{"gene":"ALG2","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":1996,"finding":"ALG-2 (PDCD6) is a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death in T cell hybridoma; loss-of-function via antisense depletion protects cells from these apoptotic stimuli.","method":"Functional genetic screen ('death trap' assay), antisense depletion, cell death assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — original discovery paper, replicated by multiple subsequent studies confirming ALG-2 requirement for apoptosis","pmids":["8560270"],"is_preprint":false},{"year":1997,"finding":"ALG-2 functions downstream of or independently of ICE/Ced-3 caspase activation during apoptosis: ALG-2-depleted T cells show normal caspase activation (PARP cleavage) yet are protected from death, placing ALG-2 downstream of caspases.","method":"Fluorogenic caspase substrate assay, PARP cleavage assay in ALG-2-depleted clones","journal":"Journal of Immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct epistasis experiment with caspase activity measurement, consistent with companion papers","pmids":["9164928"],"is_preprint":false},{"year":1999,"finding":"ALG-2 interacts with AIP1/ALIX in a strictly Ca2+-dependent manner; AIP1 and ALG-2 co-localize in the cytosol; overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death, indicating AIP1 cooperates with ALG-2 in the Ca2+-dependent cell death pathway.","method":"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence colocalization, overexpression rescue","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction confirmed by yeast two-hybrid and co-IP, replicated by independent group (PMID:10200558)","pmids":["9880530","10200558"],"is_preprint":false},{"year":1998,"finding":"ALG-2 is a penta-EF-hand Ca2+-binding protein that exposes a hydrophobic surface in a Ca2+-concentration-dependent manner (half-maximal ~6 µM Ca2+), consistent with Ca2+-dependent conformational change enabling target binding; Mg2+ is not effective.","method":"Fluorescent hydrophobicity probe (TNS), gel filtration, Ca2+-dependent precipitation assay with recombinant ALG-2","journal":"Journal of Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assay with purified recombinant protein, single lab","pmids":["9832622"],"is_preprint":false},{"year":1999,"finding":"Ca2+-free ALG-2 forms a weak homodimer; the fifth EF-hand (EF5) is required for dimerization; ALG-2 possesses two strong Ca2+-binding sites, and Ca2+ binding to both sites is required for Ca2+-induced protein aggregation.","method":"Gel filtration, chemical cross-linking, mutagenesis, fluorescence spectroscopy, circular dichroism with truncated and point mutants","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis and multiple spectroscopic methods, replicated by structural studies","pmids":["10360947"],"is_preprint":false},{"year":2000,"finding":"ALG-2 exists as two alternatively spliced isoforms (ALG-2,5 and ALG-2,1 lacking Gly121/Phe122); ALG-2,5 but not ALG-2,1 interacts with AIP1/Alix, demonstrating that these two residues are required for target recognition.","method":"Yeast two-hybrid, sequence analysis of two mRNA clones, Ca2+ binding assays","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — yeast two-hybrid with isoform comparison, Ca2+ binding measured; single lab","pmids":["10744743"],"is_preprint":false},{"year":2001,"finding":"X-ray crystal structure (2.3 Å) of Ca2+-loaded des1-20 ALG-2 revealed eight α-helices forming five EF-hands, dimer formation via EF5, and Ca2+ binding at EF1, EF3, and EF5. Ca2+ induces a rigid-body rotation between N- and C-terminal halves, exposing a hydrophobic patch and a cleft for peptide binding; a Gly/Pro-rich decapeptide occupies this cleft.","method":"X-ray crystallography at 2.3 Å resolution, limited proteolysis for crystallizable fragment","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation of conformational change mechanism","pmids":["11525164"],"is_preprint":false},{"year":2001,"finding":"ALG-2 forms a Ca2+-independent homodimer and a Ca2+-dependent heterodimer with peflin; in the presence of Ca2+, peflin dissociates from ALG-2, suggesting that peflin modulates ALG-2 function in Ca2+ signaling. In Ca2+ conditions, peflin translocates to the membrane/cytoskeletal fraction.","method":"Co-immunoprecipitation, gel filtration, immunofluorescence, subcellular fractionation","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus subcellular fractionation, replicated in multiple subsequent papers","pmids":["11278427"],"is_preprint":false},{"year":2002,"finding":"ALG-2 directly interacts with the N-terminal domains of annexin VII and annexin XI in a strictly Ca2+-dependent manner through their Pro/Gly/Ala/Tyr/Gln-rich regions, with high-affinity Kd ~40–70 nM and a low-affinity site (~500–700 nM).","method":"Yeast two-hybrid, biotin-tagged ALG-2 overlay assay, GST pulldown, surface plasmon resonance (SPR)","journal":"Biochimica et Biophysica Acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding demonstrated by overlay assay and SPR kinetics with Kd measurement, single lab","pmids":["12445460","11883939"],"is_preprint":false},{"year":2002,"finding":"ALG-2 interacts with ASK1 (apoptosis signal-regulating kinase 1) at its C-terminus (aa 941–1375); ALG-2,1 isoform (lacking Gly121/Phe122) does not bind ASK1; co-transfection of ALG-2 causes nuclear localization of ASK1 and inhibits ASK1-induced JNK activation.","method":"Co-immunoprecipitation, in vitro binding, co-transfection, immunofluorescence, JNK activity assay","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional readout (JNK activation), isoform specificity control, single lab","pmids":["12372597"],"is_preprint":false},{"year":2002,"finding":"EF5 region of both ALG-2 and peflin is essential for their dimerization and protein stability; EF5-deletion mutants are rapidly degraded by the proteasome, demonstrating that dimerization through EF5 is required for protein stability.","method":"Transient expression of deletion mutants, pulse-chase, proteasome inhibitor (MG132) treatment, Western blot","journal":"Archives of Biochemistry and Biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with proteasome inhibitor rescue, single lab","pmids":["11883899"],"is_preprint":false},{"year":2003,"finding":"Human ALG2 (hALG2, gene CDG1I/hALPG2) encodes an α1,3-mannosyltransferase (GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase) that elongates Man1GlcNAc2-PP-dolichol; deficiency causes CDG-Ii with accumulation of Man1- and Man2-GlcNAc2-PP-dolichol; wild-type but not mutant hALG2 cDNA rescues mannosyltransferase activity in patient fibroblasts and yeast alg2-1 mutant.","method":"Enzymatic activity assay in patient fibroblast extracts, complementation of yeast alg2-1 mutant, genetic analysis (heterozygous deletion/substitution), cDNA rescue","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic assay plus yeast complementation plus patient genetic validation with rescue by wild-type cDNA","pmids":["12684507"],"is_preprint":false},{"year":2004,"finding":"ALG-2 directly binds to the Pro-rich C-terminal region (aa 794–827, containing four PxY repeats) of Alix/AIP1 in a Ca2+-dependent manner; alanine substitutions of Pro and Tyr residues in PxY repeats reduce binding; ALG-2(E47A/E114A) Ca2+-binding-deficient mutant does not co-immunoprecipitate with Alix.","method":"Yeast two-hybrid, biotin-tagged ALG-2 overlay assay, co-immunoprecipitation, fluorescence microscopy","journal":"Journal of Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding established by overlay assay and mutagenesis, functional validation by co-IP with Ca2+ dependency control","pmids":["14999017"],"is_preprint":false},{"year":2004,"finding":"Yeast Alg1, Alg2, and Alg11 mannosyltransferases physically interact to form two distinct complexes in the ER; one complex contains Alg1+Alg2 and another contains Alg1+Alg11; Alg1 self-assembles through a C-terminal domain distinct from its Alg2/Alg11-interaction regions; catalytically inactive Alg1 missense mutants cause dominant-negative phenotypes, providing genetic evidence for functional Alg1-containing complexes.","method":"Biochemical co-purification, genetic epistasis (dominant-negative alleles), yeast genetics","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted complex biochemically and validated by dominant-negative genetics","pmids":["15044395"],"is_preprint":false},{"year":2005,"finding":"ALG-2 directly binds to the proline-rich region (PRR) of TSG101 (ESCRT-I component) in a Ca2+-dependent manner; association with hVps28 and hVps37A is indirect, mediated through TSG101; ALG-2 co-localizes with aberrant endosomes (SKD1/Vps4B dominant-negative) in a Ca2+-dependent manner, suggesting a role as a Ca2+-dependent accessory protein of the endosomal sorting machinery.","method":"GST pulldown, yeast two-hybrid, biotin-labeled ALG-2 overlay assay, immunofluorescence microscopy, Ca2+ chelator treatment","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding shown by overlay assay plus cellular localization with Ca2+-dependency controls, single lab","pmids":["16004603"],"is_preprint":false},{"year":2006,"finding":"ALG-2 is recruited to ER exit sites (ERES) by binding to the Pro-rich region of Sec31A (COPII outer coat) in a Ca2+-dependent manner; ALG-2 in turn stabilizes Sec31A at ERES; Ca2+-binding-deficient ALG-2 mutant fails to localize to ERES; Sec31A depletion or Ca2+ chelation (BAPTA-AM) abolishes ALG-2 localization at ERES.","method":"Co-immunoprecipitation, immunofluorescence confocal microscopy, RNAi, Ca2+ chelator treatment, GST pulldown, biotin-labeled ALG-2 overlay assay","journal":"Molecular Biology of the Cell / Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — independently replicated by two labs (PMIDs 16957052 and 17196169) using RNAi and Ca2+ chelation with direct binding assays","pmids":["16957052","17196169"],"is_preprint":false},{"year":2006,"finding":"ALG-2 subcellular distribution oscillates in synchrony with Ca2+ oscillations induced by physiological stimuli (ATP, EGF, prostaglandin, histamine), redistributing from cytosol to punctate (COPII/ERES) localization; Ca2+-binding-deficient ALG-2 mutant does not redistribute, establishing a direct link between Ca2+ signaling and ER-to-Golgi trafficking.","method":"Live-cell fluorescence imaging of tagged ALG-2 combined with Ca2+ indicators, physiological agonist stimulation","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with Ca2+-binding mutant control, single lab","pmids":["17214967"],"is_preprint":false},{"year":2006,"finding":"In Drosophila, POSH (JNK scaffold protein) forms a Ca2+-dependent complex with ALG-2 and ALIX; overexpression of ALG-2 in imaginal eye discs causes eye phenotypes; overexpression of either ALG-2 or ALIX induces ectopic JNK activation, suggesting the POSH/ALG-2/ALIX complex functions in JNK pathway regulation.","method":"Co-immunoprecipitation, genetic overexpression in Drosophila eye discs, JNK activity reporter assay","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vivo Drosophila genetics with functional readout, single lab","pmids":["16698022"],"is_preprint":false},{"year":2006,"finding":"ALG-2 interacts with RBM22 (RNA-binding protein); co-expression of RBM22 causes nuclear translocation of ALG-2 from the cytoplasm, establishing that RBM22 shuttles ALG-2 into the nucleus.","method":"Yeast two-hybrid, fluorescent fusion protein co-transfection and confocal microscopy in NIH 3T3 cells and zebrafish embryos","journal":"Biochimica et Biophysica Acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization in two model systems (mammalian cells and zebrafish), single lab","pmids":["17045351"],"is_preprint":false},{"year":2006,"finding":"In vitro reconstitution with recombinant yeast Alg2 demonstrates that it is a bifunctional enzyme catalyzing both α1,3-mannosylation and α1,6-mannosylation of the dolichylpyrophosphate-linked substrate to form the first branched pentasaccharide intermediate (Man3GlcNAc2-PP-Dol).","method":"In vitro enzymatic assay with purified recombinant Alg2 and defined Dol-PP-linked substrates, mass spectrometry product analysis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with defined substrates demonstrating dual enzymatic activity, replicated by yeast biochemical paper (PMID 19282279)","pmids":["16878994"],"is_preprint":false},{"year":2008,"finding":"ALG-2 and Alix interact with pro-caspase-8; Alix forms a complex with TNF receptor-1 (TNF-R1) dependent on ESCRT-binding capacity; Alix lacking its ALG-2-binding site fails to support TNF-R1-induced cell death, suggesting ALG-2/Alix recruit pro-caspase-8 to endosomes containing TNF-R1 for caspase activation.","method":"Mass spectrometry identification, co-immunoprecipitation, overexpression of deletion mutants, cell death assays","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional rescue with domain-deletion mutant, single lab","pmids":["18936101"],"is_preprint":false},{"year":2009,"finding":"ALG-2 directly binds the NH-terminal cytosolic tail of mucolipin-1 (MCOLN1) in a strictly Ca2+-dependent manner via a patch of charged/hydrophobic residues (MCOLN1 residues 37–49); ALG-2/MCOLN1 co-localize on enlarged endosomes; mutation of the ALG-2-binding domain in MCOLN1 reduces accumulation of aberrant endosomes, indicating ALG-2 regulates MCOLN1 function.","method":"GST pulldown, co-immunoprecipitation, immunofluorescence colocalization, site-directed mutagenesis of binding domain","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding established with mutagenesis and functional readout (endosome morphology), single lab","pmids":["19864416"],"is_preprint":false},{"year":2009,"finding":"Ca2+-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein: ALG-2-dependent Ca2+-mediated pulldown of TSG101 requires the ALG-2-binding site in Alix (not the PSAP motif); ALG-2 knockdown abolishes the Alix-TSG101 association, which is restored by adding recombinant ALG-2. The ALG-2 homodimer (not monomeric or dimerization-defective forms) is required.","method":"Strep-tag pulldown assay, ALG-2 knockdown/reconstitution, use of ALG-2 isoforms and dimerization mutants","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockdown-plus-reconstitution experiment with structural and mutant controls, demonstrates adaptor mechanism directly","pmids":["19520058"],"is_preprint":false},{"year":2009,"finding":"X-ray crystal structural analysis of Ca2+-free vs. Ca2+-bound ALG-2 and its complex with an Alix PPYP-containing peptide revealed: Ca2+ binding at EF3 repositions the Arg125 side chain to open a primary hydrophobic pocket (Pocket 1) that accepts the PPYP motif; the side chain of F122 forms Pocket 2 interactions with the Alix peptide.","method":"X-ray crystallography of Ca2+-free and Ca2+-bound ALG-2 and ALG-2/Alix peptide complex","journal":"Biochemical Society Transactions / BMC Structural Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of apo, Ca2+-bound, and ligand-bound forms with mechanistic insight into Ca2+-gated pocket opening","pmids":["19143629","20691033"],"is_preprint":false},{"year":2009,"finding":"Yeast Alg2 is a bifunctional α1,3- and α1,6-mannosyltransferase that adds both mannose residues to Man1GlcNAc2-PP-Dol on the cytosolic face of the ER membrane; Alg2 has only two functional N-terminal transmembrane segments; Lys230 (not an EX7E motif) is essential for catalytic activity, likely involved in GDP-phosphate binding.","method":"In vitro mannosyltransferase assay, site-directed mutagenesis (including K230 and EX7E motif), topology analysis, truncation mutants","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzyme assay with active-site mutagenesis identifying essential Lys230 residue","pmids":["19282279"],"is_preprint":false},{"year":2010,"finding":"The ALG-2-binding site (ABS) in Sec31A is defined as residues 839–851 in the Pro-rich region; FRAP analysis shows ABS deletion reduces the high-affinity (slow-turnover) population of Sec31A at ERES, establishing that ALG-2 binding to Sec31A determines Sec31A retention kinetics at ERES.","method":"Biotin-labeled ALG-2 overlay assay to map ABS, stable cell lines with GFP-ALG-2 and Sec31A-RFP, FRAP (fluorescence recovery after photobleaching) in live cells","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — FRAP with mutant rescue in live cells; defines ABS residues and functional consequence","pmids":["20834162"],"is_preprint":false},{"year":2011,"finding":"ALG-2 recognizes at least two distinct Pro-containing motifs in its hydrophobic pockets: type 1 (PPYPXnYP, as in ALIX) binds Pocket 1; type 2 (PXPGF, as in Sec31A) binds at a different surface. The alternatively spliced isoform ALG-2(ΔGF122) cannot bind ALIX (Pocket 1 closed) but retains Sec31A binding.","method":"X-ray crystallography, mutational analysis, binding assays summarized in review with structural validation","journal":"Science China Life Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural data with functional mutagenesis confirming two distinct binding pockets, replicating/summarizing multiple primary studies","pmids":["21786200"],"is_preprint":false},{"year":2013,"finding":"ALG-2 attenuates COPII vesicle budding in vitro through Ca2+-dependent interaction with the ALG-2-binding domain in the Pro-rich region of Sec31A; ALG-2/Ca2+ increases recruitment of COPII proteins (Sec23/24, Sec13/31A) to liposomes and mediates Sec13/31A binding to Sec23, stabilizing the outer coat assembly.","method":"In vitro COPII budding assay, liposome recruitment assay, binding assay, ALG-2 EF-hand 1 mutant","journal":"PloS ONE","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of vesicle budding with defined components, active-site (EF-hand) mutant control","pmids":["24069399"],"is_preprint":false},{"year":2013,"finding":"Nuclear ALG-2 interacts Ca2+-dependently with CHERP (Ca2+ homeostasis ER protein) at nuclear speckles; ALG-2 is recruited to CHERP-localizing speckles upon Ca2+ mobilization. Knockdown of either CHERP or ALG-2 causes generation of alternatively spliced IP3R1 isoforms (inclusion of exons 41/42), establishing that the ALG-2/CHERP complex regulates alternative splicing of IP3R1 pre-mRNA.","method":"Co-immunoprecipitation, live-cell time-lapse imaging, siRNA knockdown, RT-PCR splicing analysis, RNA immunoprecipitation","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockdown plus splicing assay plus RNA-IP plus live imaging; multiple orthogonal approaches, single lab","pmids":["24078636"],"is_preprint":false},{"year":2013,"finding":"ALG2 mutations (including p.Val68Gly severely reducing ALG2 expression) cause congenital myasthenic syndrome; reduced ALG2 expression correlates with impaired asparagine-linked glycosylation at the neuromuscular junction.","method":"Linkage analysis, whole-exome/genome sequencing, Western blot of patient muscle and transfected cells","journal":"Brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient mutation with expression analysis; functional mechanism inferred from pathway; single study","pmids":["23404334"],"is_preprint":false},{"year":2013,"finding":"ALG-2 bridges ALIX and ESCRT-I (containing VPS37B or VPS37C preferentially) as a Ca2+-dependent adaptor to form a ternary ESCRT-I/ALIX/ALG-2 complex; this was confirmed using purified recombinant proteins in in vitro binding assays.","method":"Far-Western blot with biotin-labeled ALG-2, pulldown of recombinant ESCRT-I complexes, in vitro binding with purified proteins","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins establishing ternary complex","pmids":["23924735"],"is_preprint":false},{"year":2015,"finding":"X-ray crystal structure of ALG-2 complexed with a Sec31A peptide (type 2 motif PXPGF) revealed that the peptide binds to a third hydrophobic pocket (Pocket 3), distinct from Pocket 1 (ALIX-binding); Phe85 mutation abolishes Sec31A binding without affecting ALIX binding; Tyr180 mutation abolishes ALIX but not Sec31A binding.","method":"X-ray crystallography of ALG-2/Sec31A peptide complex, site-directed mutagenesis of Pockets 1 and 3","journal":"International Journal of Molecular Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus orthogonal mutagenesis confirming distinct binding pockets for two motif types","pmids":["25667979"],"is_preprint":false},{"year":2015,"finding":"Ca2+-dependent ALG-2 interaction with ALIX relieves the intramolecular autoinhibitory interaction of ALIX, promoting CHMP4-dependent ALIX membrane association and enabling ALIX to support MVB sorting of ubiquitinated EGFR; this activation is specific to MVB sorting and does not affect cytokinetic abscission or EIAV budding.","method":"Biochemical activation assays, membrane recruitment assay, EGFR sorting assay, inhibition of ALG-2 binding domain of ALIX","journal":"Cell Discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic dissection using domain mutants showing ALG-2 relieves ALIX autoinhibition, with specific pathway controls","pmids":["27462417"],"is_preprint":false},{"year":2016,"finding":"ALG-2 promotes ER exit site (ERES) localization and Ca2+-dependent polymerization of TFG (Trk-fused gene protein); ALG-2 interacts with TFG via an ALG-2-binding motif (deletion of the motif reduces TFG half-life at ERES); overexpression of ALG-2 increases TFG accumulation at ERES; Ca2+-dependent in vitro cross-linking shows ALG-2 promotes TFG polymerization.","method":"Co-immunoprecipitation, time-lapse live-cell imaging, immunostaining, in vitro cross-linking assay, ERES half-life measurement","journal":"The FEBS Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro polymerization assay plus live-cell imaging with deletion mutant controls, multiple orthogonal methods","pmids":["27813252"],"is_preprint":false},{"year":2016,"finding":"EF5 is the high-affinity Mg2+ binding site in ALG-2; D169A mutation (EF5 +x ligand) eliminates high-affinity Mg2+ binding and also reduces Ca2+ affinity at remaining sites (EF1 and EF3); Mg2+ binding in EF5 stabilizes the ALG-2 dimer.","method":"X-ray crystallography of Mg2+-bound ALG-2, site-directed mutagenesis of D169, calorimetry-based binding analysis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis validating EF5 as Mg2+ site","pmids":["27541325"],"is_preprint":false},{"year":2017,"finding":"ALG-2 interacts with MISSL (MAPK1-interacting and spindle-stabilizing-like) protein Ca2+-dependently; MISSL relocates to ERES upon Ca2+ rise, colocalizing with ALG-2; MISSL or ALG-2 knockdown similarly attenuates SEAP secretion and delays ER-to-Golgi transport of procollagen type I; double knockdown is not additive, placing them in the same pathway. ALG-2 and MISSL interact with MAP1B, which negatively regulates secretion downstream.","method":"Co-immunoprecipitation, live-cell imaging, siRNA knockdown, secretion assay (SEAP), ER-to-Golgi transport assay (procollagen)","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis via non-additive double knockdown plus secretion assay plus transport assay, multiple orthogonal methods","pmids":["28864773"],"is_preprint":false},{"year":2018,"finding":"MAP1B binds to ALG-2 in a Ca2+-dependent manner through a region lacking canonical ABM-1/ABM-2 motifs; MAP1B binding selectively competes with ABM-2-containing proteins (including Sec31A) for ALG-2 binding; in MAP1B KO cells, ALG-2/Sec31A co-localization increases; overexpression of wild-type (but not binding-defective) MAP1B disperses ALG-2 and Sec31A localization.","method":"Pulldown assays, co-immunoprecipitation, immunofluorescence in MAP1B KO cells, mutagenesis","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cell epistasis plus mutagenesis with localization readout, single lab","pmids":["29432744"],"is_preprint":false},{"year":2018,"finding":"ALG-2 participates in plasma membrane damage repair: ALG-2 KO cells (DT-40) are more sensitive to electroporation than wild-type; wild-type ALG-2 (but not Ca2+-binding-deficient mutant) partially protects HeLa cells from digitonin-induced death; a peptide containing the ALIX ALG-2-binding sequence inhibits the protective function.","method":"PDCD6 gene knockout by homologous recombination (DT-40), electroporation sensitivity assay, digitonin cell death assay, peptide competition","journal":"PloS ONE","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with rescue plus Ca2+-mutant and peptide inhibitor controls, single lab","pmids":["30240438"],"is_preprint":false},{"year":2020,"finding":"ALG-2 directly interacts with Rpn3 (a subunit of the 26S proteasome) and regulates proteasome activity in a Ca2+-dependent manner following T cell activation; this influences MCL1 (pro-survival Bcl-2 family member) stability and accelerates T cell apoptosis during contraction.","method":"Co-immunoprecipitation (ALG-2/Rpn3), proteasome activity assay with Ca2+ modulation, MCL1 stability assay (siRNA knockdown), T cell apoptosis assay","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction plus functional assay with Ca2+ dependency, single lab","pmids":["31919392"],"is_preprint":false},{"year":2020,"finding":"ALG-2 interacts with FASLG (FAS ligand) and regulates its intracellular vesicular transport; ALG-2 involvement in FASLG trafficking contributes to T cell apoptosis pathway.","method":"Co-immunoprecipitation, vesicle transport assay","journal":"The Biochemical Journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP plus trafficking assay, limited mechanistic detail in abstract, single lab","pmids":["32766719"],"is_preprint":false},{"year":2020,"finding":"ALG-2 interacts with the cytosolic domain of SARAF (a negative SOCE regulator) Ca2+-dependently via an ABM-2 motif; ALG-2 overexpression interferes with SARAF ubiquitination in a manner requiring intact ALG-2-binding capacity (F228S mutant of SARAF abolishes this); ALG-2 dimer promotes Ca2+-dependent SARAF CytD-to-CytD bridging.","method":"Semi-quantitative in vitro binding assay, pulldown, ubiquitination analysis, half-life assay, mutagenesis","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis-supported binding and ubiquitination assay, single lab","pmids":["32878247"],"is_preprint":false},{"year":2021,"finding":"ALG-2 is required for STING trafficking from the ER: ALG-2 associates with the C-terminal tail of STING Ca2+-dependently; ALG2 knockout markedly increases type I interferon production upon cGAMP/HSV-1; ALG2 inhibits STING translocation from ER to perinuclear region; Ca2+ coordination by ALG2 is required for regulation of STING trafficking.","method":"Co-immunoprecipitation, ALG2 KO (THP-1 monocytes), interferon reporter assay, HSV-1 infection, immunofluorescence of STING trafficking","journal":"Journal of Cell Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with functional IFN readout plus co-IP and Ca2+ dependency; single lab","pmids":["34787301"],"is_preprint":false},{"year":2021,"finding":"ALG-2 and peflin together constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling: at steady-state Ca2+, ALG-2/peflin heterocomplexes at ERES confer a buffered (reduced) secretion rate, while peflin-lacking ALG-2 complexes stimulate secretion; Ca2+ signaling can either increase or decrease ER export depending on signal intensity/duration, cell type, and peflin availability.","method":"Secretion assays (constitutive and regulated), ERES localization analysis of COPII components, Ca2+ agonist stimulation in NRK and PC12 cells, ALG-2/peflin knockdown","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell types, bidirectional functional effects with mechanistic dissection of peflin/ALG-2 complexes, multiple orthogonal methods","pmids":["34762908"],"is_preprint":false},{"year":2021,"finding":"CDIP1 (pro-apoptotic p53 target) interacts with ALG-2 Ca2+-dependently; ALG-2 promotes the association between CDIP1 and ESCRT-I (preferentially containing VPS37B or VPS37C); ALG-2 and ESCRT-I together enhance CDIP1-induced caspase-3/7-mediated cell death.","method":"Co-immunoprecipitation, GFP-CDIP1 overexpression, caspase-3/7 activity assay, co-expression studies with ALG-2 and ESCRT-I variants","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus caspase assay with multiple ESCRT-I subunit controls, single lab","pmids":["33503978"],"is_preprint":false},{"year":2022,"finding":"Human Alg2 (hAlg2) has a single membrane-binding domain (not four as in yeast Alg2); under physiological conditions, hAlg2 prefers to transfer α1,3-mannose onto M1Gn2 before adding α1,6-mannose; this order preference is altered by excess GDP-Man or elevated M1Gn2, which triggers α1,6-Man addition first; both reactions occur on the cytosolic face of the ER.","method":"LC-MS quantitative kinetics assay with purified hAlg2, topology analysis, substrate competition assays","journal":"Communications Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — purified enzyme kinetics with defined substrates, topology validated, multiple substrate conditions tested","pmids":["35136180"],"is_preprint":false},{"year":2022,"finding":"Ca2+-mediated ALG-2 recruitment to lysosomes participates in a JIP4-TRPML1-ALG2 pathway controlling lysosomal retrograde transport; oxidative stress (acrolein, H2O2) activates this pathway via TRPML1-mediated Ca2+ release; phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ regulates this system.","method":"Lysosomal positioning assays, JIP4 KO cells, Ca2+ flux assays, phosphomimetic/KO mutants, pharmacological TRPML1 activation","journal":"The EMBO Journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO and phosphomimetic epistasis with lysosome positioning readout, single lab","pmids":["36394115"],"is_preprint":false},{"year":2024,"finding":"ALG-2 binds directly to acidic phospholipid membranes in a Ca2+-dependent manner through electrostatic and hydrophobic interactions; charge-reversed mutations at membrane-binding residues reduce ERES localization upon Ca2+ release but still permit lysosomal localization (rescued by ESCRT-I binding); in vitro reconstitution shows ESCRT-I binding can rescue the ALG-2 membrane-binding defect for lysosomal function.","method":"Giant unilamellar vesicle (GUV) experiments, molecular dynamics simulations, charge-reversal mutagenesis, live-cell localization assay, in vitro reconstitution with purified components","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with GUVs plus MD simulation plus mutagenesis plus live-cell imaging, multiple orthogonal approaches","pmids":["38386713"],"is_preprint":false},{"year":2024,"finding":"ALG-2 recruits ESCRT proteins to lysosomes in response to Ca2+ release (from GPN-induced osmotic stress or TRPML1 activation) and enhances lysosomal membrane resilience; ALG-2 ΔGF122 splice variant (ESCRT-binding disabled) fails to protect lysosomes; ERES microautophagy during nutrient stress (mTOR inhibition/amino acid starvation) requires ALG-2, ubiquitinated SEC31, and ALIX; ALG2 KO prevents ERES engulfment by lysosomes.","method":"Lysosomal leakage/rupture assays, Ca2+ chelation, live-cell super-resolution imaging, FIB-SEM, ALG2 KO cells, TRPML1 pharmacological activation, in vitro reconstitution with lysosomal lipid-mimicking GUVs and purified recombinant components","journal":"Proceedings of the National Academy of Sciences / Developmental Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components plus KO cells plus super-resolution imaging; replicated across two independent studies","pmids":["38781205","38593803"],"is_preprint":false}],"current_model":"ALG-2 (PDCD6) is a penta-EF-hand Ca2+-sensor that functions in two molecularly distinct capacities: (1) as the α-1,3/α-1,6-mannosyltransferase that adds the first two mannose branches to Man1GlcNAc2-PP-dolichol in ER-luminal N-glycan assembly, deficiency of which causes CDG type I; and (2) as a cytosolic Ca2+-dependent adaptor protein that, upon Ca2+ binding, undergoes a conformational change opening hydrophobic pockets (Pocket 1 for PPYP-type motifs in ALIX/TSG101; Pocket 3 for PXPGF-type motifs in Sec31A) to bridge ESCRT components (ALIX, TSG101, ESCRT-I) and COPII components (Sec31A, TFG, MISSL) on ER exit site and endolysosomal membranes, thereby regulating ER-to-Golgi vesicular transport, MVB sorting of ubiquitinated receptors, lysosomal membrane repair and resilience via ESCRT recruitment, ERES microautophagy under nutrient stress, alternative splicing of IP3R1 (via nuclear CHERP), STING trafficking, and T cell apoptosis."},"narrative":{"mechanistic_narrative":"ALG2 (PDCD6) is a penta-EF-hand Ca2+-sensor protein that couples Ca2+ signaling to membrane-trafficking and cell-death pathways by acting as a Ca2+-gated adaptor [PMID:8560270, PMID:9832622, PMID:11525164]. Ca2+ binding (half-maximal ~6 µM) drives a rigid-body conformational change that exposes a hydrophobic surface and opens distinct ligand-binding pockets: Pocket 1 accepts type-1 PPYP motifs (as in ALIX) while a separate Pocket 3 accepts type-2 PXPGF motifs (as in Sec31A), explaining how a single sensor selectively engages different partners [PMID:11525164, PMID:19143629, PMID:20691033, PMID:25667979, PMID:21786200]. Through EF5, ALG-2 forms homodimers and heterodimers with peflin; EF5-mediated dimerization is required for protein stability and underlies its bridging function [PMID:10360947, PMID:11278427, PMID:11883899, PMID:27541325]. As a Ca2+-dependent adaptor, ALG-2 bridges the ESCRT machinery — directly binding ALIX and the TSG101 proline-rich region and assembling a ternary ESCRT-I/ALIX/ALG-2 complex — and relieves ALIX autoinhibition to support MVB sorting of ubiquitinated EGFR [PMID:9880530, PMID:10200558, PMID:14999017, PMID:16004603, PMID:19520058, PMID:23924735, PMID:27462417]. At ER exit sites it binds the Sec31A proline-rich region to stabilize COPII outer-coat assembly and tune ER-to-Golgi export, with peflin availability setting whether secretion is buffered or stimulated; it also recruits TFG and MISSL to ERES [PMID:16957052, PMID:17196169, PMID:20834162, PMID:24069399, PMID:34762908, PMID:27813252, PMID:28864773]. Ca2+-dependent membrane binding to acidic phospholipids targets ALG-2 to ERES and lysosomes, where it recruits ESCRT proteins to confer lysosomal membrane resilience and to drive ERES microautophagy under nutrient stress [PMID:38386713, PMID:38781205, PMID:38593803]. In the nucleus, ALG-2 partners with CHERP to control alternative splicing of IP3R1 pre-mRNA [PMID:24078636]. ALG-2 was originally identified as a Ca2+-binding protein required for T-cell-receptor-, Fas-, and glucocorticoid-induced apoptosis, acting downstream of caspase activation [PMID:8560270, PMID:9164928]. Distinctly, the human ALG2 gene also encodes an ER mannosyltransferase activity: it is a bifunctional α1,3/α1,6-mannosyltransferase that elongates Man1GlcNAc2-PP-dolichol on the cytosolic ER face during N-glycan assembly, and loss-of-function mutations cause a congenital disorder of glycosylation and congenital myasthenic syndrome [PMID:12684507, PMID:16878994, PMID:19282279, PMID:35136180, PMID:23404334].","teleology":[{"year":1996,"claim":"Established ALG-2 as a Ca2+-binding protein functionally required for programmed cell death, defining its first biological role.","evidence":"Death-trap functional screen with antisense depletion in T cell hybridoma","pmids":["8560270"],"confidence":"High","gaps":["No molecular partner or mechanism identified","Does not place ALG-2 relative to caspase activation"]},{"year":1997,"claim":"Placed ALG-2 downstream of caspase activation, showing it is required for death execution even when caspases are normally activated.","evidence":"Caspase substrate and PARP cleavage assays in ALG-2-depleted clones","pmids":["9164928"],"confidence":"High","gaps":["Effector mechanism downstream of caspases unresolved","No biochemical target identified"]},{"year":1998,"claim":"Defined the biophysical basis of ALG-2 sensing: a Ca2+-concentration-dependent exposure of a hydrophobic surface enabling target binding.","evidence":"TNS hydrophobicity probe and Ca2+-dependent assays with recombinant protein","pmids":["9832622"],"confidence":"Medium","gaps":["Single-lab in vitro biochemistry","Physiological targets of the exposed surface not yet known"]},{"year":1999,"claim":"Identified ALIX/AIP1 as the first Ca2+-dependent ALG-2 partner and connected it to the cell-death pathway.","evidence":"Yeast two-hybrid, co-IP, colocalization and overexpression rescue, replicated independently","pmids":["9880530","10200558"],"confidence":"High","gaps":["Binding motif on ALIX not yet mapped","Functional consequence of the interaction unresolved"]},{"year":1999,"claim":"Showed EF5-mediated dimerization and dual strong Ca2+ sites define ALG-2 oligomeric state and Ca2+-induced aggregation.","evidence":"Gel filtration, cross-linking, mutagenesis and spectroscopy with truncated/point mutants","pmids":["10360947"],"confidence":"High","gaps":["Functional importance of the dimer for target bridging not yet established"]},{"year":2000,"claim":"Demonstrated alternative splicing controls target recognition: Gly121/Phe122 are required for ALIX binding.","evidence":"Yeast two-hybrid isoform comparison and Ca2+ binding assays","pmids":["10744743"],"confidence":"Medium","gaps":["Structural basis for residue requirement not yet defined","Single lab"]},{"year":2001,"claim":"Crystal structure of Ca2+-loaded ALG-2 revealed the rigid-body rotation exposing a peptide-binding cleft, defining the mechanism of Ca2+-gated target engagement.","evidence":"X-ray crystallography at 2.3 Å of a Ca2+-loaded fragment","pmids":["11525164"],"confidence":"High","gaps":["Apo vs Ca2+-bound comparison incomplete at this stage","Specific physiological ligand in the cleft not yet identified"]},{"year":2001,"claim":"Identified peflin as a Ca2+-regulated ALG-2 heterodimer partner, introducing combinatorial control of ALG-2 function.","evidence":"Co-IP, gel filtration, fractionation and immunofluorescence","pmids":["11278427"],"confidence":"High","gaps":["Downstream pathway controlled by peflin/ALG-2 not yet known"]},{"year":2002,"claim":"Expanded the Ca2+-dependent partner set (annexin VII/XI, ASK1) and linked ALG-2 to JNK signaling regulation.","evidence":"Yeast two-hybrid, overlay/GST pulldown, SPR kinetics, co-IP and JNK activity assays","pmids":["12445460","11883939","12372597"],"confidence":"Medium","gaps":["Physiological relevance of annexin and ASK1 interactions in cells limited","Mostly single-lab functional readouts"]},{"year":2002,"claim":"Showed EF5-dependent dimerization governs ALG-2 (and peflin) stability via proteasomal turnover of monomeric forms.","evidence":"Deletion-mutant pulse-chase with MG132 rescue","pmids":["11883899"],"confidence":"Medium","gaps":["E3 ligase mediating degradation not identified","Single lab"]},{"year":2003,"claim":"Established the human ALG2 gene as an α1,3-mannosyltransferase whose deficiency causes a congenital disorder of glycosylation, defining a wholly distinct enzymatic role.","evidence":"Enzyme assay in patient fibroblasts, yeast alg2-1 complementation and cDNA rescue","pmids":["12684507"],"confidence":"High","gaps":["Relationship between the mannosyltransferase and the cytosolic Ca2+-adaptor activities not reconciled","Catalytic mechanism not yet defined"]},{"year":2004,"claim":"Mapped the ALG-2 binding site to the PxY-repeat proline-rich region of ALIX and proved Ca2+-binding-deficient ALG-2 cannot bind, cementing the Ca2+-gated motif-recognition model.","evidence":"Yeast two-hybrid, overlay, co-IP and alanine-scanning mutagenesis","pmids":["14999017"],"confidence":"High","gaps":["Atomic structure of the complex not yet solved at this stage"]},{"year":2004,"claim":"Defined organization of the yeast Alg1/Alg2/Alg11 mannosyltransferase complexes, providing biochemical context for Alg2 in N-glycan assembly.","evidence":"Co-purification and dominant-negative yeast genetics","pmids":["15044395"],"confidence":"High","gaps":["Stoichiometry and human relevance of the complex unresolved"]},{"year":2005,"claim":"Extended ALG-2 adaptor function to the ESCRT-I subunit TSG101, linking ALG-2 to endosomal sorting machinery.","evidence":"GST pulldown, yeast two-hybrid, overlay and Ca2+-dependent endosomal colocalization","pmids":["16004603"],"confidence":"High","gaps":["Direct contribution to cargo sorting not yet demonstrated","Single lab"]},{"year":2006,"claim":"Established ALG-2 as a Ca2+-dependent COPII regulator at ER exit sites that binds and stabilizes Sec31A, connecting Ca2+ signaling to ER-to-Golgi trafficking.","evidence":"Co-IP, confocal imaging, RNAi, Ca2+ chelation and direct binding assays, independently replicated; live-cell imaging linked ALG-2 redistribution to Ca2+ oscillations","pmids":["16957052","17196169","17214967"],"confidence":"High","gaps":["Functional effect on vesicle budding not yet quantified at this stage","Net directional effect on secretion unresolved"]},{"year":2006,"claim":"Revealed additional ALG-2 partners (POSH/ALIX in Drosophila, RBM22) connecting ALG-2 to JNK signaling and to nuclear shuttling.","evidence":"Co-IP, Drosophila eye-disc genetics with JNK reporter, and fluorescent-fusion colocalization in cells and zebrafish","pmids":["16698022","17045351"],"confidence":"Medium","gaps":["Nuclear function of ALG-2 not yet defined","Single labs"]},{"year":2009,"claim":"Demonstrated the adaptor mechanism directly: Ca2+-loaded ALG-2 homodimer bridges ALIX and TSG101, an association lost on ALG-2 knockdown and restored by recombinant protein.","evidence":"Strep-tag pulldown with ALG-2 knockdown/reconstitution and dimerization-mutant controls","pmids":["19520058"],"confidence":"High","gaps":["Cellular cargo specificity of the bridged complex not yet defined"]},{"year":2009,"claim":"Crystal structures of apo, Ca2+-bound and ALIX-peptide-bound ALG-2 explained pocket opening: Ca2+ at EF3 repositions Arg125 to open Pocket 1 for the PPYP motif.","evidence":"X-ray crystallography of multiple ALG-2 states and a complex","pmids":["19143629","20691033"],"confidence":"High","gaps":["Structural basis for type-2 (Sec31A) motif binding not yet solved at this stage"]},{"year":2009,"claim":"Expanded membrane partners to MCOLN1 (lysosomal channel) and identified yeast Alg2 as a bifunctional α1,3/α1,6-mannosyltransferase acting on the cytosolic ER face with an essential active-site Lys.","evidence":"GST pulldown/co-IP/mutagenesis for MCOLN1; in vitro mannosyltransferase assays with topology and active-site mutagenesis for Alg2","pmids":["19864416","16878994","19282279"],"confidence":"High","gaps":["Human enzyme topology and order of mannose addition not yet established","Functional role of MCOLN1 binding in cells limited"]},{"year":2010,"claim":"Quantified the functional consequence of ALG-2–Sec31A binding: the mapped ABS determines the high-affinity, slow-turnover retention of Sec31A at ERES.","evidence":"Overlay-mapping of ABS and FRAP in live cells with deletion mutants","pmids":["20834162"],"confidence":"High","gaps":["Net effect on cargo export rate not yet resolved","Single lab"]},{"year":2011,"claim":"Unified the partner-recognition logic by defining two distinct motif classes (type-1 PPYP and type-2 PXPGF) read by separate ALG-2 surfaces, rationalizing isoform-specific partner selection.","evidence":"Structural and mutational synthesis with binding assays","pmids":["21786200"],"confidence":"High","gaps":["Crystallographic definition of the type-2 pocket still pending at this stage"]},{"year":2013,"claim":"Resolved the directional consequence of ALG-2 in COPII: Ca2+-loaded ALG-2 attenuates budding while enhancing recruitment and stabilizing outer-coat assembly, and bridges ALIX to ESCRT-I as a ternary complex.","evidence":"In vitro COPII budding and liposome recruitment assays with EF-hand mutant; in vitro reconstitution of ESCRT-I/ALIX/ALG-2 with purified proteins","pmids":["24069399","23924735"],"confidence":"High","gaps":["Reconciliation of budding attenuation with coat stabilization not fully resolved"]},{"year":2013,"claim":"Established a nuclear function: the ALG-2/CHERP complex at nuclear speckles controls Ca2+-regulated alternative splicing of IP3R1 pre-mRNA.","evidence":"Co-IP, live imaging, siRNA, RT-PCR splicing analysis and RNA-IP","pmids":["24078636"],"confidence":"High","gaps":["Breadth of ALG-2-regulated splicing events unknown","Single lab"]},{"year":2013,"claim":"Connected ALG2 enzymatic deficiency to human disease beyond CDG, implicating it in congenital myasthenic syndrome via impaired NMJ glycosylation.","evidence":"Linkage/exome sequencing and expression analysis of patient muscle and transfected cells","pmids":["23404334"],"confidence":"Medium","gaps":["Direct mechanistic link from glycosylation defect to NMJ pathology inferred, not proven","Single study"]},{"year":2015,"claim":"Defined the structural basis for type-2 motif binding: Sec31A binds a distinct Pocket 3, with Phe85 and Tyr180 mutations dissociating Sec31A and ALIX binding.","evidence":"Crystal structure of ALG-2/Sec31A peptide complex with pocket mutagenesis","pmids":["25667979"],"confidence":"High","gaps":["Whether both pockets can be occupied simultaneously in trafficking not addressed"]},{"year":2015,"claim":"Showed ALG-2 activates ALIX by relieving its autoinhibition, specifically enabling CHMP4-dependent membrane recruitment and MVB sorting of ubiquitinated EGFR.","evidence":"Biochemical activation, membrane recruitment and EGFR sorting assays with pathway-specific controls","pmids":["27462417"],"confidence":"High","gaps":["Why activation is selective for MVB sorting vs abscission/budding not fully explained"]},{"year":2016,"claim":"Extended ERES regulation to TFG polymerization and defined EF5 as the high-affinity Mg2+ site stabilizing the ALG-2 dimer.","evidence":"Co-IP, live imaging, in vitro cross-linking for TFG; crystallography and calorimetry with D169A mutant for Mg2+","pmids":["27813252","27541325"],"confidence":"High","gaps":["Physiological role of Mg2+ occupancy versus Ca2+ in cells unresolved"]},{"year":2017,"claim":"Placed MISSL in the ALG-2 ERES pathway and identified MAP1B as a downstream negative regulator of secretion, building a multi-component secretion-control module.","evidence":"Co-IP, live imaging, non-additive double knockdown, SEAP and procollagen transport assays","pmids":["28864773"],"confidence":"High","gaps":["Mechanism by which MAP1B negatively regulates secretion not fully defined"]},{"year":2018,"claim":"Showed competitive partner regulation: MAP1B binds ALG-2 and selectively competes off ABM-2 partners such as Sec31A, providing a tuning mechanism for ALG-2 localization.","evidence":"Pulldown, co-IP and immunofluorescence in MAP1B KO cells with mutagenesis","pmids":["29432744"],"confidence":"Medium","gaps":["Physiological trigger for MAP1B competition unknown","Single lab"]},{"year":2018,"claim":"Demonstrated a role in plasma membrane damage repair, dependent on Ca2+ binding and ALIX engagement.","evidence":"PDCD6 KO DT-40 cells, electroporation/digitonin sensitivity assays and peptide competition","pmids":["30240438"],"confidence":"Medium","gaps":["Direct ESCRT recruitment to wounds not yet visualized","Single lab"]},{"year":2020,"claim":"Linked ALG-2 back to apoptosis mechanistically through proteasome regulation (Rpn3/MCL1) and FASLG trafficking, and to SOCE regulation via SARAF.","evidence":"Co-IP, proteasome activity and MCL1 stability assays; FASLG trafficking co-IP; SARAF binding/ubiquitination assays","pmids":["31919392","32766719","32878247"],"confidence":"Medium","gaps":["FASLG interaction rests on a single low-detail Co-IP/trafficking assay","Integration of these activities with the COPII/ESCRT roles unclear"]},{"year":2021,"claim":"Established ALG-2 as a Ca2+-dependent regulator of STING ER-to-perinuclear trafficking that restrains type I interferon, and as a hetero-bifunctional COPII rheostat with peflin.","evidence":"ALG2 KO THP-1 with IFN reporter and STING imaging; secretion assays in NRK/PC12 with ALG-2/peflin knockdown","pmids":["34787301","34762908"],"confidence":"High","gaps":["How peflin availability is set physiologically not defined","STING mechanism: direct trafficking step controlled not resolved"]},{"year":2021,"claim":"Connected the ESCRT adaptor function to apoptosis by showing ALG-2 promotes CDIP1–ESCRT-I association to enhance caspase-dependent death.","evidence":"Co-IP, caspase-3/7 assays and co-expression with ESCRT-I subunit variants","pmids":["33503978"],"confidence":"Medium","gaps":["Endogenous-level relevance limited by overexpression approach","Single lab"]},{"year":2022,"claim":"Defined human Alg2 enzyme architecture and kinetics, showing a single membrane-binding domain and a substrate-dependent order of α1,3- then α1,6-mannose addition on the cytosolic ER face.","evidence":"LC-MS quantitative kinetics with purified hAlg2, topology and substrate competition","pmids":["35136180"],"confidence":"High","gaps":["In vivo regulation of reaction order not established"]},{"year":2022,"claim":"Embedded ALG-2 in a lysosomal stress pathway (JIP4–TRPML1–ALG2) controlling retrograde lysosome transport under oxidative stress.","evidence":"Lysosomal positioning assays, JIP4 KO, Ca2+ flux, phosphomimetics and TRPML1 activation","pmids":["36394115"],"confidence":"Medium","gaps":["Direct ALG-2 effector at lysosomes in this pathway not pinpointed","Single lab"]},{"year":2024,"claim":"Resolved how ALG-2 reaches its membrane targets: direct Ca2+-dependent acidic-phospholipid binding drives ERES localization, while ESCRT-I binding can substitute for membrane binding at lysosomes; ALG-2 recruits ESCRT to confer lysosomal membrane resilience and to drive ERES microautophagy.","evidence":"GUV reconstitution, MD simulation, charge-reversal mutagenesis, super-resolution imaging, FIB-SEM and ALG2 KO across two independent studies","pmids":["38386713","38781205","38593803"],"confidence":"High","gaps":["Quantitative contribution of lipid vs protein recruitment in different compartments not fully partitioned"]},{"year":null,"claim":"How the ER-luminal/cytosolic mannosyltransferase activity and the cytosolic Ca2+-adaptor activities are encoded, partitioned, and regulated within cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No study reconciles the glycosyltransferase and adaptor functions of the same gene","Tissue- and stimulus-specific selection among ESCRT, COPII, splicing and lysosomal roles not defined","Determinants of partner choice in vivo beyond motif type and isoform poorly characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,14,22,30,32]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[11,19,24,44]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[46]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[3,16,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,7,16]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[15,25,27,35,46]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[14,21]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[45,46,47]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[18,28]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[14,15,27,35,42]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,38,43]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,19,24,44]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[47]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[28]}],"complexes":["ESCRT-I/ALIX/ALG-2 ternary complex","ALG-2/peflin heterodimer","COPII outer coat (Sec13/31A) at ERES","Alg1/Alg2/Alg11 mannosyltransferase complex"],"partners":["ALIX","TSG101","SEC31A","PEF1","TFG","MCOLN1","CHERP","MAP1B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75340","full_name":"Programmed cell death protein 6","aliases":["Apoptosis-linked gene 2 protein homolog","ALG-2"],"length_aa":191,"mass_kda":21.9,"function":"Calcium sensor that plays a key role in processes such as endoplasmic reticulum (ER)-Golgi vesicular transport, endosomal biogenesis or membrane repair. Acts as an adapter that bridges unrelated proteins or stabilizes weak protein-protein complexes in response to calcium: calcium-binding triggers exposure of apolar surface, promoting interaction with different sets of proteins thanks to 3 different hydrophobic pockets, leading to translocation to membranes (PubMed:20691033, PubMed:25667979). Involved in ER-Golgi transport by promoting the association between PDCD6IP and TSG101, thereby bridging together the ESCRT-III and ESCRT-I complexes (PubMed:19520058). Together with PEF1, acts as a calcium-dependent adapter for the BCR(KLHL12) complex, a complex involved in ER-Golgi transport by regulating the size of COPII coats (PubMed:27716508). In response to cytosolic calcium increase, the heterodimer formed with PEF1 interacts with, and bridges together the BCR(KLHL12) complex and SEC31 (SEC31A or SEC31B), promoting monoubiquitination of SEC31 and subsequent collagen export, which is required for neural crest specification (PubMed:27716508). Involved in the regulation of the distribution and function of MCOLN1 in the endosomal pathway (PubMed:19864416). Promotes localization and polymerization of TFG at endoplasmic reticulum exit site (PubMed:27813252). Required for T-cell receptor-, Fas-, and glucocorticoid-induced apoptosis (By similarity). May mediate Ca(2+)-regulated signals along the death pathway: interaction with DAPK1 can accelerate apoptotic cell death by increasing caspase-3 activity (PubMed:16132846). Its role in apoptosis may however be indirect, as suggested by knockout experiments (By similarity). May inhibit KDR/VEGFR2-dependent angiogenesis; the function involves inhibition of VEGF-induced phosphorylation of the Akt signaling pathway (PubMed:21893193). In case of infection by HIV-1 virus, indirectly inhibits HIV-1 production by affecting viral Gag expression and distribution (PubMed:27784779) Has a lower Ca(2+) affinity than isoform 1 (By similarity)","subcellular_location":"Endoplasmic reticulum membrane; Cytoplasmic vesicle, COPII-coated vesicle membrane; Cytoplasm; Nucleus; Endosome","url":"https://www.uniprot.org/uniprotkb/O75340/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ALG2","classification":"Common 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biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/29432744","citation_count":8,"is_preprint":false},{"pmid":"30259798","id":"PMC_30259798","title":"Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum.","date":"2018","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30259798","citation_count":8,"is_preprint":false},{"pmid":"34723149","id":"PMC_34723149","title":"The Argonaute Proteins ALG-1 and ALG-2 Are Linked to Stress Resistance and Proteostasis.","date":"2021","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/34723149","citation_count":8,"is_preprint":false},{"pmid":"22837710","id":"PMC_22837710","title":"Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.","date":"2012","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/22837710","citation_count":8,"is_preprint":false},{"pmid":"34106226","id":"PMC_34106226","title":"A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.","date":"2021","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/34106226","citation_count":7,"is_preprint":false},{"pmid":"33644825","id":"PMC_33644825","title":"Mass spectrometry glycophenotype characterization of ALG2-CDG in Argentinean patients with a new genetic variant in homozygosis.","date":"2021","source":"Glycoconjugate journal","url":"https://pubmed.ncbi.nlm.nih.gov/33644825","citation_count":7,"is_preprint":false},{"pmid":"10561453","id":"PMC_10561453","title":"Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus.","date":"1999","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/10561453","citation_count":7,"is_preprint":false},{"pmid":"23296612","id":"PMC_23296612","title":"Biochemical and immunological detection of physical interactions between penta-EF-hand protein ALG-2 and its binding partners.","date":"2013","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/23296612","citation_count":7,"is_preprint":false},{"pmid":"11804327","id":"PMC_11804327","title":"Mapping of the interaction sites between apoptosis linked gene ALG-2 and HEED.","date":"2001","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/11804327","citation_count":7,"is_preprint":false},{"pmid":"35136180","id":"PMC_35136180","title":"Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.","date":"2022","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/35136180","citation_count":6,"is_preprint":false},{"pmid":"12845606","id":"PMC_12845606","title":"ALG2, the Hansenula polymorpha isocitrate lyase gene.","date":"2003","source":"Yeast 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Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/18997320","citation_count":3,"is_preprint":false},{"pmid":"9674578","id":"PMC_9674578","title":"Localization of mRNA for the apoptosis-linked gene ALG-2 in young and aged rat brain.","date":"1998","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/9674578","citation_count":3,"is_preprint":false},{"pmid":"11468406","id":"PMC_11468406","title":"Crystallization and preliminary crystallographic studies of an apoptosis-linked calcium-binding protein ALG-2.","date":"2001","source":"Acta crystallographica. Section D, Biological crystallography","url":"https://pubmed.ncbi.nlm.nih.gov/11468406","citation_count":3,"is_preprint":false},{"pmid":"29758467","id":"PMC_29758467","title":"Ligation events influence ALG-2 dimerization.","date":"2018","source":"Biophysical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29758467","citation_count":2,"is_preprint":false},{"pmid":"38352356","id":"PMC_38352356","title":"Ca 2+ -sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38352356","citation_count":2,"is_preprint":false},{"pmid":"27541325","id":"PMC_27541325","title":"EF5 Is the High-Affinity Mg(2+) Site in ALG-2.","date":"2016","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27541325","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48224,"output_tokens":12146,"usd":0.163431,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23325,"output_tokens":9241,"usd":0.173825,"stage2_stop_reason":"end_turn"},"total_usd":0.337256,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"ALG-2 (PDCD6) is a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death in T cell hybridoma; loss-of-function via antisense depletion protects cells from these apoptotic stimuli.\",\n      \"method\": \"Functional genetic screen ('death trap' assay), antisense depletion, cell death assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — original discovery paper, replicated by multiple subsequent studies confirming ALG-2 requirement for apoptosis\",\n      \"pmids\": [\"8560270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"ALG-2 functions downstream of or independently of ICE/Ced-3 caspase activation during apoptosis: ALG-2-depleted T cells show normal caspase activation (PARP cleavage) yet are protected from death, placing ALG-2 downstream of caspases.\",\n      \"method\": \"Fluorogenic caspase substrate assay, PARP cleavage assay in ALG-2-depleted clones\",\n      \"journal\": \"Journal of Immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct epistasis experiment with caspase activity measurement, consistent with companion papers\",\n      \"pmids\": [\"9164928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 interacts with AIP1/ALIX in a strictly Ca2+-dependent manner; AIP1 and ALG-2 co-localize in the cytosol; overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death, indicating AIP1 cooperates with ALG-2 in the Ca2+-dependent cell death pathway.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence colocalization, overexpression rescue\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction confirmed by yeast two-hybrid and co-IP, replicated by independent group (PMID:10200558)\",\n      \"pmids\": [\"9880530\", \"10200558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"ALG-2 is a penta-EF-hand Ca2+-binding protein that exposes a hydrophobic surface in a Ca2+-concentration-dependent manner (half-maximal ~6 µM Ca2+), consistent with Ca2+-dependent conformational change enabling target binding; Mg2+ is not effective.\",\n      \"method\": \"Fluorescent hydrophobicity probe (TNS), gel filtration, Ca2+-dependent precipitation assay with recombinant ALG-2\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assay with purified recombinant protein, single lab\",\n      \"pmids\": [\"9832622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Ca2+-free ALG-2 forms a weak homodimer; the fifth EF-hand (EF5) is required for dimerization; ALG-2 possesses two strong Ca2+-binding sites, and Ca2+ binding to both sites is required for Ca2+-induced protein aggregation.\",\n      \"method\": \"Gel filtration, chemical cross-linking, mutagenesis, fluorescence spectroscopy, circular dichroism with truncated and point mutants\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis and multiple spectroscopic methods, replicated by structural studies\",\n      \"pmids\": [\"10360947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"ALG-2 exists as two alternatively spliced isoforms (ALG-2,5 and ALG-2,1 lacking Gly121/Phe122); ALG-2,5 but not ALG-2,1 interacts with AIP1/Alix, demonstrating that these two residues are required for target recognition.\",\n      \"method\": \"Yeast two-hybrid, sequence analysis of two mRNA clones, Ca2+ binding assays\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — yeast two-hybrid with isoform comparison, Ca2+ binding measured; single lab\",\n      \"pmids\": [\"10744743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"X-ray crystal structure (2.3 Å) of Ca2+-loaded des1-20 ALG-2 revealed eight α-helices forming five EF-hands, dimer formation via EF5, and Ca2+ binding at EF1, EF3, and EF5. Ca2+ induces a rigid-body rotation between N- and C-terminal halves, exposing a hydrophobic patch and a cleft for peptide binding; a Gly/Pro-rich decapeptide occupies this cleft.\",\n      \"method\": \"X-ray crystallography at 2.3 Å resolution, limited proteolysis for crystallizable fragment\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation of conformational change mechanism\",\n      \"pmids\": [\"11525164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ALG-2 forms a Ca2+-independent homodimer and a Ca2+-dependent heterodimer with peflin; in the presence of Ca2+, peflin dissociates from ALG-2, suggesting that peflin modulates ALG-2 function in Ca2+ signaling. In Ca2+ conditions, peflin translocates to the membrane/cytoskeletal fraction.\",\n      \"method\": \"Co-immunoprecipitation, gel filtration, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus subcellular fractionation, replicated in multiple subsequent papers\",\n      \"pmids\": [\"11278427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 directly interacts with the N-terminal domains of annexin VII and annexin XI in a strictly Ca2+-dependent manner through their Pro/Gly/Ala/Tyr/Gln-rich regions, with high-affinity Kd ~40–70 nM and a low-affinity site (~500–700 nM).\",\n      \"method\": \"Yeast two-hybrid, biotin-tagged ALG-2 overlay assay, GST pulldown, surface plasmon resonance (SPR)\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding demonstrated by overlay assay and SPR kinetics with Kd measurement, single lab\",\n      \"pmids\": [\"12445460\", \"11883939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 interacts with ASK1 (apoptosis signal-regulating kinase 1) at its C-terminus (aa 941–1375); ALG-2,1 isoform (lacking Gly121/Phe122) does not bind ASK1; co-transfection of ALG-2 causes nuclear localization of ASK1 and inhibits ASK1-induced JNK activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding, co-transfection, immunofluorescence, JNK activity assay\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional readout (JNK activation), isoform specificity control, single lab\",\n      \"pmids\": [\"12372597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EF5 region of both ALG-2 and peflin is essential for their dimerization and protein stability; EF5-deletion mutants are rapidly degraded by the proteasome, demonstrating that dimerization through EF5 is required for protein stability.\",\n      \"method\": \"Transient expression of deletion mutants, pulse-chase, proteasome inhibitor (MG132) treatment, Western blot\",\n      \"journal\": \"Archives of Biochemistry and Biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with proteasome inhibitor rescue, single lab\",\n      \"pmids\": [\"11883899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human ALG2 (hALG2, gene CDG1I/hALPG2) encodes an α1,3-mannosyltransferase (GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase) that elongates Man1GlcNAc2-PP-dolichol; deficiency causes CDG-Ii with accumulation of Man1- and Man2-GlcNAc2-PP-dolichol; wild-type but not mutant hALG2 cDNA rescues mannosyltransferase activity in patient fibroblasts and yeast alg2-1 mutant.\",\n      \"method\": \"Enzymatic activity assay in patient fibroblast extracts, complementation of yeast alg2-1 mutant, genetic analysis (heterozygous deletion/substitution), cDNA rescue\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic assay plus yeast complementation plus patient genetic validation with rescue by wild-type cDNA\",\n      \"pmids\": [\"12684507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALG-2 directly binds to the Pro-rich C-terminal region (aa 794–827, containing four PxY repeats) of Alix/AIP1 in a Ca2+-dependent manner; alanine substitutions of Pro and Tyr residues in PxY repeats reduce binding; ALG-2(E47A/E114A) Ca2+-binding-deficient mutant does not co-immunoprecipitate with Alix.\",\n      \"method\": \"Yeast two-hybrid, biotin-tagged ALG-2 overlay assay, co-immunoprecipitation, fluorescence microscopy\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding established by overlay assay and mutagenesis, functional validation by co-IP with Ca2+ dependency control\",\n      \"pmids\": [\"14999017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast Alg1, Alg2, and Alg11 mannosyltransferases physically interact to form two distinct complexes in the ER; one complex contains Alg1+Alg2 and another contains Alg1+Alg11; Alg1 self-assembles through a C-terminal domain distinct from its Alg2/Alg11-interaction regions; catalytically inactive Alg1 missense mutants cause dominant-negative phenotypes, providing genetic evidence for functional Alg1-containing complexes.\",\n      \"method\": \"Biochemical co-purification, genetic epistasis (dominant-negative alleles), yeast genetics\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted complex biochemically and validated by dominant-negative genetics\",\n      \"pmids\": [\"15044395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ALG-2 directly binds to the proline-rich region (PRR) of TSG101 (ESCRT-I component) in a Ca2+-dependent manner; association with hVps28 and hVps37A is indirect, mediated through TSG101; ALG-2 co-localizes with aberrant endosomes (SKD1/Vps4B dominant-negative) in a Ca2+-dependent manner, suggesting a role as a Ca2+-dependent accessory protein of the endosomal sorting machinery.\",\n      \"method\": \"GST pulldown, yeast two-hybrid, biotin-labeled ALG-2 overlay assay, immunofluorescence microscopy, Ca2+ chelator treatment\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding shown by overlay assay plus cellular localization with Ca2+-dependency controls, single lab\",\n      \"pmids\": [\"16004603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 is recruited to ER exit sites (ERES) by binding to the Pro-rich region of Sec31A (COPII outer coat) in a Ca2+-dependent manner; ALG-2 in turn stabilizes Sec31A at ERES; Ca2+-binding-deficient ALG-2 mutant fails to localize to ERES; Sec31A depletion or Ca2+ chelation (BAPTA-AM) abolishes ALG-2 localization at ERES.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence confocal microscopy, RNAi, Ca2+ chelator treatment, GST pulldown, biotin-labeled ALG-2 overlay assay\",\n      \"journal\": \"Molecular Biology of the Cell / Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independently replicated by two labs (PMIDs 16957052 and 17196169) using RNAi and Ca2+ chelation with direct binding assays\",\n      \"pmids\": [\"16957052\", \"17196169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 subcellular distribution oscillates in synchrony with Ca2+ oscillations induced by physiological stimuli (ATP, EGF, prostaglandin, histamine), redistributing from cytosol to punctate (COPII/ERES) localization; Ca2+-binding-deficient ALG-2 mutant does not redistribute, establishing a direct link between Ca2+ signaling and ER-to-Golgi trafficking.\",\n      \"method\": \"Live-cell fluorescence imaging of tagged ALG-2 combined with Ca2+ indicators, physiological agonist stimulation\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with Ca2+-binding mutant control, single lab\",\n      \"pmids\": [\"17214967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In Drosophila, POSH (JNK scaffold protein) forms a Ca2+-dependent complex with ALG-2 and ALIX; overexpression of ALG-2 in imaginal eye discs causes eye phenotypes; overexpression of either ALG-2 or ALIX induces ectopic JNK activation, suggesting the POSH/ALG-2/ALIX complex functions in JNK pathway regulation.\",\n      \"method\": \"Co-immunoprecipitation, genetic overexpression in Drosophila eye discs, JNK activity reporter assay\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vivo Drosophila genetics with functional readout, single lab\",\n      \"pmids\": [\"16698022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 interacts with RBM22 (RNA-binding protein); co-expression of RBM22 causes nuclear translocation of ALG-2 from the cytoplasm, establishing that RBM22 shuttles ALG-2 into the nucleus.\",\n      \"method\": \"Yeast two-hybrid, fluorescent fusion protein co-transfection and confocal microscopy in NIH 3T3 cells and zebrafish embryos\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization in two model systems (mammalian cells and zebrafish), single lab\",\n      \"pmids\": [\"17045351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In vitro reconstitution with recombinant yeast Alg2 demonstrates that it is a bifunctional enzyme catalyzing both α1,3-mannosylation and α1,6-mannosylation of the dolichylpyrophosphate-linked substrate to form the first branched pentasaccharide intermediate (Man3GlcNAc2-PP-Dol).\",\n      \"method\": \"In vitro enzymatic assay with purified recombinant Alg2 and defined Dol-PP-linked substrates, mass spectrometry product analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with defined substrates demonstrating dual enzymatic activity, replicated by yeast biochemical paper (PMID 19282279)\",\n      \"pmids\": [\"16878994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 and Alix interact with pro-caspase-8; Alix forms a complex with TNF receptor-1 (TNF-R1) dependent on ESCRT-binding capacity; Alix lacking its ALG-2-binding site fails to support TNF-R1-induced cell death, suggesting ALG-2/Alix recruit pro-caspase-8 to endosomes containing TNF-R1 for caspase activation.\",\n      \"method\": \"Mass spectrometry identification, co-immunoprecipitation, overexpression of deletion mutants, cell death assays\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional rescue with domain-deletion mutant, single lab\",\n      \"pmids\": [\"18936101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 directly binds the NH-terminal cytosolic tail of mucolipin-1 (MCOLN1) in a strictly Ca2+-dependent manner via a patch of charged/hydrophobic residues (MCOLN1 residues 37–49); ALG-2/MCOLN1 co-localize on enlarged endosomes; mutation of the ALG-2-binding domain in MCOLN1 reduces accumulation of aberrant endosomes, indicating ALG-2 regulates MCOLN1 function.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, immunofluorescence colocalization, site-directed mutagenesis of binding domain\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding established with mutagenesis and functional readout (endosome morphology), single lab\",\n      \"pmids\": [\"19864416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ca2+-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein: ALG-2-dependent Ca2+-mediated pulldown of TSG101 requires the ALG-2-binding site in Alix (not the PSAP motif); ALG-2 knockdown abolishes the Alix-TSG101 association, which is restored by adding recombinant ALG-2. The ALG-2 homodimer (not monomeric or dimerization-defective forms) is required.\",\n      \"method\": \"Strep-tag pulldown assay, ALG-2 knockdown/reconstitution, use of ALG-2 isoforms and dimerization mutants\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockdown-plus-reconstitution experiment with structural and mutant controls, demonstrates adaptor mechanism directly\",\n      \"pmids\": [\"19520058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"X-ray crystal structural analysis of Ca2+-free vs. Ca2+-bound ALG-2 and its complex with an Alix PPYP-containing peptide revealed: Ca2+ binding at EF3 repositions the Arg125 side chain to open a primary hydrophobic pocket (Pocket 1) that accepts the PPYP motif; the side chain of F122 forms Pocket 2 interactions with the Alix peptide.\",\n      \"method\": \"X-ray crystallography of Ca2+-free and Ca2+-bound ALG-2 and ALG-2/Alix peptide complex\",\n      \"journal\": \"Biochemical Society Transactions / BMC Structural Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of apo, Ca2+-bound, and ligand-bound forms with mechanistic insight into Ca2+-gated pocket opening\",\n      \"pmids\": [\"19143629\", \"20691033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Yeast Alg2 is a bifunctional α1,3- and α1,6-mannosyltransferase that adds both mannose residues to Man1GlcNAc2-PP-Dol on the cytosolic face of the ER membrane; Alg2 has only two functional N-terminal transmembrane segments; Lys230 (not an EX7E motif) is essential for catalytic activity, likely involved in GDP-phosphate binding.\",\n      \"method\": \"In vitro mannosyltransferase assay, site-directed mutagenesis (including K230 and EX7E motif), topology analysis, truncation mutants\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzyme assay with active-site mutagenesis identifying essential Lys230 residue\",\n      \"pmids\": [\"19282279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ALG-2-binding site (ABS) in Sec31A is defined as residues 839–851 in the Pro-rich region; FRAP analysis shows ABS deletion reduces the high-affinity (slow-turnover) population of Sec31A at ERES, establishing that ALG-2 binding to Sec31A determines Sec31A retention kinetics at ERES.\",\n      \"method\": \"Biotin-labeled ALG-2 overlay assay to map ABS, stable cell lines with GFP-ALG-2 and Sec31A-RFP, FRAP (fluorescence recovery after photobleaching) in live cells\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRAP with mutant rescue in live cells; defines ABS residues and functional consequence\",\n      \"pmids\": [\"20834162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ALG-2 recognizes at least two distinct Pro-containing motifs in its hydrophobic pockets: type 1 (PPYPXnYP, as in ALIX) binds Pocket 1; type 2 (PXPGF, as in Sec31A) binds at a different surface. The alternatively spliced isoform ALG-2(ΔGF122) cannot bind ALIX (Pocket 1 closed) but retains Sec31A binding.\",\n      \"method\": \"X-ray crystallography, mutational analysis, binding assays summarized in review with structural validation\",\n      \"journal\": \"Science China Life Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural data with functional mutagenesis confirming two distinct binding pockets, replicating/summarizing multiple primary studies\",\n      \"pmids\": [\"21786200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALG-2 attenuates COPII vesicle budding in vitro through Ca2+-dependent interaction with the ALG-2-binding domain in the Pro-rich region of Sec31A; ALG-2/Ca2+ increases recruitment of COPII proteins (Sec23/24, Sec13/31A) to liposomes and mediates Sec13/31A binding to Sec23, stabilizing the outer coat assembly.\",\n      \"method\": \"In vitro COPII budding assay, liposome recruitment assay, binding assay, ALG-2 EF-hand 1 mutant\",\n      \"journal\": \"PloS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of vesicle budding with defined components, active-site (EF-hand) mutant control\",\n      \"pmids\": [\"24069399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nuclear ALG-2 interacts Ca2+-dependently with CHERP (Ca2+ homeostasis ER protein) at nuclear speckles; ALG-2 is recruited to CHERP-localizing speckles upon Ca2+ mobilization. Knockdown of either CHERP or ALG-2 causes generation of alternatively spliced IP3R1 isoforms (inclusion of exons 41/42), establishing that the ALG-2/CHERP complex regulates alternative splicing of IP3R1 pre-mRNA.\",\n      \"method\": \"Co-immunoprecipitation, live-cell time-lapse imaging, siRNA knockdown, RT-PCR splicing analysis, RNA immunoprecipitation\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockdown plus splicing assay plus RNA-IP plus live imaging; multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"24078636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALG2 mutations (including p.Val68Gly severely reducing ALG2 expression) cause congenital myasthenic syndrome; reduced ALG2 expression correlates with impaired asparagine-linked glycosylation at the neuromuscular junction.\",\n      \"method\": \"Linkage analysis, whole-exome/genome sequencing, Western blot of patient muscle and transfected cells\",\n      \"journal\": \"Brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient mutation with expression analysis; functional mechanism inferred from pathway; single study\",\n      \"pmids\": [\"23404334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALG-2 bridges ALIX and ESCRT-I (containing VPS37B or VPS37C preferentially) as a Ca2+-dependent adaptor to form a ternary ESCRT-I/ALIX/ALG-2 complex; this was confirmed using purified recombinant proteins in in vitro binding assays.\",\n      \"method\": \"Far-Western blot with biotin-labeled ALG-2, pulldown of recombinant ESCRT-I complexes, in vitro binding with purified proteins\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins establishing ternary complex\",\n      \"pmids\": [\"23924735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray crystal structure of ALG-2 complexed with a Sec31A peptide (type 2 motif PXPGF) revealed that the peptide binds to a third hydrophobic pocket (Pocket 3), distinct from Pocket 1 (ALIX-binding); Phe85 mutation abolishes Sec31A binding without affecting ALIX binding; Tyr180 mutation abolishes ALIX but not Sec31A binding.\",\n      \"method\": \"X-ray crystallography of ALG-2/Sec31A peptide complex, site-directed mutagenesis of Pockets 1 and 3\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus orthogonal mutagenesis confirming distinct binding pockets for two motif types\",\n      \"pmids\": [\"25667979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ca2+-dependent ALG-2 interaction with ALIX relieves the intramolecular autoinhibitory interaction of ALIX, promoting CHMP4-dependent ALIX membrane association and enabling ALIX to support MVB sorting of ubiquitinated EGFR; this activation is specific to MVB sorting and does not affect cytokinetic abscission or EIAV budding.\",\n      \"method\": \"Biochemical activation assays, membrane recruitment assay, EGFR sorting assay, inhibition of ALG-2 binding domain of ALIX\",\n      \"journal\": \"Cell Discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic dissection using domain mutants showing ALG-2 relieves ALIX autoinhibition, with specific pathway controls\",\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 (Trk-fused gene protein); ALG-2 interacts with TFG via an ALG-2-binding motif (deletion of the motif reduces TFG half-life at ERES); overexpression of ALG-2 increases TFG accumulation at ERES; Ca2+-dependent in vitro cross-linking shows ALG-2 promotes TFG polymerization.\",\n      \"method\": \"Co-immunoprecipitation, time-lapse live-cell imaging, immunostaining, in vitro cross-linking assay, ERES half-life measurement\",\n      \"journal\": \"The FEBS Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro polymerization assay plus live-cell imaging with deletion mutant controls, multiple orthogonal methods\",\n      \"pmids\": [\"27813252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EF5 is the high-affinity Mg2+ binding site in ALG-2; D169A mutation (EF5 +x ligand) eliminates high-affinity Mg2+ binding and also reduces Ca2+ affinity at remaining sites (EF1 and EF3); Mg2+ binding in EF5 stabilizes the ALG-2 dimer.\",\n      \"method\": \"X-ray crystallography of Mg2+-bound ALG-2, site-directed mutagenesis of D169, calorimetry-based binding analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis validating EF5 as Mg2+ site\",\n      \"pmids\": [\"27541325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALG-2 interacts with MISSL (MAPK1-interacting and spindle-stabilizing-like) protein Ca2+-dependently; MISSL relocates to ERES upon Ca2+ rise, colocalizing with ALG-2; MISSL or ALG-2 knockdown similarly attenuates SEAP secretion and delays ER-to-Golgi transport of procollagen type I; double knockdown is not additive, placing them in the same pathway. ALG-2 and MISSL interact with MAP1B, which negatively regulates secretion downstream.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging, siRNA knockdown, secretion assay (SEAP), ER-to-Golgi transport assay (procollagen)\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis via non-additive double knockdown plus secretion assay plus transport assay, multiple orthogonal methods\",\n      \"pmids\": [\"28864773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MAP1B binds to ALG-2 in a Ca2+-dependent manner through a region lacking canonical ABM-1/ABM-2 motifs; MAP1B binding selectively competes with ABM-2-containing proteins (including Sec31A) for ALG-2 binding; in MAP1B KO cells, ALG-2/Sec31A co-localization increases; overexpression of wild-type (but not binding-defective) MAP1B disperses ALG-2 and Sec31A localization.\",\n      \"method\": \"Pulldown assays, co-immunoprecipitation, immunofluorescence in MAP1B KO cells, mutagenesis\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cell epistasis plus mutagenesis with localization readout, single lab\",\n      \"pmids\": [\"29432744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALG-2 participates in plasma membrane damage repair: ALG-2 KO cells (DT-40) are more sensitive to electroporation than wild-type; wild-type ALG-2 (but not Ca2+-binding-deficient mutant) partially protects HeLa cells from digitonin-induced death; a peptide containing the ALIX ALG-2-binding sequence inhibits the protective function.\",\n      \"method\": \"PDCD6 gene knockout by homologous recombination (DT-40), electroporation sensitivity assay, digitonin cell death assay, peptide competition\",\n      \"journal\": \"PloS ONE\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with rescue plus Ca2+-mutant and peptide inhibitor controls, single lab\",\n      \"pmids\": [\"30240438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 directly interacts with Rpn3 (a subunit of the 26S proteasome) and regulates proteasome activity in a Ca2+-dependent manner following T cell activation; this influences MCL1 (pro-survival Bcl-2 family member) stability and accelerates T cell apoptosis during contraction.\",\n      \"method\": \"Co-immunoprecipitation (ALG-2/Rpn3), proteasome activity assay with Ca2+ modulation, MCL1 stability assay (siRNA knockdown), T cell apoptosis assay\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction plus functional assay with Ca2+ dependency, single lab\",\n      \"pmids\": [\"31919392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 interacts with FASLG (FAS ligand) and regulates its intracellular vesicular transport; ALG-2 involvement in FASLG trafficking contributes to T cell apoptosis pathway.\",\n      \"method\": \"Co-immunoprecipitation, vesicle transport assay\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP plus trafficking assay, limited mechanistic detail in abstract, single lab\",\n      \"pmids\": [\"32766719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 interacts with the cytosolic domain of SARAF (a negative SOCE regulator) Ca2+-dependently via an ABM-2 motif; ALG-2 overexpression interferes with SARAF ubiquitination in a manner requiring intact ALG-2-binding capacity (F228S mutant of SARAF abolishes this); ALG-2 dimer promotes Ca2+-dependent SARAF CytD-to-CytD bridging.\",\n      \"method\": \"Semi-quantitative in vitro binding assay, pulldown, ubiquitination analysis, half-life assay, mutagenesis\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis-supported binding and ubiquitination assay, single lab\",\n      \"pmids\": [\"32878247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG-2 is required for STING trafficking from the ER: ALG-2 associates with the C-terminal tail of STING Ca2+-dependently; ALG2 knockout markedly increases type I interferon production upon cGAMP/HSV-1; ALG2 inhibits STING translocation from ER to perinuclear region; Ca2+ coordination by ALG2 is required for regulation of STING trafficking.\",\n      \"method\": \"Co-immunoprecipitation, ALG2 KO (THP-1 monocytes), interferon reporter assay, HSV-1 infection, immunofluorescence of STING trafficking\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with functional IFN readout plus co-IP and Ca2+ dependency; single lab\",\n      \"pmids\": [\"34787301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG-2 and peflin together constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling: at steady-state Ca2+, ALG-2/peflin heterocomplexes at ERES confer a buffered (reduced) secretion rate, while peflin-lacking ALG-2 complexes stimulate secretion; Ca2+ signaling can either increase or decrease ER export depending on signal intensity/duration, cell type, and peflin availability.\",\n      \"method\": \"Secretion assays (constitutive and regulated), ERES localization analysis of COPII components, Ca2+ agonist stimulation in NRK and PC12 cells, ALG-2/peflin knockdown\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell types, bidirectional functional effects with mechanistic dissection of peflin/ALG-2 complexes, multiple orthogonal methods\",\n      \"pmids\": [\"34762908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CDIP1 (pro-apoptotic p53 target) interacts with ALG-2 Ca2+-dependently; ALG-2 promotes the association between CDIP1 and ESCRT-I (preferentially containing VPS37B or VPS37C); ALG-2 and ESCRT-I together enhance CDIP1-induced caspase-3/7-mediated cell death.\",\n      \"method\": \"Co-immunoprecipitation, GFP-CDIP1 overexpression, caspase-3/7 activity assay, co-expression studies with ALG-2 and ESCRT-I variants\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus caspase assay with multiple ESCRT-I subunit controls, single lab\",\n      \"pmids\": [\"33503978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human Alg2 (hAlg2) has a single membrane-binding domain (not four as in yeast Alg2); under physiological conditions, hAlg2 prefers to transfer α1,3-mannose onto M1Gn2 before adding α1,6-mannose; this order preference is altered by excess GDP-Man or elevated M1Gn2, which triggers α1,6-Man addition first; both reactions occur on the cytosolic face of the ER.\",\n      \"method\": \"LC-MS quantitative kinetics assay with purified hAlg2, topology analysis, substrate competition assays\",\n      \"journal\": \"Communications Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purified enzyme kinetics with defined substrates, topology validated, multiple substrate conditions tested\",\n      \"pmids\": [\"35136180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ca2+-mediated ALG-2 recruitment to lysosomes participates in a JIP4-TRPML1-ALG2 pathway controlling lysosomal retrograde transport; oxidative stress (acrolein, H2O2) activates this pathway via TRPML1-mediated Ca2+ release; phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ regulates this system.\",\n      \"method\": \"Lysosomal positioning assays, JIP4 KO cells, Ca2+ flux assays, phosphomimetic/KO mutants, pharmacological TRPML1 activation\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO and phosphomimetic epistasis with lysosome positioning readout, single lab\",\n      \"pmids\": [\"36394115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG-2 binds directly to acidic phospholipid membranes in a Ca2+-dependent manner through electrostatic and hydrophobic interactions; charge-reversed mutations at membrane-binding residues reduce ERES localization upon Ca2+ release but still permit lysosomal localization (rescued by ESCRT-I binding); in vitro reconstitution shows ESCRT-I binding can rescue the ALG-2 membrane-binding defect for lysosomal function.\",\n      \"method\": \"Giant unilamellar vesicle (GUV) experiments, molecular dynamics simulations, charge-reversal mutagenesis, live-cell localization assay, in vitro reconstitution with purified components\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with GUVs plus MD simulation plus mutagenesis plus live-cell imaging, multiple orthogonal approaches\",\n      \"pmids\": [\"38386713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG-2 recruits ESCRT proteins to lysosomes in response to Ca2+ release (from GPN-induced osmotic stress or TRPML1 activation) and enhances lysosomal membrane resilience; ALG-2 ΔGF122 splice variant (ESCRT-binding disabled) fails to protect lysosomes; ERES microautophagy during nutrient stress (mTOR inhibition/amino acid starvation) requires ALG-2, ubiquitinated SEC31, and ALIX; ALG2 KO prevents ERES engulfment by lysosomes.\",\n      \"method\": \"Lysosomal leakage/rupture assays, Ca2+ chelation, live-cell super-resolution imaging, FIB-SEM, ALG2 KO cells, TRPML1 pharmacological activation, in vitro reconstitution with lysosomal lipid-mimicking GUVs and purified recombinant components\",\n      \"journal\": \"Proceedings of the National Academy of Sciences / Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components plus KO cells plus super-resolution imaging; replicated across two independent studies\",\n      \"pmids\": [\"38781205\", \"38593803\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALG-2 (PDCD6) is a penta-EF-hand Ca2+-sensor that functions in two molecularly distinct capacities: (1) as the α-1,3/α-1,6-mannosyltransferase that adds the first two mannose branches to Man1GlcNAc2-PP-dolichol in ER-luminal N-glycan assembly, deficiency of which causes CDG type I; and (2) as a cytosolic Ca2+-dependent adaptor protein that, upon Ca2+ binding, undergoes a conformational change opening hydrophobic pockets (Pocket 1 for PPYP-type motifs in ALIX/TSG101; Pocket 3 for PXPGF-type motifs in Sec31A) to bridge ESCRT components (ALIX, TSG101, ESCRT-I) and COPII components (Sec31A, TFG, MISSL) on ER exit site and endolysosomal membranes, thereby regulating ER-to-Golgi vesicular transport, MVB sorting of ubiquitinated receptors, lysosomal membrane repair and resilience via ESCRT recruitment, ERES microautophagy under nutrient stress, alternative splicing of IP3R1 (via nuclear CHERP), STING trafficking, and T cell apoptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALG2 (PDCD6) is a penta-EF-hand Ca2+-sensor protein that couples Ca2+ signaling to membrane-trafficking and cell-death pathways by acting as a Ca2+-gated adaptor [#0, #3, #6]. Ca2+ binding (half-maximal ~6 µM) drives a rigid-body conformational change that exposes a hydrophobic surface and opens distinct ligand-binding pockets: Pocket 1 accepts type-1 PPYP motifs (as in ALIX) while a separate Pocket 3 accepts type-2 PXPGF motifs (as in Sec31A), explaining how a single sensor selectively engages different partners [#6, #23, #31, #26]. Through EF5, ALG-2 forms homodimers and heterodimers with peflin; EF5-mediated dimerization is required for protein stability and underlies its bridging function [#4, #7, #10, #34]. As a Ca2+-dependent adaptor, ALG-2 bridges the ESCRT machinery — directly binding ALIX and the TSG101 proline-rich region and assembling a ternary ESCRT-I/ALIX/ALG-2 complex — and relieves ALIX autoinhibition to support MVB sorting of ubiquitinated EGFR [#2, #12, #14, #22, #30, #32]. At ER exit sites it binds the Sec31A proline-rich region to stabilize COPII outer-coat assembly and tune ER-to-Golgi export, with peflin availability setting whether secretion is buffered or stimulated; it also recruits TFG and MISSL to ERES [#15, #25, #27, #42, #33, #35]. Ca2+-dependent membrane binding to acidic phospholipids targets ALG-2 to ERES and lysosomes, where it recruits ESCRT proteins to confer lysosomal membrane resilience and to drive ERES microautophagy under nutrient stress [#46, #47]. In the nucleus, ALG-2 partners with CHERP to control alternative splicing of IP3R1 pre-mRNA [#28]. ALG-2 was originally identified as a Ca2+-binding protein required for T-cell-receptor-, Fas-, and glucocorticoid-induced apoptosis, acting downstream of caspase activation [#0, #1]. Distinctly, the human ALG2 gene also encodes an ER mannosyltransferase activity: it is a bifunctional α1,3/α1,6-mannosyltransferase that elongates Man1GlcNAc2-PP-dolichol on the cytosolic ER face during N-glycan assembly, and loss-of-function mutations cause a congenital disorder of glycosylation and congenital myasthenic syndrome [#11, #19, #24, #44, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established ALG-2 as a Ca2+-binding protein functionally required for programmed cell death, defining its first biological role.\",\n      \"evidence\": \"Death-trap functional screen with antisense depletion in T cell hybridoma\",\n      \"pmids\": [\"8560270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No molecular partner or mechanism identified\", \"Does not place ALG-2 relative to caspase activation\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placed ALG-2 downstream of caspase activation, showing it is required for death execution even when caspases are normally activated.\",\n      \"evidence\": \"Caspase substrate and PARP cleavage assays in ALG-2-depleted clones\",\n      \"pmids\": [\"9164928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effector mechanism downstream of caspases unresolved\", \"No biochemical target identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined the biophysical basis of ALG-2 sensing: a Ca2+-concentration-dependent exposure of a hydrophobic surface enabling target binding.\",\n      \"evidence\": \"TNS hydrophobicity probe and Ca2+-dependent assays with recombinant protein\",\n      \"pmids\": [\"9832622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vitro biochemistry\", \"Physiological targets of the exposed surface not yet known\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified ALIX/AIP1 as the first Ca2+-dependent ALG-2 partner and connected it to the cell-death pathway.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, colocalization and overexpression rescue, replicated independently\",\n      \"pmids\": [\"9880530\", \"10200558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding motif on ALIX not yet mapped\", \"Functional consequence of the interaction unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Showed EF5-mediated dimerization and dual strong Ca2+ sites define ALG-2 oligomeric state and Ca2+-induced aggregation.\",\n      \"evidence\": \"Gel filtration, cross-linking, mutagenesis and spectroscopy with truncated/point mutants\",\n      \"pmids\": [\"10360947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional importance of the dimer for target bridging not yet established\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated alternative splicing controls target recognition: Gly121/Phe122 are required for ALIX binding.\",\n      \"evidence\": \"Yeast two-hybrid isoform comparison and Ca2+ binding assays\",\n      \"pmids\": [\"10744743\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for residue requirement not yet defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Crystal structure of Ca2+-loaded ALG-2 revealed the rigid-body rotation exposing a peptide-binding cleft, defining the mechanism of Ca2+-gated target engagement.\",\n      \"evidence\": \"X-ray crystallography at 2.3 Å of a Ca2+-loaded fragment\",\n      \"pmids\": [\"11525164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Apo vs Ca2+-bound comparison incomplete at this stage\", \"Specific physiological ligand in the cleft not yet identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified peflin as a Ca2+-regulated ALG-2 heterodimer partner, introducing combinatorial control of ALG-2 function.\",\n      \"evidence\": \"Co-IP, gel filtration, fractionation and immunofluorescence\",\n      \"pmids\": [\"11278427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream pathway controlled by peflin/ALG-2 not yet known\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Expanded the Ca2+-dependent partner set (annexin VII/XI, ASK1) and linked ALG-2 to JNK signaling regulation.\",\n      \"evidence\": \"Yeast two-hybrid, overlay/GST pulldown, SPR kinetics, co-IP and JNK activity assays\",\n      \"pmids\": [\"12445460\", \"11883939\", \"12372597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of annexin and ASK1 interactions in cells limited\", \"Mostly single-lab functional readouts\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed EF5-dependent dimerization governs ALG-2 (and peflin) stability via proteasomal turnover of monomeric forms.\",\n      \"evidence\": \"Deletion-mutant pulse-chase with MG132 rescue\",\n      \"pmids\": [\"11883899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating degradation not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the human ALG2 gene as an α1,3-mannosyltransferase whose deficiency causes a congenital disorder of glycosylation, defining a wholly distinct enzymatic role.\",\n      \"evidence\": \"Enzyme assay in patient fibroblasts, yeast alg2-1 complementation and cDNA rescue\",\n      \"pmids\": [\"12684507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between the mannosyltransferase and the cytosolic Ca2+-adaptor activities not reconciled\", \"Catalytic mechanism not yet defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapped the ALG-2 binding site to the PxY-repeat proline-rich region of ALIX and proved Ca2+-binding-deficient ALG-2 cannot bind, cementing the Ca2+-gated motif-recognition model.\",\n      \"evidence\": \"Yeast two-hybrid, overlay, co-IP and alanine-scanning mutagenesis\",\n      \"pmids\": [\"14999017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the complex not yet solved at this stage\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined organization of the yeast Alg1/Alg2/Alg11 mannosyltransferase complexes, providing biochemical context for Alg2 in N-glycan assembly.\",\n      \"evidence\": \"Co-purification and dominant-negative yeast genetics\",\n      \"pmids\": [\"15044395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and human relevance of the complex unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended ALG-2 adaptor function to the ESCRT-I subunit TSG101, linking ALG-2 to endosomal sorting machinery.\",\n      \"evidence\": \"GST pulldown, yeast two-hybrid, overlay and Ca2+-dependent endosomal colocalization\",\n      \"pmids\": [\"16004603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct contribution to cargo sorting not yet demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established ALG-2 as a Ca2+-dependent COPII regulator at ER exit sites that binds and stabilizes Sec31A, connecting Ca2+ signaling to ER-to-Golgi trafficking.\",\n      \"evidence\": \"Co-IP, confocal imaging, RNAi, Ca2+ chelation and direct binding assays, independently replicated; live-cell imaging linked ALG-2 redistribution to Ca2+ oscillations\",\n      \"pmids\": [\"16957052\", \"17196169\", \"17214967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional effect on vesicle budding not yet quantified at this stage\", \"Net directional effect on secretion unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed additional ALG-2 partners (POSH/ALIX in Drosophila, RBM22) connecting ALG-2 to JNK signaling and to nuclear shuttling.\",\n      \"evidence\": \"Co-IP, Drosophila eye-disc genetics with JNK reporter, and fluorescent-fusion colocalization in cells and zebrafish\",\n      \"pmids\": [\"16698022\", \"17045351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear function of ALG-2 not yet defined\", \"Single labs\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated the adaptor mechanism directly: Ca2+-loaded ALG-2 homodimer bridges ALIX and TSG101, an association lost on ALG-2 knockdown and restored by recombinant protein.\",\n      \"evidence\": \"Strep-tag pulldown with ALG-2 knockdown/reconstitution and dimerization-mutant controls\",\n      \"pmids\": [\"19520058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular cargo specificity of the bridged complex not yet defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structures of apo, Ca2+-bound and ALIX-peptide-bound ALG-2 explained pocket opening: Ca2+ at EF3 repositions Arg125 to open Pocket 1 for the PPYP motif.\",\n      \"evidence\": \"X-ray crystallography of multiple ALG-2 states and a complex\",\n      \"pmids\": [\"19143629\", \"20691033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for type-2 (Sec31A) motif binding not yet solved at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Expanded membrane partners to MCOLN1 (lysosomal channel) and identified yeast Alg2 as a bifunctional α1,3/α1,6-mannosyltransferase acting on the cytosolic ER face with an essential active-site Lys.\",\n      \"evidence\": \"GST pulldown/co-IP/mutagenesis for MCOLN1; in vitro mannosyltransferase assays with topology and active-site mutagenesis for Alg2\",\n      \"pmids\": [\"19864416\", \"16878994\", \"19282279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human enzyme topology and order of mannose addition not yet established\", \"Functional role of MCOLN1 binding in cells limited\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Quantified the functional consequence of ALG-2–Sec31A binding: the mapped ABS determines the high-affinity, slow-turnover retention of Sec31A at ERES.\",\n      \"evidence\": \"Overlay-mapping of ABS and FRAP in live cells with deletion mutants\",\n      \"pmids\": [\"20834162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Net effect on cargo export rate not yet resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Unified the partner-recognition logic by defining two distinct motif classes (type-1 PPYP and type-2 PXPGF) read by separate ALG-2 surfaces, rationalizing isoform-specific partner selection.\",\n      \"evidence\": \"Structural and mutational synthesis with binding assays\",\n      \"pmids\": [\"21786200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystallographic definition of the type-2 pocket still pending at this stage\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the directional consequence of ALG-2 in COPII: Ca2+-loaded ALG-2 attenuates budding while enhancing recruitment and stabilizing outer-coat assembly, and bridges ALIX to ESCRT-I as a ternary complex.\",\n      \"evidence\": \"In vitro COPII budding and liposome recruitment assays with EF-hand mutant; in vitro reconstitution of ESCRT-I/ALIX/ALG-2 with purified proteins\",\n      \"pmids\": [\"24069399\", \"23924735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of budding attenuation with coat stabilization not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a nuclear function: the ALG-2/CHERP complex at nuclear speckles controls Ca2+-regulated alternative splicing of IP3R1 pre-mRNA.\",\n      \"evidence\": \"Co-IP, live imaging, siRNA, RT-PCR splicing analysis and RNA-IP\",\n      \"pmids\": [\"24078636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Breadth of ALG-2-regulated splicing events unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected ALG2 enzymatic deficiency to human disease beyond CDG, implicating it in congenital myasthenic syndrome via impaired NMJ glycosylation.\",\n      \"evidence\": \"Linkage/exome sequencing and expression analysis of patient muscle and transfected cells\",\n      \"pmids\": [\"23404334\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanistic link from glycosylation defect to NMJ pathology inferred, not proven\", \"Single study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the structural basis for type-2 motif binding: Sec31A binds a distinct Pocket 3, with Phe85 and Tyr180 mutations dissociating Sec31A and ALIX binding.\",\n      \"evidence\": \"Crystal structure of ALG-2/Sec31A peptide complex with pocket mutagenesis\",\n      \"pmids\": [\"25667979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether both pockets can be occupied simultaneously in trafficking not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed ALG-2 activates ALIX by relieving its autoinhibition, specifically enabling CHMP4-dependent membrane recruitment and MVB sorting of ubiquitinated EGFR.\",\n      \"evidence\": \"Biochemical activation, membrane recruitment and EGFR sorting assays with pathway-specific controls\",\n      \"pmids\": [\"27462417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why activation is selective for MVB sorting vs abscission/budding not fully explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended ERES regulation to TFG polymerization and defined EF5 as the high-affinity Mg2+ site stabilizing the ALG-2 dimer.\",\n      \"evidence\": \"Co-IP, live imaging, in vitro cross-linking for TFG; crystallography and calorimetry with D169A mutant for Mg2+\",\n      \"pmids\": [\"27813252\", \"27541325\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological role of Mg2+ occupancy versus Ca2+ in cells unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed MISSL in the ALG-2 ERES pathway and identified MAP1B as a downstream negative regulator of secretion, building a multi-component secretion-control module.\",\n      \"evidence\": \"Co-IP, live imaging, non-additive double knockdown, SEAP and procollagen transport assays\",\n      \"pmids\": [\"28864773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MAP1B negatively regulates secretion not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed competitive partner regulation: MAP1B binds ALG-2 and selectively competes off ABM-2 partners such as Sec31A, providing a tuning mechanism for ALG-2 localization.\",\n      \"evidence\": \"Pulldown, co-IP and immunofluorescence in MAP1B KO cells with mutagenesis\",\n      \"pmids\": [\"29432744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger for MAP1B competition unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated a role in plasma membrane damage repair, dependent on Ca2+ binding and ALIX engagement.\",\n      \"evidence\": \"PDCD6 KO DT-40 cells, electroporation/digitonin sensitivity assays and peptide competition\",\n      \"pmids\": [\"30240438\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ESCRT recruitment to wounds not yet visualized\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked ALG-2 back to apoptosis mechanistically through proteasome regulation (Rpn3/MCL1) and FASLG trafficking, and to SOCE regulation via SARAF.\",\n      \"evidence\": \"Co-IP, proteasome activity and MCL1 stability assays; FASLG trafficking co-IP; SARAF binding/ubiquitination assays\",\n      \"pmids\": [\"31919392\", \"32766719\", \"32878247\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FASLG interaction rests on a single low-detail Co-IP/trafficking assay\", \"Integration of these activities with the COPII/ESCRT roles unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established ALG-2 as a Ca2+-dependent regulator of STING ER-to-perinuclear trafficking that restrains type I interferon, and as a hetero-bifunctional COPII rheostat with peflin.\",\n      \"evidence\": \"ALG2 KO THP-1 with IFN reporter and STING imaging; secretion assays in NRK/PC12 with ALG-2/peflin knockdown\",\n      \"pmids\": [\"34787301\", \"34762908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How peflin availability is set physiologically not defined\", \"STING mechanism: direct trafficking step controlled not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected the ESCRT adaptor function to apoptosis by showing ALG-2 promotes CDIP1–ESCRT-I association to enhance caspase-dependent death.\",\n      \"evidence\": \"Co-IP, caspase-3/7 assays and co-expression with ESCRT-I subunit variants\",\n      \"pmids\": [\"33503978\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous-level relevance limited by overexpression approach\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined human Alg2 enzyme architecture and kinetics, showing a single membrane-binding domain and a substrate-dependent order of α1,3- then α1,6-mannose addition on the cytosolic ER face.\",\n      \"evidence\": \"LC-MS quantitative kinetics with purified hAlg2, topology and substrate competition\",\n      \"pmids\": [\"35136180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulation of reaction order not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Embedded ALG-2 in a lysosomal stress pathway (JIP4–TRPML1–ALG2) controlling retrograde lysosome transport under oxidative stress.\",\n      \"evidence\": \"Lysosomal positioning assays, JIP4 KO, Ca2+ flux, phosphomimetics and TRPML1 activation\",\n      \"pmids\": [\"36394115\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ALG-2 effector at lysosomes in this pathway not pinpointed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved how ALG-2 reaches its membrane targets: direct Ca2+-dependent acidic-phospholipid binding drives ERES localization, while ESCRT-I binding can substitute for membrane binding at lysosomes; ALG-2 recruits ESCRT to confer lysosomal membrane resilience and to drive ERES microautophagy.\",\n      \"evidence\": \"GUV reconstitution, MD simulation, charge-reversal mutagenesis, super-resolution imaging, FIB-SEM and ALG2 KO across two independent studies\",\n      \"pmids\": [\"38386713\", \"38781205\", \"38593803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of lipid vs protein recruitment in different compartments not fully partitioned\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the ER-luminal/cytosolic mannosyltransferase activity and the cytosolic Ca2+-adaptor activities are encoded, partitioned, and regulated within cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No study reconciles the glycosyltransferase and adaptor functions of the same gene\", \"Tissue- and stimulus-specific selection among ESCRT, COPII, splicing and lysosomal roles not defined\", \"Determinants of partner choice in vivo beyond motif type and isoform poorly characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 14, 22, 30, 32]},\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": [3, 4, 6, 23, 34]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [11, 19, 24, 44]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [46]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [3, 16, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 7, 16]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [15, 25, 27, 35, 46]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [14, 21]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [45, 46, 47]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [18, 28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [14, 15, 27, 35, 42]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 38, 43]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 19, 24, 44]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [47]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"complexes\": [\n      \"ESCRT-I/ALIX/ALG-2 ternary complex\",\n      \"ALG-2/peflin heterodimer\",\n      \"COPII outer coat (Sec13/31A) at ERES\",\n      \"Alg1/Alg2/Alg11 mannosyltransferase complex\"\n    ],\n    \"partners\": [\n      \"ALIX\",\n      \"TSG101\",\n      \"SEC31A\",\n      \"PEF1\",\n      \"TFG\",\n      \"MCOLN1\",\n      \"CHERP\",\n      \"MAP1B\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"ALG2","tier":"IDENTITY","verdict":"Identity concern","subtype":"paralog","uniprot_band":"rich","rules_fired":"R3","issue":"R3: opener equates ALG2 to different HGNC gene PDCD6"},"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}