preprint":false},{"year":2009,"finding":"Biochemical characterization of yeast Alg2 establishes it as a bifunctional α1,3- and α1,6-mannosyltransferase in ER LLO biosynthesis; topology analysis shows two N-terminal transmembrane spans anchor it to the ER; Lys230 is essential for catalytic activity while an EX7E motif is dispensable.","method":"In vitro mannosyltransferase assay, topology analysis, site-directed mutagenesis (K230, EX7E motif)","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro assay with site-directed mutagenesis identifying catalytic residue","pmids":["19282279"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the ALG-2(ΔGF122) isoform explains its inability to bind Alix: deletion of Gly121-Phe122 deforms the main-chain wall facing pockets 1 and 2 and repositions Arg125 to occlude pocket 1, not loss of the F122 side chain per se. F122 substitution with Ala or Gly (not Trp) actually increases Alix binding.","method":"X-ray crystallography, pulldown assays with F122 substitution mutants","journal":"BMC Structural Biology","confidence":"High","confidence_rationale":"Tier 1 — X-ray structure combined with mutagenesis/binding assays","pmids":["20691033"],"is_preprint":false},{"year":2010,"finding":"The ALG-2-binding site (ABS, residues 839–851) in Sec31A is necessary and sufficient for direct ALG-2 binding; FRAP analysis reveals that ABS deletion reduces the high-affinity population of Sec31A at ERES, indicating ALG-2 controls Sec31A retention kinetics.","method":"Biotin-ALG-2 overlay assay, stable GFP-ALG-2/Sec31A-RFP cell lines, Ca2+ mobilization, FRAP","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding motif defined, retention kinetics measured by FRAP","pmids":["20834162"],"is_preprint":false},{"year":2013,"finding":"ALG-2 attenuates COPII vesicle budding in vitro by binding to the Pro-rich region of Sec31A; this requires an intact EF-hand 1 Ca2+-binding site in ALG-2. ALG-2/Ca2+ increases recruitment of Sec23/24 and Sec13/31A to liposomes and mediates Sec31A-Sec23 bridging.","method":"In vitro COPII budding assay, liposome recruitment assay, EF1 mutant ALG-2","journal":"PLoS ONE","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstituted COPII budding assay with mutagenesis","pmids":["24069399"],"is_preprint":false},{"year":2013,"finding":"Nuclear ALG-2 interacts with CHERP (Ca2+ homeostasis ER protein) in a Ca2+-dependent manner at nuclear speckles; knockdown of CHERP or ALG-2 alters alternative splicing of IP3R1 pre-mRNA, and CHERP binds IP3R1 RNA. ALG-2 is recruited to CHERP-localizing nuclear speckles upon Ca2+ mobilization.","method":"Co-immunoprecipitation, live-cell time-lapse imaging, siRNA knockdown, RNA immunoprecipitation, RT-PCR for splicing isoforms","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, nuclear function linked to splicing regulation","pmids":["24078636"],"is_preprint":false},{"year":2013,"finding":"Mutations in ALG2 cause congenital myasthenic syndrome; the ALG2 p.Val68Gly mutation severely reduces ALG2 protein expression in patient muscle and cell cultures. ALG2 functions as an α1,3-mannosyltransferase in early steps of asparagine-linked glycosylation, critical for proper NMJ function.","method":"Linkage analysis, exome/genome sequencing, Western blot of patient tissue and transfected cells, SiRNA knockdown of ALG14 with AChR surface expression readout","journal":"Brain","confidence":"High","confidence_rationale":"Tier 2 — patient genetics with functional protein expression validation in cells and tissue","pmids":["23404334"],"is_preprint":false},{"year":2013,"finding":"VPS37B and VPS37C isoforms of ESCRT-I interact more strongly with ALG-2 than TSG101; purified recombinant ESCRT-I/ALIX/ALG-2 can form a ternary complex in vitro in a Ca2+-dependent manner.","method":"Far-Western blot with biotin-ALG-2, pulldown with recombinant proteins, in vitro reconstitution","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstitution of ternary complex, single lab","pmids":["23924735"],"is_preprint":false},{"year":2015,"finding":"X-ray crystal structure of ALG-2 bound to a Sec31A peptide (type 2 motif, PXPGF) shows binding to a third hydrophobic pocket (Pocket 3), distinct from the Pocket 1/2 used by Alix. F85A mutation abrogates Sec31A binding but not Alix binding; Y180A eliminates Alix binding but not Sec31A binding.","method":"X-ray crystallography of ALG-2/Sec31A peptide complex, alanine mutagenesis of pocket residues, pulldown binding assays","journal":"International Journal of Molecular Sciences","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis defining two independent binding surfaces","pmids":["25667979"],"is_preprint":false},{"year":2015,"finding":"Ca2+-dependent ALG-2 interaction with ALIX relieves ALIX intramolecular autoinhibition and promotes CHMP4-dependent ALIX membrane association, thereby enabling ALIX function in MVB sorting of ubiquitinated EGFR specifically (not cytokinesis or EIAV budding).","method":"Co-immunoprecipitation, membrane fractionation, EGFR MVB sorting assay, ALIX deletion and CHMP4-binding mutants, siRNA knockdown","journal":"Cell Discovery","confidence":"High","confidence_rationale":"Tier 2 — multiple functional assays distinguishing ALIX activities, mechanistic model with mutant validation","pmids":["27462417"],"is_preprint":false},{"year":2016,"finding":"ALG-2 promotes ER exit site (ERES) localization and Ca2+-dependent polymerization of TFG protein; ALG-2 overexpression accumulates TFG at ERES; deletion of TFG's ALG-2-binding motif shortens TFG half-life at ERES.","method":"Immunostaining, live-cell time-lapse imaging with Ca2+ mobilization, in vitro cross-linking polymerization assay, Co-IP","journal":"The FEBS Journal","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro polymerization assay plus live-cell imaging and binding motif mutant","pmids":["27813252"],"is_preprint":false},{"year":2017,"finding":"ALG-2 interacts Ca2+-dependently with MISSL, and together they regulate ER-to-Golgi transport; knockdown of either ALG-2 or MISSL similarly attenuates SEAP secretion and delays procollagen I ER-to-Golgi transport. ALG-2/MISSL interact with MAP1B, which negatively regulates this secretory pathway.","method":"Co-IP, live-cell imaging, siRNA knockdown, secreted alkaline phosphatase (SEAP) secretion assay, procollagen transport assay","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple functional readouts, pathway placement by double-knockdown epistasis","pmids":["28864773"],"is_preprint":false},{"year":2018,"finding":"MAP1B interacts with ALG-2 Ca2+-dependently via a unique mode (no canonical ABM-1/ABM-2 motif); MAP1B competes with ABM-2-containing proteins such as Sec31A for ALG-2 binding, and MAP1B overexpression disperses ALG-2 and Sec31A from ERES.","method":"Co-IP, pulldown with MAP1B mutants, co-localization microscopy, MAP1B knockout cells","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 — competitive binding shown with mutants, KO cell phenotype, single lab","pmids":["29432744"],"is_preprint":false},{"year":2018,"finding":"ALG-2 participates in plasma membrane repair after damage by electroporation or digitonin; ALG-2 KO cells are more sensitive to electroporation, reversible by ALG-2 re-expression; Ca2+-binding-deficient ALG-2 does not protect, and a blocking ALIX peptide abolishes protection.","method":"ALG-2 gene knockout (DT-40 cells), electroporation and digitonin survival assays, Ca2+-binding mutant and ALIX-peptide blocking experiments","journal":"PLoS ONE","confidence":"High","confidence_rationale":"Tier 2 — KO with rescue, Ca2+-binding requirement established, mechanism linked to ALIX interaction","pmids":["30240438"],"is_preprint":false},{"year":2020,"finding":"ALG-2 directly interacts with Rpn3, a component of the 26S proteasome; upon Ca2+ influx following T cell activation, ALG-2 regulates proteasome activity, which controls MCL1 stability and accelerates T cell apoptosis during immune contraction.","method":"Co-immunoprecipitation, proteasome activity assay, MCL1 stability assay, ALG-2 knockdown in T cells","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 — direct interaction identified, functional pathway placement in T cell apoptosis, single lab","pmids":["31919392"],"is_preprint":false},{"year":2020,"finding":"ALG-2 interacts with SARAF (a negative regulator of SOCE) Ca2+-dependently; ALG-2 overexpression interferes with SARAF ubiquitination, stabilizing SARAF, and promotes Ca2+-dependent SARAF dimerization through its ABM-2 motif.","method":"Pulldown assay, co-IP, ubiquitination assay, half-life analysis, site-directed mutagenesis of ABM-2","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — interaction and ubiquitination interference established with mutagenesis, single lab","pmids":["32878247"],"is_preprint":false},{"year":2020,"finding":"ALG-2 interacts with FasL (FASLG) and regulates its intracellular vesicle-mediated transport, thereby influencing T cell apoptosis via the FasL–Fas pathway.","method":"Co-immunoprecipitation, subcellular localization microscopy, ALG-2 knockdown with FasL trafficking readout","journal":"The Biochemical Journal","confidence":"Medium","confidence_rationale":"Tier 2 — interaction confirmed by Co-IP, vesicle trafficking phenotype shown, single lab","pmids":["32766719"],"is_preprint":false},{"year":2021,"finding":"ALG-2 suppresses STING-mediated type I interferon signaling by associating with the C-terminal tail of STING and inhibiting its trafficking from the ER to perinuclear vesicles; this requires intact Ca2+-coordinating residues in ALG-2; ALG-2 knockout increases IFN expression upon HSV-1 or cGAMP stimulation.","method":"ALG-2 knockout (THP-1 cells), co-immunoprecipitation, immunofluorescence trafficking assay, Ca2+-binding mutant ALG-2","journal":"Journal of Cell Science","confidence":"High","confidence_rationale":"Tier 2 — KO with defined trafficking phenotype, Ca2+-requirement established, direct interaction shown","pmids":["34787301"],"is_preprint":false},{"year":2021,"finding":"CDIP1 interacts with ALG-2 in a Ca2+-dependent manner; ALG-2 promotes CDIP1 association with ESCRT-I (specifically VPS37B/C-containing complexes); co-expression of ALG-2 and ESCRT-I enhances CDIP1-induced caspase-3/7 cell death.","method":"Co-immunoprecipitation, caspase-3/7 activity assay, ALG-2 and ESCRT-I co-expression experiments","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — interaction and functional consequence shown, single lab","pmids":["33503978"],"is_preprint":false},{"year":2021,"finding":"ALG-2 and peflin form a hetero-bifunctional COPII regulator; at steady-state Ca2+, ALG-2/peflin heterocomplexes bind ERES and buffer secretion; Ca2+ signaling shifts balance toward peflin-lacking ALG-2 complexes that stimulate secretion; ALG-2-dependent effects on secretion can be opposing depending on signal intensity and cell type.","method":"Live-cell imaging, siRNA knockdown, secretion assays (SEAP, collagen I), ERES fractionation, Ca2+ agonist treatments in NRK and PC12 cells","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple cell types, multiple cargos, mechanistic model supported by peflin/ALG-2 double-knockdown epistasis","pmids":["34762908"],"is_preprint":false},{"year":2022,"finding":"Oxidative stress-induced Ca2+ flux triggers JIP4 phosphorylation at T217 by CaMK2G, which activates a JIP4–TRPML1–ALG-2 pathway that promotes lysosomal retrograde transport (clustering near MTOC) and autophagy as a protective response.","method":"Knockout cells, phospho-specific mutants, lysosomal positioning assay, Ca2+ flux measurement, CaMK2G kinase assay","journal":"The EMBO Journal","confidence":"Medium","confidence_rationale":"Tier 2 — pathway placement by KO and phospho-mutants, single lab","pmids":["36394115"],"is_preprint":false},{"year":2022,"finding":"Human ALG2 prefers to transfer α1,3-mannose before α1,6-mannose onto M1GlcNAc2-PP-Dol under physiological conditions, but excess GDP-Man donor or elevated M1Gn2 substrate can shift it toward first producing the M2Gn2(α1,6) intermediate; single membrane-binding domain anchors hAlg2 to ER (differs from yeast 4-domain topology).","method":"LC-MS quantitative kinetics assay with purified hAlg2, topology analysis, defined LLO substrates","journal":"Communications Biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinetics with purified human enzyme and defined substrates, topology established","pmids":["35136180"],"is_preprint":false},{"year":2024,"finding":"Ca2+-dependent direct membrane binding by ALG-2 is mediated by electrostatic and hydrophobic interactions; charge-reversed ALG-2 mutants reduce ERES localization after thapsigargin-induced Ca2+ release but still localize to lysosomes (rescued by ESCRT-I binding); direct membrane binding and protein binding are mechanistically distinct pathways.","method":"GUV-based membrane binding experiments, molecular dynamics simulations, charge-reversal mutagenesis, in vitro reconstitution with ESCRT-I, live-cell ERES localization","journal":"Proceedings of the National Academy of Sciences of the USA","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution, MD simulations, mutagenesis and cellular validation","pmids":["38386713"],"is_preprint":false},{"year":2024,"finding":"ALG-2, upon lysosomal Ca2+ release, recruits ESCRT proteins to lysosomes and enhances lysosomal membrane resilience to osmotic stress; the ΔGF122 splice variant of ALG-2 that cannot bind ESCRTs does not confer this protection; activating TRPML1 without membrane damage is sufficient to recruit ALG-2/ESCRTs and protect lysosomes.","method":"Lysosomal leakage/rupture assays, Ca2+ chelation (BAPTA), ΔGF122 ALG-2 mutant, TRPML1 activation, immunofluorescence redistribution assay","journal":"Proceedings of the National Academy of Sciences of the USA","confidence":"High","confidence_rationale":"Tier 2 — functional KO-equivalent (splice-variant mutant), multiple mechanistic controls, replicated across two papers (peer-reviewed + preprint)","pmids":["38781205","38352356"],"is_preprint":false},{"year":2024,"finding":"ERESs undergo lysosome-dependent microautophagy triggered by nutrient stress (mTOR inhibition, amino acid starvation); this requires ubiquitinated SEC31, ALG-2, and ALIX; ALG-2 knockout or function-blocking ALIX mutations prevent ERES engulfment by lysosomes; the pathway was reconstituted in vitro with purified components on lysosomal lipid-mimicking GUVs.","method":"Super-resolution live-cell imaging, FIB-SEM, ALG-2 knockout, ALIX function-blocking mutants, in vitro GUV reconstitution","journal":"Developmental Cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus super-resolution imaging and KO, multiple orthogonal methods","pmids":["38593803"],"is_preprint":false}],"current_model":"ALG-2 (PDCD6) is a penta-EF-hand Ca2+-sensor protein that, upon Ca2+ binding, undergoes a conformational change exposing hydrophobic pockets and directly membrane-binding surfaces, enabling it to function as a Ca2+-dependent adaptor bridging COPII components (Sec31A, TFG, MISSL) at ER exit sites to regulate ER-to-Golgi secretion, to bridge ESCRT proteins (ALIX, TSG101, ESCRT-I) at endosomes and lysosomes to control MVB sorting, membrane repair, and lysosomal resilience, to activate ALIX for EGFR MVB sorting by relieving its autoinhibition, to regulate STING trafficking and innate immune signaling, to control alternative splicing of IP3R1 pre-mRNA via nuclear interaction with CHERP, and to modulate T cell apoptosis through interactions with caspase-8, FasL trafficking, and proteasome-regulated MCL1 stability; in addition, its glycosyltransferase function (human ALG2) catalyzes sequential α1,3- and α1,6-mannosylation steps in dolichol-linked oligosaccharide biosynthesis in the ER, and mutations in this activity cause congenital disorders of glycosylation and congenital myasthenic syndrome."},"narrative":{"teleology":[{"year":1996,"claim":"Establishing that ALG-2 is a Ca²⁺-binding protein required for programmed cell death resolved a key unknown—whether Ca²⁺-sensing proteins mediate receptor-triggered apoptosis—by showing that ALG-2 depletion protects T cells from TCR-, Fas-, and glucocorticoid-induced death.","evidence":"Functional death-trap selection and antisense depletion in T cell hybridoma with apoptosis readout","pmids":["8560270"],"confidence":"High","gaps":["Downstream targets of ALG-2 in apoptosis were unknown","Whether ALG-2 acts cell-autonomously or through secreted signals was unclear"]},{"year":1997,"claim":"Placing ALG-2 genetically downstream of caspase activation answered whether ALG-2 operated in the canonical caspase cascade, establishing it as a post-caspase or parallel effector rather than a caspase activator.","evidence":"ALG-2 antisense depletion with PARP cleavage and fluorogenic caspase substrate assays showing intact caspase activity but blocked death","pmids":["9164928"],"confidence":"High","gaps":["Identity of ALG-2's downstream effectors remained unknown","Whether ALG-2 acts on membrane integrity or nuclear apoptosis was unresolved"]},{"year":1999,"claim":"Discovery of the Ca²⁺-dependent ALG-2–ALIX interaction and ALG-2 homodimerization via the fifth EF-hand established the first protein partner and the oligomeric framework for ALG-2 function, answering how Ca²⁺ is transduced into protein–protein interactions.","evidence":"Yeast two-hybrid, co-immunoprecipitation, gel filtration, and Ca²⁺-binding mutagenesis (E47A/E114A)","pmids":["9880530","10200558","10360947"],"confidence":"High","gaps":["Structural basis of Ca²⁺-induced conformational change was unknown","Cellular function of the ALG-2–ALIX complex was not established"]},{"year":1998,"claim":"Demonstration that Ca²⁺ binding induces hydrophobic surface exposure on ALG-2 provided the biophysical mechanism by which Ca²⁺ triggers partner recruitment, answering why ALG-2 interactions are Ca²⁺-dependent.","evidence":"TNS fluorescence spectroscopy with recombinant ALG-2, showing half-maximal hydrophobic exposure at ~6 µM Ca²⁺","pmids":["9832622"],"confidence":"High","gaps":["Which specific hydrophobic surfaces were exposed was unresolved until crystallography"]},{"year":2001,"claim":"The crystal structure of Ca²⁺-loaded ALG-2 revealed Ca²⁺ occupancy at EF1, EF3, and EF5 and a rigid-body rotation exposing a hydrophobic cleft, providing the atomic-level explanation for Ca²⁺-dependent target recognition.","evidence":"X-ray crystallography at 2.3 Å resolution","pmids":["11525164"],"confidence":"High","gaps":["No co-crystal with any binding partner was available","How ALG-2 discriminates between different targets structurally was unknown"]},{"year":2001,"claim":"Discovery that peflin forms a Ca²⁺-sensitive heterodimer with ALG-2 that dissociates upon Ca²⁺ addition established an additional layer of regulation—ALG-2 is sequestered by peflin at low Ca²⁺ and liberated for target engagement at high Ca²⁺.","evidence":"Co-immunoprecipitation, gel filtration, and subcellular fractionation in Jurkat cells","pmids":["11278427"],"confidence":"High","gaps":["Functional consequence of peflin–ALG-2 heterodimer dissociation for any specific cellular process was unknown"]},{"year":2003,"claim":"Identification of hALG2 as an α1,3-mannosyltransferase deficient in CDG-Ii patients—rescued by wild-type expression in both human fibroblasts and yeast—established the enzymatic function and disease relevance of the glycosyltransferase ALG2, distinct from the PDCD6 Ca²⁺-sensor.","evidence":"Mannosyltransferase activity assay in patient fibroblast extracts and complementation of yeast alg2-1","pmids":["12684507"],"confidence":"High","gaps":["Whether ALG2 catalyzes one or both mannosylation steps was unresolved","Structural basis of catalysis was unknown"]},{"year":2004,"claim":"Mapping the ALG-2–ALIX binding interface to four PxY repeats in ALIX's Pro-rich region, and showing ALG-2 binds TSG101 and co-localizes with aberrant endosomes, connected ALG-2 to the ESCRT pathway for the first time and answered how ALG-2 accesses endosomal function.","evidence":"Yeast two-hybrid, biotin-ALG-2 overlay, GST pulldown, alanine mutagenesis of PxY motifs, immunofluorescence with BAPTA","pmids":["14999017","16004603"],"confidence":"High","gaps":["Whether ALG-2 actively bridges ALIX and TSG101 or binds them independently was unclear","Functional role in MVB sorting was not yet tested"]},{"year":2006,"claim":"Discovery that ALG-2 is recruited to ER exit sites by binding Sec31A and stabilizes COPII coat retention established a major new cellular role for ALG-2 in ER-to-Golgi transport, answering how Ca²⁺ signaling intersects with secretory pathway regulation.","evidence":"Co-IP, RNAi, Ca²⁺ ionophore/chelator treatments, confocal microscopy, and live-cell imaging showing ALG-2 dynamics track Ca²⁺ oscillations","pmids":["16957052","17196169","17214967"],"confidence":"High","gaps":["Whether ALG-2 promotes or inhibits COPII budding was unresolved","Identity of additional ERES partners was unknown"]},{"year":2006,"claim":"In vitro reconstitution proved yeast Alg2 is a bifunctional enzyme catalyzing sequential α1,3- then α1,6-mannosylation to form the first branched Man₃GlcNAc₂-PP-Dol, resolving a longstanding ambiguity about whether one or two enzymes perform these steps.","evidence":"In vitro mannosyltransferase assay with recombinant Alg2 and defined dolichylpyrophosphate-linked substrates","pmids":["16878994"],"confidence":"High","gaps":["Human ALG2 kinetic order preference was not yet established","Structural basis of bifunctionality unknown"]},{"year":2009,"claim":"Reconstitution showing ALG-2 dimers physically bridge ALIX and TSG101 answered whether ALG-2 is a passive Ca²⁺-sensor or an active scaffold, establishing its adaptor function within the ESCRT network.","evidence":"Strep-tag pulldown, ALG-2 knockdown, recombinant ALG-2 add-back, dimerization-defective mutant analysis","pmids":["19520058"],"confidence":"High","gaps":["Which ESCRT-I subunits are primarily contacted was not fully resolved","In vivo relevance for MVB cargo sorting was untested"]},{"year":2013,"claim":"Multiple discoveries simultaneously expanded ALG-2's functional reach: nuclear ALG-2 interacts with CHERP to regulate IP3R1 alternative splicing, ALG2 mutations cause congenital myasthenic syndrome, and an in vitro COPII budding assay showed ALG-2/Ca²⁺ attenuates vesicle budding by bridging Sec31A to Sec23—collectively establishing ALG-2 as both a secretory pathway regulator and a nuclear splicing co-factor, and linking the glycosyltransferase to neuromuscular disease.","evidence":"In vitro COPII budding assay with EF1 mutant; co-IP plus siRNA knockdown with RT-PCR splicing readout; patient exome sequencing with protein expression validation","pmids":["24069399","24078636","23404334"],"confidence":"High","gaps":["How ALG-2 is imported into the nucleus mechanistically was unclear","Whether splicing regulation requires ALIX was not tested","ALG2 CMS therapeutic rescue was not demonstrated"]},{"year":2015,"claim":"Crystal structures of ALG-2 bound to Sec31A versus ALIX peptides revealed that these partners bind to structurally distinct hydrophobic pockets (Pocket 3 vs. Pockets 1/2), with separation-of-function mutations (F85A, Y180A) proving target discrimination—answering how one small adaptor binds multiple partners selectively.","evidence":"X-ray crystallography of ALG-2–Sec31A peptide complex, alanine mutagenesis of pocket residues, pulldown assays","pmids":["25667979","27462417"],"confidence":"High","gaps":["Whether simultaneous binding of two different targets occurs on a single dimer was untested","Structural basis of ESCRT-I binding not resolved"]},{"year":2016,"claim":"Identification of TFG and MISSL as additional ALG-2 partners at ERES that regulate ER-to-Golgi transport demonstrated that ALG-2 coordinates a network of ERES factors rather than acting solely through Sec31A.","evidence":"Live-cell imaging, Ca²⁺ mobilization, in vitro cross-linking polymerization of TFG, SEAP secretion and procollagen transport assays with siRNA knockdown","pmids":["27813252","28864773"],"confidence":"High","gaps":["Hierarchy among ALG-2 ERES partners unknown","Whether peflin regulates TFG/MISSL interactions was untested"]},{"year":2018,"claim":"Demonstrating that ALG-2 knockout cells are sensitized to plasma membrane damage and that protection requires both Ca²⁺-binding and ALIX interaction established ALG-2 as a Ca²⁺-triggered membrane repair factor, expanding its role beyond trafficking.","evidence":"ALG-2 gene knockout in DT-40 cells, electroporation and digitonin survival assays, Ca²⁺-binding mutant and ALIX-blocking peptide","pmids":["30240438"],"confidence":"High","gaps":["Whether ESCRT-III is the downstream effector of repair was not directly shown","Relevance in mammalian primary cells not tested"]},{"year":2020,"claim":"Multiple studies connected ALG-2 to T cell immune regulation through distinct mechanisms—proteasome-mediated MCL1 degradation (via Rpn3 binding) and FasL vesicular transport—explaining how ALG-2 promotes apoptosis during immune contraction.","evidence":"Co-IP of ALG-2 with Rpn3, proteasome activity and MCL1 stability assays, ALG-2 knockdown in T cells; Co-IP of ALG-2 with FasL, FasL trafficking readout","pmids":["31919392","32766719"],"confidence":"Medium","gaps":["Relative contribution of proteasome vs. FasL pathways to T cell death not quantified","In vivo immune phenotype of ALG-2 deficiency not established","Rpn3 interaction awaits independent replication"]},{"year":2021,"claim":"Establishing that ALG-2 suppresses STING ER-to-vesicle trafficking in a Ca²⁺-dependent manner, with knockout cells showing enhanced type I IFN signaling, placed ALG-2 as a negative regulator of innate immunity and answered how Ca²⁺ sensing intersects with antiviral defense.","evidence":"ALG-2 knockout in THP-1 cells, co-IP, immunofluorescence trafficking assay, Ca²⁺-binding mutant, HSV-1 and cGAMP stimulation","pmids":["34787301"],"confidence":"High","gaps":["Mechanism by which ALG-2 retains STING in the ER is unclear","Whether ALIX or ESCRT is involved was not tested"]},{"year":2021,"claim":"Demonstrating that the ALG-2/peflin heterodimer buffers secretion at steady state while Ca²⁺ signals shift the balance toward peflin-free ALG-2 complexes that stimulate secretion resolved the apparent contradiction of ALG-2 sometimes promoting and sometimes inhibiting COPII function.","evidence":"Peflin/ALG-2 double-knockdown epistasis, SEAP and collagen I secretion assays, ERES fractionation in NRK and PC12 cells","pmids":["34762908"],"confidence":"High","gaps":["Quantitative Ca²⁺ thresholds for heterodimer vs. homodimer switching unknown","Whether peflin regulates ESCRT-related functions was untested"]},{"year":2022,"claim":"Kinetic analysis of purified human ALG2 confirmed the α1,3-before-α1,6 mannosylation order under physiological conditions and revealed a single membrane-anchoring domain (unlike yeast), resolving species-specific topology differences.","evidence":"LC-MS quantitative kinetics with purified hAlg2 and defined LLO substrates, topology analysis","pmids":["35136180"],"confidence":"High","gaps":["No crystal structure of human ALG2 mannosyltransferase available","Regulation of catalytic activity in vivo is unknown"]},{"year":2024,"claim":"Three landmark studies resolved how ALG-2 reaches membranes—via direct electrostatic/hydrophobic membrane binding distinct from protein-mediated recruitment—and established new functional roles at lysosomes (ESCRT-dependent resilience to osmotic stress) and in nutrient-stress-triggered ERES microautophagy requiring ALG-2, ALIX, and ubiquitinated SEC31.","evidence":"GUV membrane binding, MD simulations, charge-reversal mutagenesis, lysosomal leakage assays with ΔGF122 variant, super-resolution imaging, FIB-SEM, in vitro GUV reconstitution of ERES microautophagy, ALG-2 knockout","pmids":["38386713","38781205","38593803"],"confidence":"High","gaps":["Whether ERES microautophagy is selective for specific cargoes is unknown","Relative contribution of direct membrane binding vs. protein-mediated recruitment in vivo is unquantified","Whether lysosomal resilience function is relevant in disease settings remains untested"]},{"year":null,"claim":"Key unresolved questions include: the structural basis of ALG-2 bridging ESCRT-I subunits (no ESCRT-I co-crystal), whether ALG-2 functions overlap or are separable in vivo (no animal knockout phenotype published), and how ALG-2 nuclear import is regulated.","evidence":"","pmids":[],"confidence":"Low","gaps":["No ALG-2 knockout mouse or in vivo phenotyping reported","No co-crystal structure of ALG-2 with full-length ALIX or ESCRT-I","Mechanism of ALG-2 nuclear import unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[21,26,31,33,45]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[44]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[12,17,23,43]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,8,18]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[16,25,26,32,41]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[13,31]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[19,27]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[42,44,45]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[35,44]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[16,26,32,33,41,46]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,20,36]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[12,17,23,43]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[39]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[42,46]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[12,28]}],"complexes":["ALG-2/peflin heterodimer","ALG-2 homodimer","Alg1/Alg2/Alg11 mannosyltransferase complex"],"partners":["PDCD6IP","SEC31A","TSG101","PEF1","TFG","STING1","CHERP","MCOLN1"],"other_free_text":[]},"mechanistic_narrative":"ALG2/PDCD6 encodes a penta-EF-hand calcium-sensor protein that functions as a Ca²⁺-dependent adaptor linking membrane-trafficking machinery to diverse cellular processes including ER-to-Golgi secretion, endosomal sorting, lysosomal resilience, membrane repair, innate immune signaling, and apoptosis. Upon Ca²⁺ binding, ALG-2 undergoes a conformational change exposing hydrophobic pockets and membrane-binding surfaces that enable it to bridge COPII coat components (Sec31A, TFG, MISSL) at ER exit sites—stabilizing Sec31A retention and modulating vesicle budding—and to bridge ESCRT machinery (ALIX, TSG101, VPS37B/C) at endosomes and lysosomes, where it relieves ALIX autoinhibition to promote ubiquitinated EGFR MVB sorting and recruits ESCRT proteins to maintain lysosomal membrane integrity [PMID:16957052, PMID:24069399, PMID:27462417, PMID:38781205]. ALG-2 also enters the nucleus where it interacts with CHERP at nuclear speckles to regulate alternative splicing of IP3R1 pre-mRNA, suppresses STING ER-to-vesicle trafficking to attenuate type I interferon responses, and participates in T cell apoptosis through proteasome-mediated MCL1 degradation and FasL vesicular transport [PMID:24078636, PMID:34787301, PMID:31919392, PMID:32766719]. Separately, the ALG2 mannosyltransferase (same gene symbol, distinct protein) catalyzes sequential α1,3- and α1,6-mannosylation in dolichol-linked oligosaccharide biosynthesis, and loss-of-function mutations cause congenital disorder of glycosylation type Ii and congenital myasthenic syndrome [PMID:12684507, PMID:16878994, PMID:23404334]."},"prefetch_data":{"uniprot":{"accession":"Q9H553","full_name":"Alpha-1,3/1,6-mannosyltransferase ALG2","aliases":["Asparagine-linked glycosylation protein 2 homolog","GDP-Man:Man(1)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase","GDP-Man:Man(1)GlcNAc(2)-PP-dolichol mannosyltransferase","GDP-Man:Man(2)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase"],"length_aa":416,"mass_kda":47.1,"function":"Mannosyltransferase that operates in the biosynthetic pathway of dolichol-linked oligosaccharides, the glycan precursors employed in protein asparagine (N)-glycosylation. The assembly of dolichol-linked oligosaccharides begins on the cytosolic side of the endoplasmic reticulum membrane and finishes in its lumen. The sequential addition of sugars to dolichol pyrophosphate produces dolichol-linked oligosaccharides containing fourteen sugars, including two GlcNAcs, nine mannoses and three glucoses. Once assembled, the oligosaccharide is transferred from the lipid to nascent proteins by oligosaccharyltransferases. Catalyzes, on the cytoplasmic face of the endoplasmic reticulum, the addition of the second and third mannose residues to the dolichol-linked oligosaccharide chain, to produce Man3GlcNAc(2)-PP-dolichol core oligosaccharide. Man3GlcNAc(2)-PP-dolichol is a substrate for ALG11, the following enzyme in the biosynthetic pathway (PubMed:12684507, PubMed:35136180). While both alpha 1,3 and alpha 1,6 linkages are possible, the sequential addition of alpha 1,3 followed by alpha 1,6 is probably the preferred route (PubMed:35136180)","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9H553/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ALG2","classification":"Common Essential","n_dependent_lines":1188,"n_total_lines":1208,"dependency_fraction":0.9834437086092715},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000119523","cell_line_id":"CID000957","localizations":[{"compartment":"er","grade":3}],"interactors":[{"gene":"LDHA","stoichiometry":0.2},{"gene":"TECR","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000957","total_profiled":1310},"omim":[{"mim_id":"618539","title":"CALCIUM HOMEOSTASIS ENDOPLASMIC RETICULUM PROTEIN; CHERP","url":"https://www.omim.org/entry/618539"},{"mim_id":"616228","title":"MYASTHENIC SYNDROME, CONGENITAL, 14; CMS14","url":"https://www.omim.org/entry/616228"},{"mim_id":"616227","title":"MYASTHENIC SYNDROME, CONGENITAL, 15; CMS15","url":"https://www.omim.org/entry/616227"},{"mim_id":"612866","title":"ALG14 UDP-N-ACETYLGLUCOSAMINYLTRANSFERASE SUBUNIT; ALG14","url":"https://www.omim.org/entry/612866"},{"mim_id":"612430","title":"RNA-BINDING MOTIF PROTEIN 22; RBM22","url":"https://www.omim.org/entry/612430"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ALG2"},"hgnc":{"alias_symbol":["CDGIi","FLJ14511","hALPG2","NET38","CDG1I"],"prev_symbol":[]},"alphafold":{"accession":"O75340","domains":[{"cath_id":"1.10.238.10","chopping":"24-188","consensus_level":"medium","plddt":94.7142,"start":24,"end":188}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75340","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75340-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75340-F1-predicted_aligned_error_v6.png","plddt_mean":89.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALG2","jax_strain_url":"https://www.jax.org/strain/search?query=ALG2"},"sequence":{"accession":"O75340","fasta_url":"https://rest.uniprot.org/uniprotkb/O75340.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75340/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75340"}},"corpus_meta":[{"pmid":"8560270","id":"PMC_8560270","title":"Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3.","date":"1996","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8560270","citation_count":450,"is_preprint":false},{"pmid":"10200558","id":"PMC_10200558","title":"Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein.","date":"1999","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/10200558","citation_count":214,"is_preprint":false},{"pmid":"9880530","id":"PMC_9880530","title":"Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9880530","citation_count":201,"is_preprint":false},{"pmid":"10858458","id":"PMC_10858458","title":"The glioma-associated protein SETA interacts with AIP1/Alix and ALG-2 and modulates apoptosis in astrocytes.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10858458","citation_count":111,"is_preprint":false},{"pmid":"23404334","id":"PMC_23404334","title":"Congenital myasthenic syndromes due to mutations in ALG2 and ALG14.","date":"2013","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/23404334","citation_count":107,"is_preprint":false},{"pmid":"12684507","id":"PMC_12684507","title":"A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12684507","citation_count":94,"is_preprint":false},{"pmid":"16957052","id":"PMC_16957052","title":"The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16957052","citation_count":86,"is_preprint":false},{"pmid":"19864416","id":"PMC_19864416","title":"Identification of the penta-EF-hand protein ALG-2 as a Ca2+-dependent interactor of mucolipin-1.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19864416","citation_count":79,"is_preprint":false},{"pmid":"17196169","id":"PMC_17196169","title":"ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17196169","citation_count":76,"is_preprint":false},{"pmid":"11525164","id":"PMC_11525164","title":"Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins.","date":"2001","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/11525164","citation_count":74,"is_preprint":false},{"pmid":"10360947","id":"PMC_10360947","title":"Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins.","date":"1999","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10360947","citation_count":71,"is_preprint":false},{"pmid":"11278427","id":"PMC_11278427","title":"Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11278427","citation_count":71,"is_preprint":false},{"pmid":"17584079","id":"PMC_17584079","title":"Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond.","date":"2007","source":"Current molecular 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depletion of ALG-2 protects T cell hybridoma from apoptosis, placing ALG-2 as a required mediator of Ca2+-regulated death signals.\",\n      \"method\": \"Functional selection/death trap assay, antisense depletion with apoptosis readout\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational loss-of-function study with defined cellular phenotype, highly cited, replicated\",\n      \"pmids\": [\"8560270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"ALG-2 functions downstream of ICE/Ced-3 caspase activation in apoptosis; ALG-2-depleted cells still activate caspases normally (cleave PARP) yet do not die, placing ALG-2 downstream of or independent of caspases.\",\n      \"method\": \"ALG-2 antisense depletion combined with fluorogenic and PARP substrate caspase activity assays\",\n      \"journal\": \"Journal of Immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined biochemical readouts, replicated in original lab\",\n      \"pmids\": [\"9164928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 directly interacts with AIP1 (Alix) in a strictly Ca2+-dependent manner; both proteins co-localize in the cytosol, and overexpression of a truncated AIP1 protects cells from trophic-factor withdrawal death.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, subcellular co-localization\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction validated by multiple methods, Ca2+ dependence established, replicated in subsequent papers\",\n      \"pmids\": [\"9880530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 binds to itself (Ca2+-independent homodimerization) and to Alix (Ca2+-dependent); the fifth EF-hand participates in dimer formation as shown by biochemical and gel-filtration analysis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, gel filtration\",\n      \"journal\": \"Cell Death and Differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two orthogonal methods, confirmed by subsequent structural studies\",\n      \"pmids\": [\"10200558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Ca2+ binding induces exposure of a hydrophobic surface on ALG-2 in a concentration-dependent manner (~6 µM half-maximal), a conformational change that is Mg2+-insensitive and drives association with macromolecules in cells.\",\n      \"method\": \"Fluorescent hydrophobicity probe (TNS) spectroscopy, recombinant protein characterization\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical reconstitution with recombinant protein\",\n      \"pmids\": [\"9832622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 possesses two high-affinity Ca2+-binding sites and one low-affinity site; Ca2+ binding to both strong sites is required for Ca2+-induced aggregation; the fifth EF-hand mediates homodimerization.\",\n      \"method\": \"Gel filtration, chemical cross-linking, mutagenesis (E47A/E114A), fluorescence spectroscopy, circular dichroism\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay with mutagenesis and multiple spectroscopic methods\",\n      \"pmids\": [\"10360947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Two alternatively spliced isoforms of ALG-2 exist (ALG-2,5 and ALG-2,1, differing by Gly121-Phe122); ALG-2,1 lacks the ability to interact with AIP1 and has a higher Ca2+-binding affinity, demonstrating that two residues govern target specificity.\",\n      \"method\": \"Yeast two-hybrid, Ca2+ binding assays, sequence analysis of isoforms\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical assays plus mutagenic isoform comparison, confirmed by structural studies\",\n      \"pmids\": [\"10744743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"X-ray crystal structure of Ca2+-loaded ALG-2 (des1-20, 2.3 Å) reveals five EF-hand fold with Ca2+ bound to EF1, EF3, and EF5; Ca2+ induces a rigid-body rotation of EF1-2 relative to EF4-5, exposing a hydrophobic cleft that accepts a Gly/Pro-rich peptide.\",\n      \"method\": \"X-ray crystallography at 2.3 Å resolution\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure\",\n      \"pmids\": [\"11525164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Peflin forms a heterodimer with ALG-2 in Jurkat cells; the heterodimer dissociates upon Ca2+ addition, and peflin translocates to membranes/cytoskeleton in the presence of Ca2+, while ALG-2 persists in both cytoplasm and nucleus.\",\n      \"method\": \"Co-immunoprecipitation, gel filtration, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, endogenous protein interaction\",\n      \"pmids\": [\"11278427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 directly interacts with the N-terminal domain of annexin XI (and similarly annexin VII) in a Ca2+-dependent manner; dissociation constants ~70 nM (high affinity) and ~500–700 nM (low affinity) were measured.\",\n      \"method\": \"Yeast two-hybrid, biotin-ALG-2 overlay assay, surface plasmon resonance (SPR)\",\n      \"journal\": \"Biochemical and Biophysical Research Communications / Biochimica et Biophysica Acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding demonstrated by SPR with kinetics and mutagenesis\",\n      \"pmids\": [\"11883939\", \"12445460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 interacts with the C-terminus of ASK1 in a Ca2+-dependent and isoform-specific manner (ALG-2,1 does not bind); co-transfection of ALG-2 causes nuclear redistribution of ASK1 and suppresses ASK1-mediated JNK activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding, cotransfection/subcellular localization, JNK reporter assay\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction plus functional readout, single lab\",\n      \"pmids\": [\"12372597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The fifth EF-hand of ALG-2 (and peflin) is essential for dimerization and protein stability; deletion of EF5 leads to rapid proteasomal degradation of the monomer.\",\n      \"method\": \"Exogenous expression of deletion mutants, pulse-chase, proteasome inhibitor (MG132) rescue, Western blot\",\n      \"journal\": \"Archives of Biochemistry and Biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with defined degradation phenotype, single lab\",\n      \"pmids\": [\"11883899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human ALG2 (hALG2) is an α1,3-mannosyltransferase that elongates Man1GlcNAc2-PP-dolichol; deficiency causes CDG-Ii with accumulation of Man1 and Man2 LLO intermediates. Expression of wild-type but not mutant hALG2 rescues both patient fibroblasts and yeast alg2-1 cells.\",\n      \"method\": \"Mannosyltransferase activity assay in patient fibroblast extracts, complementation of yeast alg2-1, genetic analysis of patient mutations\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — enzymatic assay with patient mutations and genetic complementation in two systems\",\n      \"pmids\": [\"12684507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALG-2 interacts with the ESCRT-I component TSG101 directly via its Pro-rich region in a Ca2+-dependent manner; indirect Ca2+-dependent association with hVps28 and hVps37A occurs through TSG101. ALG-2 co-localizes with aberrant endosomes (SKD1 dominant-negative) in a Ca2+-dependent fashion.\",\n      \"method\": \"GST pulldown, yeast two-hybrid, biotin-ALG-2 overlay assay, immunofluorescence with Ca2+ chelator (BAPTA)\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding demonstrated by overlay assay plus multiple interaction methods, subcellular localization linked to Ca2+\",\n      \"pmids\": [\"16004603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALG-2 interacts directly with the Alix C-terminal Pro-rich region (residues 794–827) via four tandem PxY repeats; alanine substitution of critical Pro and Tyr residues ablates binding. ALG-2 is required for the punctate subcellular distribution of Alix.\",\n      \"method\": \"Yeast two-hybrid, biotin-ALG-2 overlay, co-immunoprecipitation, Ca2+-binding-deficient mutant, fluorescence microscopy\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding motif defined by mutagenesis, confirmed by multiple methods\",\n      \"pmids\": [\"14999017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast Alg1, Alg2, and Alg11 mannosyltransferases form distinct physical complexes in the ER membrane; Alg1 self-assembles via a C-terminal domain distinct from its Alg2/Alg11 interaction domain; catalytically inactive Alg1 alleles cause dominant-negative phenotypes consistent with functional complex assembly.\",\n      \"method\": \"Co-immunoprecipitation, genetic complementation, dominant-negative analysis in S. cerevisiae\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical and genetic epistasis in multiple assays, yeast ortholog of human ALG2\",\n      \"pmids\": [\"15044395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 is recruited to ER exit sites (ERES) by Ca2+-dependent binding to Sec31A (a COPII outer coat component); ALG-2 in turn stabilizes Sec31A at ERES. Ca2+-binding-deficient ALG-2 (E47A/E114A) fails to localize to ERES and fails to stabilize Sec31A.\",\n      \"method\": \"Co-immunoprecipitation, RNAi, Ca2+ ionophore (A23187) and chelator (BAPTA-AM) treatment, immunofluorescence confocal microscopy, GST pulldown\",\n      \"journal\": \"Molecular Biology of the Cell / Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, functional consequence of localization established, replicated across two labs\",\n      \"pmids\": [\"16957052\", \"17196169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In vitro reconstitution demonstrates that S. cerevisiae Alg2 is a bifunctional enzyme catalyzing sequential α1,3- then α1,6-mannosylation to form the first branched pentasaccharide (Man3GlcNAc2-PP-Dol) intermediate in the dolichol pathway.\",\n      \"method\": \"In vitro mannosyltransferase assay with recombinant Alg2 and defined dolichylpyrophosphate-linked substrates from E. coli overexpression\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified enzyme and defined substrates\",\n      \"pmids\": [\"16878994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 subcellular localization oscillates in synchrony with intracellular Ca2+ oscillations induced by physiological stimuli (ATP, EGF, prostaglandin, histamine); Ca2+-binding-deficient ALG-2 does not redistribute, linking ALG-2 dynamics to Ca2+ signaling.\",\n      \"method\": \"Live-cell imaging of tagged ALG-2 with simultaneous Ca2+ indicator, Ca2+-binding mutant comparison\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging with functional mutant control, directly links localization to Ca2+ signaling\",\n      \"pmids\": [\"17214967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Nuclear translocation of ALG-2 is induced by the RNA-binding protein RBM22; when co-expressed, ALG-2 translocates to the nucleus where RBM22 is located, indicating RBM22 drives ALG-2 nuclear import.\",\n      \"method\": \"Fluorescent fusion protein co-expression, confocal microscopy in NIH 3T3 cells and zebrafish embryos\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging with two model systems, single lab\",\n      \"pmids\": [\"17045351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Alix and ALG-2 interact with pro-caspase-8 in a Ca2+-dependent manner and form a complex with TNF-R1 on endosomes; deletion of the ALG-2-binding site in Alix significantly reduces TNF-R1-induced cell death without affecting receptor endocytosis.\",\n      \"method\": \"Mass spectrometry of co-immunoprecipitates, co-immunoprecipitation, deletion mutant Alix (ΔAlg-2 binding site), cell death assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complex formation identified by MS and confirmed by co-IP, functional consequence established by deletion mutant\",\n      \"pmids\": [\"18936101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101: Alix requires its ALG-2-binding site (not its PSAP-TSG101 binding motif) for Ca2+-dependent pulldown of TSG101; ALG-2 knockdown abolishes the association; the ALG-2 dimer bridges the two binding partners.\",\n      \"method\": \"Strep-tag pulldown assays, ALG-2 knockdown, recombinant ALG-2 add-back, dimerization-defective mutant\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstitution with purified protein, knockdown rescue, mutant analysis\",\n      \"pmids\": [\"19520058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 directly binds the NH2-terminal cytosolic tail of mucolipin-1 (MCOLN1) in a strictly Ca2+-dependent manner via residues 37–49 of MCOLN1; ALG-2 regulates MCOLN1 function as mutation of the ALG-2-binding domain in MCOLN1 reduces MCOLN1-induced aberrant endosome aggregation.\",\n      \"method\": \"In vitro binding assay, co-localization, mutagenesis of MCOLN1 ALG-2-binding domain\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding demonstrated, functional mutagenesis with endosome phenotype readout\",\n      \"pmids\": [\"19864416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Biochemical characterization of yeast Alg2 establishes it as a bifunctional α1,3- and α1,6-mannosyltransferase in ER LLO biosynthesis; topology analysis shows two N-terminal transmembrane spans anchor it to the ER; Lys230 is essential for catalytic activity while an EX7E motif is dispensable.\",\n      \"method\": \"In vitro mannosyltransferase assay, topology analysis, site-directed mutagenesis (K230, EX7E motif)\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay with site-directed mutagenesis identifying catalytic residue\",\n      \"pmids\": [\"19282279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the ALG-2(ΔGF122) isoform explains its inability to bind Alix: deletion of Gly121-Phe122 deforms the main-chain wall facing pockets 1 and 2 and repositions Arg125 to occlude pocket 1, not loss of the F122 side chain per se. F122 substitution with Ala or Gly (not Trp) actually increases Alix binding.\",\n      \"method\": \"X-ray crystallography, pulldown assays with F122 substitution mutants\",\n      \"journal\": \"BMC Structural Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — X-ray structure combined with mutagenesis/binding assays\",\n      \"pmids\": [\"20691033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ALG-2-binding site (ABS, residues 839–851) in Sec31A is necessary and sufficient for direct ALG-2 binding; FRAP analysis reveals that ABS deletion reduces the high-affinity population of Sec31A at ERES, indicating ALG-2 controls Sec31A retention kinetics.\",\n      \"method\": \"Biotin-ALG-2 overlay assay, stable GFP-ALG-2/Sec31A-RFP cell lines, Ca2+ mobilization, FRAP\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding motif defined, retention kinetics measured by FRAP\",\n      \"pmids\": [\"20834162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALG-2 attenuates COPII vesicle budding in vitro by binding to the Pro-rich region of Sec31A; this requires an intact EF-hand 1 Ca2+-binding site in ALG-2. ALG-2/Ca2+ increases recruitment of Sec23/24 and Sec13/31A to liposomes and mediates Sec31A-Sec23 bridging.\",\n      \"method\": \"In vitro COPII budding assay, liposome recruitment assay, EF1 mutant ALG-2\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted COPII budding assay with mutagenesis\",\n      \"pmids\": [\"24069399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nuclear ALG-2 interacts with CHERP (Ca2+ homeostasis ER protein) in a Ca2+-dependent manner at nuclear speckles; knockdown of CHERP or ALG-2 alters alternative splicing of IP3R1 pre-mRNA, and CHERP binds IP3R1 RNA. ALG-2 is recruited to CHERP-localizing nuclear speckles upon Ca2+ mobilization.\",\n      \"method\": \"Co-immunoprecipitation, live-cell time-lapse imaging, siRNA knockdown, RNA immunoprecipitation, RT-PCR for splicing isoforms\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, nuclear function linked to splicing regulation\",\n      \"pmids\": [\"24078636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mutations in ALG2 cause congenital myasthenic syndrome; the ALG2 p.Val68Gly mutation severely reduces ALG2 protein expression in patient muscle and cell cultures. ALG2 functions as an α1,3-mannosyltransferase in early steps of asparagine-linked glycosylation, critical for proper NMJ function.\",\n      \"method\": \"Linkage analysis, exome/genome sequencing, Western blot of patient tissue and transfected cells, SiRNA knockdown of ALG14 with AChR surface expression readout\",\n      \"journal\": \"Brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — patient genetics with functional protein expression validation in cells and tissue\",\n      \"pmids\": [\"23404334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VPS37B and VPS37C isoforms of ESCRT-I interact more strongly with ALG-2 than TSG101; purified recombinant ESCRT-I/ALIX/ALG-2 can form a ternary complex in vitro in a Ca2+-dependent manner.\",\n      \"method\": \"Far-Western blot with biotin-ALG-2, pulldown with recombinant proteins, in vitro reconstitution\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of ternary complex, single lab\",\n      \"pmids\": [\"23924735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray crystal structure of ALG-2 bound to a Sec31A peptide (type 2 motif, PXPGF) shows binding to a third hydrophobic pocket (Pocket 3), distinct from the Pocket 1/2 used by Alix. F85A mutation abrogates Sec31A binding but not Alix binding; Y180A eliminates Alix binding but not Sec31A binding.\",\n      \"method\": \"X-ray crystallography of ALG-2/Sec31A peptide complex, alanine mutagenesis of pocket residues, pulldown binding assays\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis defining two independent binding surfaces\",\n      \"pmids\": [\"25667979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ca2+-dependent ALG-2 interaction with ALIX relieves ALIX intramolecular autoinhibition and promotes CHMP4-dependent ALIX membrane association, thereby enabling ALIX function in MVB sorting of ubiquitinated EGFR specifically (not cytokinesis or EIAV budding).\",\n      \"method\": \"Co-immunoprecipitation, membrane fractionation, EGFR MVB sorting assay, ALIX deletion and CHMP4-binding mutants, siRNA knockdown\",\n      \"journal\": \"Cell Discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays distinguishing ALIX activities, mechanistic model with mutant validation\",\n      \"pmids\": [\"27462417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALG-2 promotes ER exit site (ERES) localization and Ca2+-dependent polymerization of TFG protein; ALG-2 overexpression accumulates TFG at ERES; deletion of TFG's ALG-2-binding motif shortens TFG half-life at ERES.\",\n      \"method\": \"Immunostaining, live-cell time-lapse imaging with Ca2+ mobilization, in vitro cross-linking polymerization assay, Co-IP\",\n      \"journal\": \"The FEBS Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro polymerization assay plus live-cell imaging and binding motif mutant\",\n      \"pmids\": [\"27813252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALG-2 interacts Ca2+-dependently with MISSL, and together they regulate ER-to-Golgi transport; knockdown of either ALG-2 or MISSL similarly attenuates SEAP secretion and delays procollagen I ER-to-Golgi transport. ALG-2/MISSL interact with MAP1B, which negatively regulates this secretory pathway.\",\n      \"method\": \"Co-IP, live-cell imaging, siRNA knockdown, secreted alkaline phosphatase (SEAP) secretion assay, procollagen transport assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts, pathway placement by double-knockdown epistasis\",\n      \"pmids\": [\"28864773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MAP1B interacts with ALG-2 Ca2+-dependently via a unique mode (no canonical ABM-1/ABM-2 motif); MAP1B competes with ABM-2-containing proteins such as Sec31A for ALG-2 binding, and MAP1B overexpression disperses ALG-2 and Sec31A from ERES.\",\n      \"method\": \"Co-IP, pulldown with MAP1B mutants, co-localization microscopy, MAP1B knockout cells\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — competitive binding shown with mutants, KO cell phenotype, single lab\",\n      \"pmids\": [\"29432744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALG-2 participates in plasma membrane repair after damage by electroporation or digitonin; ALG-2 KO cells are more sensitive to electroporation, reversible by ALG-2 re-expression; Ca2+-binding-deficient ALG-2 does not protect, and a blocking ALIX peptide abolishes protection.\",\n      \"method\": \"ALG-2 gene knockout (DT-40 cells), electroporation and digitonin survival assays, Ca2+-binding mutant and ALIX-peptide blocking experiments\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with rescue, Ca2+-binding requirement established, mechanism linked to ALIX interaction\",\n      \"pmids\": [\"30240438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 directly interacts with Rpn3, a component of the 26S proteasome; upon Ca2+ influx following T cell activation, ALG-2 regulates proteasome activity, which controls MCL1 stability and accelerates T cell apoptosis during immune contraction.\",\n      \"method\": \"Co-immunoprecipitation, proteasome activity assay, MCL1 stability assay, ALG-2 knockdown in T cells\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction identified, functional pathway placement in T cell apoptosis, single lab\",\n      \"pmids\": [\"31919392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 interacts with SARAF (a negative regulator of SOCE) Ca2+-dependently; ALG-2 overexpression interferes with SARAF ubiquitination, stabilizing SARAF, and promotes Ca2+-dependent SARAF dimerization through its ABM-2 motif.\",\n      \"method\": \"Pulldown assay, co-IP, ubiquitination assay, half-life analysis, site-directed mutagenesis of ABM-2\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — interaction and ubiquitination interference established with mutagenesis, single lab\",\n      \"pmids\": [\"32878247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 interacts with FasL (FASLG) and regulates its intracellular vesicle-mediated transport, thereby influencing T cell apoptosis via the FasL–Fas pathway.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization microscopy, ALG-2 knockdown with FasL trafficking readout\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — interaction confirmed by Co-IP, vesicle trafficking phenotype shown, single lab\",\n      \"pmids\": [\"32766719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG-2 suppresses STING-mediated type I interferon signaling by associating with the C-terminal tail of STING and inhibiting its trafficking from the ER to perinuclear vesicles; this requires intact Ca2+-coordinating residues in ALG-2; ALG-2 knockout increases IFN expression upon HSV-1 or cGAMP stimulation.\",\n      \"method\": \"ALG-2 knockout (THP-1 cells), co-immunoprecipitation, immunofluorescence trafficking assay, Ca2+-binding mutant ALG-2\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined trafficking phenotype, Ca2+-requirement established, direct interaction shown\",\n      \"pmids\": [\"34787301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CDIP1 interacts with ALG-2 in a Ca2+-dependent manner; ALG-2 promotes CDIP1 association with ESCRT-I (specifically VPS37B/C-containing complexes); co-expression of ALG-2 and ESCRT-I enhances CDIP1-induced caspase-3/7 cell death.\",\n      \"method\": \"Co-immunoprecipitation, caspase-3/7 activity assay, ALG-2 and ESCRT-I co-expression experiments\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — interaction and functional consequence shown, single lab\",\n      \"pmids\": [\"33503978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG-2 and peflin form a hetero-bifunctional COPII regulator; at steady-state Ca2+, ALG-2/peflin heterocomplexes bind ERES and buffer secretion; Ca2+ signaling shifts balance toward peflin-lacking ALG-2 complexes that stimulate secretion; ALG-2-dependent effects on secretion can be opposing depending on signal intensity and cell type.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, secretion assays (SEAP, collagen I), ERES fractionation, Ca2+ agonist treatments in NRK and PC12 cells\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell types, multiple cargos, mechanistic model supported by peflin/ALG-2 double-knockdown epistasis\",\n      \"pmids\": [\"34762908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Oxidative stress-induced Ca2+ flux triggers JIP4 phosphorylation at T217 by CaMK2G, which activates a JIP4–TRPML1–ALG-2 pathway that promotes lysosomal retrograde transport (clustering near MTOC) and autophagy as a protective response.\",\n      \"method\": \"Knockout cells, phospho-specific mutants, lysosomal positioning assay, Ca2+ flux measurement, CaMK2G kinase assay\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway placement by KO and phospho-mutants, single lab\",\n      \"pmids\": [\"36394115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human ALG2 prefers to transfer α1,3-mannose before α1,6-mannose onto M1GlcNAc2-PP-Dol under physiological conditions, but excess GDP-Man donor or elevated M1Gn2 substrate can shift it toward first producing the M2Gn2(α1,6) intermediate; single membrane-binding domain anchors hAlg2 to ER (differs from yeast 4-domain topology).\",\n      \"method\": \"LC-MS quantitative kinetics assay with purified hAlg2, topology analysis, defined LLO substrates\",\n      \"journal\": \"Communications Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinetics with purified human enzyme and defined substrates, topology established\",\n      \"pmids\": [\"35136180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ca2+-dependent direct membrane binding by ALG-2 is mediated by electrostatic and hydrophobic interactions; charge-reversed ALG-2 mutants reduce ERES localization after thapsigargin-induced Ca2+ release but still localize to lysosomes (rescued by ESCRT-I binding); direct membrane binding and protein binding are mechanistically distinct pathways.\",\n      \"method\": \"GUV-based membrane binding experiments, molecular dynamics simulations, charge-reversal mutagenesis, in vitro reconstitution with ESCRT-I, live-cell ERES localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the USA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution, MD simulations, mutagenesis and cellular validation\",\n      \"pmids\": [\"38386713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG-2, upon lysosomal Ca2+ release, recruits ESCRT proteins to lysosomes and enhances lysosomal membrane resilience to osmotic stress; the ΔGF122 splice variant of ALG-2 that cannot bind ESCRTs does not confer this protection; activating TRPML1 without membrane damage is sufficient to recruit ALG-2/ESCRTs and protect lysosomes.\",\n      \"method\": \"Lysosomal leakage/rupture assays, Ca2+ chelation (BAPTA), ΔGF122 ALG-2 mutant, TRPML1 activation, immunofluorescence redistribution assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the USA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional KO-equivalent (splice-variant mutant), multiple mechanistic controls, replicated across two papers (peer-reviewed + preprint)\",\n      \"pmids\": [\"38781205\", \"38352356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ERESs undergo lysosome-dependent microautophagy triggered by nutrient stress (mTOR inhibition, amino acid starvation); this requires ubiquitinated SEC31, ALG-2, and ALIX; ALG-2 knockout or function-blocking ALIX mutations prevent ERES engulfment by lysosomes; the pathway was reconstituted in vitro with purified components on lysosomal lipid-mimicking GUVs.\",\n      \"method\": \"Super-resolution live-cell imaging, FIB-SEM, ALG-2 knockout, ALIX function-blocking mutants, in vitro GUV reconstitution\",\n      \"journal\": \"Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus super-resolution imaging and KO, multiple orthogonal methods\",\n      \"pmids\": [\"38593803\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALG-2 (PDCD6) is a penta-EF-hand Ca2+-sensor protein that, upon Ca2+ binding, undergoes a conformational change exposing hydrophobic pockets and directly membrane-binding surfaces, enabling it to function as a Ca2+-dependent adaptor bridging COPII components (Sec31A, TFG, MISSL) at ER exit sites to regulate ER-to-Golgi secretion, to bridge ESCRT proteins (ALIX, TSG101, ESCRT-I) at endosomes and lysosomes to control MVB sorting, membrane repair, and lysosomal resilience, to activate ALIX for EGFR MVB sorting by relieving its autoinhibition, to regulate STING trafficking and innate immune signaling, to control alternative splicing of IP3R1 pre-mRNA via nuclear interaction with CHERP, and to modulate T cell apoptosis through interactions with caspase-8, FasL trafficking, and proteasome-regulated MCL1 stability; in addition, its glycosyltransferase function (human ALG2) catalyzes sequential α1,3- and α1,6-mannosylation steps in dolichol-linked oligosaccharide biosynthesis in the ER, and mutations in this activity cause congenital disorders of glycosylation and congenital myasthenic syndrome.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ALG2/PDCD6 encodes a penta-EF-hand calcium-sensor protein that functions as a Ca²⁺-dependent adaptor linking membrane-trafficking machinery to diverse cellular processes including ER-to-Golgi secretion, endosomal sorting, lysosomal resilience, membrane repair, innate immune signaling, and apoptosis. Upon Ca²⁺ binding, ALG-2 undergoes a conformational change exposing hydrophobic pockets and membrane-binding surfaces that enable it to bridge COPII coat components (Sec31A, TFG, MISSL) at ER exit sites—stabilizing Sec31A retention and modulating vesicle budding—and to bridge ESCRT machinery (ALIX, TSG101, VPS37B/C) at endosomes and lysosomes, where it relieves ALIX autoinhibition to promote ubiquitinated EGFR MVB sorting and recruits ESCRT proteins to maintain lysosomal membrane integrity [PMID:16957052, PMID:24069399, PMID:27462417, PMID:38781205]. ALG-2 also enters the nucleus where it interacts with CHERP at nuclear speckles to regulate alternative splicing of IP3R1 pre-mRNA, suppresses STING ER-to-vesicle trafficking to attenuate type I interferon responses, and participates in T cell apoptosis through proteasome-mediated MCL1 degradation and FasL vesicular transport [PMID:24078636, PMID:34787301, PMID:31919392, PMID:32766719]. Separately, the ALG2 mannosyltransferase (same gene symbol, distinct protein) catalyzes sequential α1,3- and α1,6-mannosylation in dolichol-linked oligosaccharide biosynthesis, and loss-of-function mutations cause congenital disorder of glycosylation type Ii and congenital myasthenic syndrome [PMID:12684507, PMID:16878994, PMID:23404334].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing that ALG-2 is a Ca²⁺-binding protein required for programmed cell death resolved a key unknown—whether Ca²⁺-sensing proteins mediate receptor-triggered apoptosis—by showing that ALG-2 depletion protects T cells from TCR-, Fas-, and glucocorticoid-induced death.\",\n      \"evidence\": \"Functional death-trap selection and antisense depletion in T cell hybridoma with apoptosis readout\",\n      \"pmids\": [\"8560270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream targets of ALG-2 in apoptosis were unknown\", \"Whether ALG-2 acts cell-autonomously or through secreted signals was unclear\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placing ALG-2 genetically downstream of caspase activation answered whether ALG-2 operated in the canonical caspase cascade, establishing it as a post-caspase or parallel effector rather than a caspase activator.\",\n      \"evidence\": \"ALG-2 antisense depletion with PARP cleavage and fluorogenic caspase substrate assays showing intact caspase activity but blocked death\",\n      \"pmids\": [\"9164928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of ALG-2's downstream effectors remained unknown\", \"Whether ALG-2 acts on membrane integrity or nuclear apoptosis was unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery of the Ca²⁺-dependent ALG-2–ALIX interaction and ALG-2 homodimerization via the fifth EF-hand established the first protein partner and the oligomeric framework for ALG-2 function, answering how Ca²⁺ is transduced into protein–protein interactions.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, gel filtration, and Ca²⁺-binding mutagenesis (E47A/E114A)\",\n      \"pmids\": [\"9880530\", \"10200558\", \"10360947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Ca²⁺-induced conformational change was unknown\", \"Cellular function of the ALG-2–ALIX complex was not established\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstration that Ca²⁺ binding induces hydrophobic surface exposure on ALG-2 provided the biophysical mechanism by which Ca²⁺ triggers partner recruitment, answering why ALG-2 interactions are Ca²⁺-dependent.\",\n      \"evidence\": \"TNS fluorescence spectroscopy with recombinant ALG-2, showing half-maximal hydrophobic exposure at ~6 µM Ca²⁺\",\n      \"pmids\": [\"9832622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific hydrophobic surfaces were exposed was unresolved until crystallography\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"The crystal structure of Ca²⁺-loaded ALG-2 revealed Ca²⁺ occupancy at EF1, EF3, and EF5 and a rigid-body rotation exposing a hydrophobic cleft, providing the atomic-level explanation for Ca²⁺-dependent target recognition.\",\n      \"evidence\": \"X-ray crystallography at 2.3 Å resolution\",\n      \"pmids\": [\"11525164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal with any binding partner was available\", \"How ALG-2 discriminates between different targets structurally was unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery that peflin forms a Ca²⁺-sensitive heterodimer with ALG-2 that dissociates upon Ca²⁺ addition established an additional layer of regulation—ALG-2 is sequestered by peflin at low Ca²⁺ and liberated for target engagement at high Ca²⁺.\",\n      \"evidence\": \"Co-immunoprecipitation, gel filtration, and subcellular fractionation in Jurkat cells\",\n      \"pmids\": [\"11278427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of peflin–ALG-2 heterodimer dissociation for any specific cellular process was unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of hALG2 as an α1,3-mannosyltransferase deficient in CDG-Ii patients—rescued by wild-type expression in both human fibroblasts and yeast—established the enzymatic function and disease relevance of the glycosyltransferase ALG2, distinct from the PDCD6 Ca²⁺-sensor.\",\n      \"evidence\": \"Mannosyltransferase activity assay in patient fibroblast extracts and complementation of yeast alg2-1\",\n      \"pmids\": [\"12684507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALG2 catalyzes one or both mannosylation steps was unresolved\", \"Structural basis of catalysis was unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapping the ALG-2–ALIX binding interface to four PxY repeats in ALIX's Pro-rich region, and showing ALG-2 binds TSG101 and co-localizes with aberrant endosomes, connected ALG-2 to the ESCRT pathway for the first time and answered how ALG-2 accesses endosomal function.\",\n      \"evidence\": \"Yeast two-hybrid, biotin-ALG-2 overlay, GST pulldown, alanine mutagenesis of PxY motifs, immunofluorescence with BAPTA\",\n      \"pmids\": [\"14999017\", \"16004603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALG-2 actively bridges ALIX and TSG101 or binds them independently was unclear\", \"Functional role in MVB sorting was not yet tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that ALG-2 is recruited to ER exit sites by binding Sec31A and stabilizes COPII coat retention established a major new cellular role for ALG-2 in ER-to-Golgi transport, answering how Ca²⁺ signaling intersects with secretory pathway regulation.\",\n      \"evidence\": \"Co-IP, RNAi, Ca²⁺ ionophore/chelator treatments, confocal microscopy, and live-cell imaging showing ALG-2 dynamics track Ca²⁺ oscillations\",\n      \"pmids\": [\"16957052\", \"17196169\", \"17214967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALG-2 promotes or inhibits COPII budding was unresolved\", \"Identity of additional ERES partners was unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"In vitro reconstitution proved yeast Alg2 is a bifunctional enzyme catalyzing sequential α1,3- then α1,6-mannosylation to form the first branched Man₃GlcNAc₂-PP-Dol, resolving a longstanding ambiguity about whether one or two enzymes perform these steps.\",\n      \"evidence\": \"In vitro mannosyltransferase assay with recombinant Alg2 and defined dolichylpyrophosphate-linked substrates\",\n      \"pmids\": [\"16878994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human ALG2 kinetic order preference was not yet established\", \"Structural basis of bifunctionality unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Reconstitution showing ALG-2 dimers physically bridge ALIX and TSG101 answered whether ALG-2 is a passive Ca²⁺-sensor or an active scaffold, establishing its adaptor function within the ESCRT network.\",\n      \"evidence\": \"Strep-tag pulldown, ALG-2 knockdown, recombinant ALG-2 add-back, dimerization-defective mutant analysis\",\n      \"pmids\": [\"19520058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which ESCRT-I subunits are primarily contacted was not fully resolved\", \"In vivo relevance for MVB cargo sorting was untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Multiple discoveries simultaneously expanded ALG-2's functional reach: nuclear ALG-2 interacts with CHERP to regulate IP3R1 alternative splicing, ALG2 mutations cause congenital myasthenic syndrome, and an in vitro COPII budding assay showed ALG-2/Ca²⁺ attenuates vesicle budding by bridging Sec31A to Sec23—collectively establishing ALG-2 as both a secretory pathway regulator and a nuclear splicing co-factor, and linking the glycosyltransferase to neuromuscular disease.\",\n      \"evidence\": \"In vitro COPII budding assay with EF1 mutant; co-IP plus siRNA knockdown with RT-PCR splicing readout; patient exome sequencing with protein expression validation\",\n      \"pmids\": [\"24069399\", \"24078636\", \"23404334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ALG-2 is imported into the nucleus mechanistically was unclear\", \"Whether splicing regulation requires ALIX was not tested\", \"ALG2 CMS therapeutic rescue was not demonstrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Crystal structures of ALG-2 bound to Sec31A versus ALIX peptides revealed that these partners bind to structurally distinct hydrophobic pockets (Pocket 3 vs. Pockets 1/2), with separation-of-function mutations (F85A, Y180A) proving target discrimination—answering how one small adaptor binds multiple partners selectively.\",\n      \"evidence\": \"X-ray crystallography of ALG-2–Sec31A peptide complex, alanine mutagenesis of pocket residues, pulldown assays\",\n      \"pmids\": [\"25667979\", \"27462417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether simultaneous binding of two different targets occurs on a single dimer was untested\", \"Structural basis of ESCRT-I binding not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of TFG and MISSL as additional ALG-2 partners at ERES that regulate ER-to-Golgi transport demonstrated that ALG-2 coordinates a network of ERES factors rather than acting solely through Sec31A.\",\n      \"evidence\": \"Live-cell imaging, Ca²⁺ mobilization, in vitro cross-linking polymerization of TFG, SEAP secretion and procollagen transport assays with siRNA knockdown\",\n      \"pmids\": [\"27813252\", \"28864773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy among ALG-2 ERES partners unknown\", \"Whether peflin regulates TFG/MISSL interactions was untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that ALG-2 knockout cells are sensitized to plasma membrane damage and that protection requires both Ca²⁺-binding and ALIX interaction established ALG-2 as a Ca²⁺-triggered membrane repair factor, expanding its role beyond trafficking.\",\n      \"evidence\": \"ALG-2 gene knockout in DT-40 cells, electroporation and digitonin survival assays, Ca²⁺-binding mutant and ALIX-blocking peptide\",\n      \"pmids\": [\"30240438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ESCRT-III is the downstream effector of repair was not directly shown\", \"Relevance in mammalian primary cells not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple studies connected ALG-2 to T cell immune regulation through distinct mechanisms—proteasome-mediated MCL1 degradation (via Rpn3 binding) and FasL vesicular transport—explaining how ALG-2 promotes apoptosis during immune contraction.\",\n      \"evidence\": \"Co-IP of ALG-2 with Rpn3, proteasome activity and MCL1 stability assays, ALG-2 knockdown in T cells; Co-IP of ALG-2 with FasL, FasL trafficking readout\",\n      \"pmids\": [\"31919392\", \"32766719\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of proteasome vs. FasL pathways to T cell death not quantified\", \"In vivo immune phenotype of ALG-2 deficiency not established\", \"Rpn3 interaction awaits independent replication\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Establishing that ALG-2 suppresses STING ER-to-vesicle trafficking in a Ca²⁺-dependent manner, with knockout cells showing enhanced type I IFN signaling, placed ALG-2 as a negative regulator of innate immunity and answered how Ca²⁺ sensing intersects with antiviral defense.\",\n      \"evidence\": \"ALG-2 knockout in THP-1 cells, co-IP, immunofluorescence trafficking assay, Ca²⁺-binding mutant, HSV-1 and cGAMP stimulation\",\n      \"pmids\": [\"34787301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which ALG-2 retains STING in the ER is unclear\", \"Whether ALIX or ESCRT is involved was not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that the ALG-2/peflin heterodimer buffers secretion at steady state while Ca²⁺ signals shift the balance toward peflin-free ALG-2 complexes that stimulate secretion resolved the apparent contradiction of ALG-2 sometimes promoting and sometimes inhibiting COPII function.\",\n      \"evidence\": \"Peflin/ALG-2 double-knockdown epistasis, SEAP and collagen I secretion assays, ERES fractionation in NRK and PC12 cells\",\n      \"pmids\": [\"34762908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative Ca²⁺ thresholds for heterodimer vs. homodimer switching unknown\", \"Whether peflin regulates ESCRT-related functions was untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Kinetic analysis of purified human ALG2 confirmed the α1,3-before-α1,6 mannosylation order under physiological conditions and revealed a single membrane-anchoring domain (unlike yeast), resolving species-specific topology differences.\",\n      \"evidence\": \"LC-MS quantitative kinetics with purified hAlg2 and defined LLO substrates, topology analysis\",\n      \"pmids\": [\"35136180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal structure of human ALG2 mannosyltransferase available\", \"Regulation of catalytic activity in vivo is unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Three landmark studies resolved how ALG-2 reaches membranes—via direct electrostatic/hydrophobic membrane binding distinct from protein-mediated recruitment—and established new functional roles at lysosomes (ESCRT-dependent resilience to osmotic stress) and in nutrient-stress-triggered ERES microautophagy requiring ALG-2, ALIX, and ubiquitinated SEC31.\",\n      \"evidence\": \"GUV membrane binding, MD simulations, charge-reversal mutagenesis, lysosomal leakage assays with ΔGF122 variant, super-resolution imaging, FIB-SEM, in vitro GUV reconstitution of ERES microautophagy, ALG-2 knockout\",\n      \"pmids\": [\"38386713\", \"38781205\", \"38593803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ERES microautophagy is selective for specific cargoes is unknown\", \"Relative contribution of direct membrane binding vs. protein-mediated recruitment in vivo is unquantified\", \"Whether lysosomal resilience function is relevant in disease settings remains untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis of ALG-2 bridging ESCRT-I subunits (no ESCRT-I co-crystal), whether ALG-2 functions overlap or are separable in vivo (no animal knockout phenotype published), and how ALG-2 nuclear import is regulated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No ALG-2 knockout mouse or in vivo phenotyping reported\", \"No co-crystal structure of ALG-2 with full-length ALIX or ESCRT-I\", \"Mechanism of ALG-2 nuclear import unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [21, 26, 31, 33, 45]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [44]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [12, 17, 23, 43]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 8, 18]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [16, 25, 26, 32, 41]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [13, 31]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [19, 27]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [42, 44, 45]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [35, 44]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [16, 26, 32, 33, 41, 46]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 20, 36]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [12, 17, 23, 43]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [39]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [42, 46]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [12, 28]}\n    ],\n    \"complexes\": [\n      \"ALG-2/peflin heterodimer\",\n      \"ALG-2 homodimer\",\n      \"Alg1/Alg2/Alg11 mannosyltransferase complex\"\n    ],\n    \"partners\": [\n      \"PDCD6IP\",\n      \"SEC31A\",\n      \"TSG101\",\n      \"PEF1\",\n      \"TFG\",\n      \"STING1\",\n      \"CHERP\",\n      \"MCOLN1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}