{"gene":"PLEKHA3","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2000,"finding":"The PH domain of FAPP1 (PLEKHA3) specifically binds phosphatidylinositol-4-phosphate (PtdIns(4)P) in vitro, with distinct selectivity from PtdIns(3,4,5)P3 and other phosphoinositides.","method":"In vitro phosphoinositide-binding assay using recombinant PH domains","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro binding assay with specificity determination, foundational result replicated extensively in subsequent studies across multiple labs","pmids":["11001876"],"is_preprint":false},{"year":2004,"finding":"FAPP1 localizes to the trans-Golgi network (TGN) on nascent carriers and interacts with both PtdIns(4)P and the small GTPase ARF through its PH domain; knockdown or displacement of FAPP1 inhibits cargo transfer to the plasma membrane, and overexpression of FAPP-PH impairs carrier fission, establishing FAPP1 as an essential component of a PtdIns(4)P- and ARF-regulated machinery controlling post-Golgi carrier generation.","method":"Subcellular localization (fluorescence microscopy), siRNA knockdown, overexpression with cargo transport assays, co-immunoprecipitation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (localization, knockdown, overexpression, binding), replicated across subsequent studies","pmids":["15107860"],"is_preprint":false},{"year":2005,"finding":"The FAPP1 PH domain detects PI(4)P in distinct cellular compartments including the Golgi and plasma membrane; PI4KIIalpha and PI4KIIIbeta regulate Golgi PI(4)P pools sensed by FAPP1-PH, whereas plasma membrane PI(4)P pool detected by FAPP1-PH is specifically regulated by PI4KIIIalpha.","method":"GFP-fusion live-cell imaging, siRNA knockdown of individual PI4 kinases, pharmacological inhibition (wortmannin, PAO)","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with genetic and pharmacological perturbations, single lab, multiple orthogonal approaches","pmids":["15635101"],"is_preprint":false},{"year":2010,"finding":"The FAPP1 PH domain contains a prominent hydrophobic wedge that independently tubulates Golgi membranes by penetrating into the lipid leaflet; the mechanism involves electrostatic approach, phosphoinositide sampling, and perpendicular bilayer penetration.","method":"Solution NMR structure of micelle-bound FAPP1-PH domain, mutagenesis of hydrophobic wedge residues, membrane tubulation assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis and functional membrane tubulation assay in a single study","pmids":["20300118"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of the FAPP1 PH domain at 1.9 Å resolution reveals a seven-stranded β-barrel with a lipid-binding pocket; PtdIns(4)P binding is enhanced in acidic environment and required for membrane penetration and tubulation; ARF1 binds to the outer side of the β-barrel at a site distinct from the lipid-binding pocket, allowing simultaneous and independent binding of both ligands.","method":"X-ray crystallography (1.9 Å), NMR resonance perturbation analysis, site-directed mutagenesis, binding affinity measurements, membrane tubulation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with NMR, mutagenesis, and functional assays in a single rigorous study","pmids":["21454700"],"is_preprint":false},{"year":2011,"finding":"Metabolically stabilized analogs of PtdIns(4)P (methylenephosphonate and phosphorothioate derivatives) are recognized by the FAPP1 PH domain and stimulate membrane insertion and tubulation activity of the PH domain, demonstrating that the head group recognition drives membrane remodeling.","method":"Chemical synthesis of PtdIns(4)P analogs, binding assays, membrane tubulation assay","journal":"Chemistry & biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with synthetic analogs, single lab, single study","pmids":["22035800"],"is_preprint":false},{"year":2014,"finding":"At a model membrane surface, yeast Arf1 (yArf1) interacts with the FAPP1-PH domain primarily through contacts between switch I residues of Arf1 and the C-terminal extension of the PH domain; the Arf1 binding site is distinct from the PI4P binding site, supporting coincidence detection of activated ARF and PI4P as the membrane recruitment mechanism.","method":"NMR chemical shift perturbation analysis at model membrane surface, structural modeling","journal":"Structure","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR-based structural mapping at membrane surface, single lab, consistent with crystal structure data from independent study","pmids":["24462251"],"is_preprint":false},{"year":2015,"finding":"The FAPP1-PH domain preferentially targets PI4P-containing liquid disordered membranes over liquid ordered membranes; membrane penetration is mediated by an exposed conserved hydrophobic wedge adjacent to the PI4P recognition site, providing selectivity for dynamic TGN membrane zones.","method":"Liposome sedimentation assays, membrane partitioning assays, NMR spectroscopy with PI4P-containing bicelles","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods (sedimentation, NMR) in single lab, consistent with prior structural studies","pmids":["25579996"],"is_preprint":false},{"year":2019,"finding":"FAPP1 localizes at ER-TGN contact sites (ERTGoCS), physically interacts with the ER phosphatase Sac1, and promotes Sac1's in-trans dephosphorylation of PI4P at the TGN in vitro; depletion of FAPP1 increases TGN PI4P levels and increases secretion of selected cargoes (e.g., ApoB100), establishing FAPP1 as a PI4P detector/adaptor that positions Sac1 to consume TGN PI4P and thereby acts as a gatekeeper of Golgi exit.","method":"Co-immunoprecipitation (FAPP1–Sac1 interaction), in vitro phosphatase assay (Sac1 activity with/without FAPP1), FAPP1 depletion (siRNA) with PI4P level measurement and cargo secretion assay, super-resolution microscopy for ERTGoCS localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, in vitro reconstitution of phosphatase activity, genetic depletion with defined biochemical and secretory phenotypes, multiple orthogonal methods","pmids":["30659099"],"is_preprint":false},{"year":2005,"finding":"FAPP1 depletion (siRNA) had no effect on apical or basolateral transport in polarized MDCK cells, in contrast to FAPP2 depletion which specifically reduced apical transport.","method":"RNA interference, adenovirus-mediated siRNA, transport assays in polarized MDCK cells","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — negative finding from clean siRNA knockdown in defined transport assay; single lab but rigorous transport readout","pmids":["16103222"],"is_preprint":false},{"year":2005,"finding":"A protein named FASP1 (FAPP1-associated protein-1) was identified as a binding partner of pp5644; FASP1 is described as being associated with FAPP1, and co-localizes with pp5644 in the cytoplasm of HeLa cells.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, co-localization by fluorescence microscopy","journal":"Molecular and cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — interaction identified for FASP1 (a FAPP1-associated protein), not directly establishing new mechanism for FAPP1 itself; single lab, limited follow-up","pmids":["15881666"],"is_preprint":false}],"current_model":"FAPP1/PLEKHA3 is a PH domain-containing adaptor protein that is recruited to the trans-Golgi network through coincidence detection of PI(4)P and GTP-bound ARF1 via distinct sites on its PH domain; once at the TGN, FAPP1 promotes post-Golgi carrier formation through membrane tubulation via a hydrophobic wedge, and at ER-TGN contact sites it positions the ER phosphatase Sac1 to dephosphorylate TGN PI(4)P in trans, thereby acting as a gatekeeper that regulates PI(4)P levels and the rate of selective cargo secretion."},"narrative":{"mechanistic_narrative":"PLEKHA3 (FAPP1) is a pleckstrin-homology (PH) domain adaptor that controls PI(4)P homeostasis and selective cargo exit at the trans-Golgi network (TGN) [PMID:15107860, PMID:30659099]. Its PH domain is recruited to the TGN by coincidence detection of two ligands engaged at structurally distinct sites: PtdIns(4)P bound in a lipid-binding pocket and GTP-bound ARF engaged on the outer face of the β-barrel, allowing simultaneous and independent binding of both [PMID:11001876, PMID:21454700, PMID:24462251]. Membrane recognition is sharpened by preferential targeting of PI(4)P-containing liquid-disordered bilayers and by a conserved hydrophobic wedge adjacent to the lipid pocket that penetrates the leaflet and can independently tubulate Golgi membranes, linking head-group recognition to membrane remodeling [PMID:20300118, PMID:22035800, PMID:25579996]. Functionally, FAPP1 is required for post-Golgi carrier generation, as its displacement blocks cargo transfer to the plasma membrane and PH-domain overexpression impairs carrier fission [PMID:15107860]. At ER-TGN contact sites FAPP1 binds the ER phosphatase Sac1 and positions it to dephosphorylate TGN PI(4)P in trans; FAPP1 depletion raises TGN PI(4)P and increases secretion of selected cargoes, defining FAPP1 as a gatekeeper of Golgi exit [PMID:30659099].","teleology":[{"year":2000,"claim":"Established the molecular ligand of FAPP1 by showing its PH domain is a specific PtdIns(4)P reader, distinguishing it from PIP3-binding PH domains and providing the lipid-recognition basis for its function.","evidence":"In vitro phosphoinositide-binding assay with recombinant PH domains","pmids":["11001876"],"confidence":"High","gaps":["Did not address subcellular localization or cellular function","No structural basis for the selectivity"]},{"year":2004,"claim":"Placed FAPP1 in a cellular pathway by showing it localizes to TGN carriers, binds both PI(4)P and ARF, and is required for post-Golgi cargo transport and carrier fission.","evidence":"Fluorescence localization, siRNA knockdown, PH-domain overexpression with cargo transport assays, co-IP","pmids":["15107860"],"confidence":"High","gaps":["Did not resolve how PI(4)P and ARF are simultaneously engaged","Molecular mechanism of carrier fission unresolved"]},{"year":2005,"claim":"Defined which kinases generate the PI(4)P pools FAPP1 senses, distinguishing Golgi pools (PI4KIIalpha/PI4KIIIbeta) from a plasma-membrane pool (PI4KIIIalpha).","evidence":"GFP-fusion live-cell imaging with PI4-kinase siRNA and pharmacological inhibition","pmids":["15635101"],"confidence":"Medium","gaps":["Functional consequence of plasma-membrane PI(4)P sensing not established","Single-lab imaging-based assignment"]},{"year":2005,"claim":"Clarified FAPP1's transport specificity by showing, in contrast to FAPP2, that FAPP1 depletion does not affect apical/basolateral transport in polarized cells.","evidence":"Adenovirus-mediated siRNA with transport assays in polarized MDCK cells","pmids":["16103222"],"confidence":"Medium","gaps":["Negative result; does not exclude roles in non-polarized secretion","Cargo-specific dependencies not tested"]},{"year":2010,"claim":"Revealed how lipid recognition is coupled to membrane remodeling, showing a hydrophobic wedge in the PH domain that penetrates the bilayer and independently tubulates Golgi membranes.","evidence":"Solution NMR of micelle-bound PH domain, wedge mutagenesis, membrane tubulation assay","pmids":["20300118"],"confidence":"High","gaps":["Tubulation shown in vitro; in-cell contribution to fission not directly quantified","Role of ARF in wedge insertion not addressed"]},{"year":2011,"claim":"Provided the high-resolution structural basis for coincidence detection, showing PI(4)P binds a defined pocket while ARF1 binds a distinct outer β-barrel site, permitting independent simultaneous engagement.","evidence":"1.9 Å crystal structure with NMR perturbation, mutagenesis, affinity measurements, tubulation assay","pmids":["21454700"],"confidence":"High","gaps":["Membrane-bound ternary geometry not directly visualized","Regulation of binding by ARF nucleotide state in cells not shown"]},{"year":2011,"claim":"Demonstrated that head-group recognition itself drives remodeling, since stabilized PtdIns(4)P analogs are recognized and stimulate PH-domain membrane insertion and tubulation.","evidence":"Synthetic PI(4)P analogs with binding and membrane tubulation assays","pmids":["22035800"],"confidence":"Medium","gaps":["In vitro analog system; physiological relevance of analogs limited","Single-lab study"]},{"year":2014,"claim":"Mapped the ARF-binding interface at a membrane surface, showing Arf1 switch I contacts the PH domain C-terminal extension at a site distinct from the PI(4)P pocket, confirming the coincidence-detection recruitment model.","evidence":"NMR chemical-shift perturbation at model membrane surface with structural modeling","pmids":["24462251"],"confidence":"Medium","gaps":["Used yeast Arf1; human ARF1 contacts inferred","Affinity and stoichiometry on cellular membranes not measured"]},{"year":2015,"claim":"Refined membrane targeting selectivity, showing preference for PI(4)P in liquid-disordered membranes via a hydrophobic wedge adjacent to the recognition site, tuning FAPP1 to dynamic TGN zones.","evidence":"Liposome sedimentation, membrane partitioning, NMR with PI(4)P bicelles","pmids":["25579996"],"confidence":"Medium","gaps":["Lipid-phase preference shown in reconstituted membranes","Functional impact on cargo selection not tested"]},{"year":2019,"claim":"Redefined FAPP1 as a PI(4)P-consuming gatekeeper rather than only a carrier-forming factor, showing it operates at ER-TGN contacts to position Sac1 for in-trans PI(4)P dephosphorylation and to limit secretion of selected cargoes.","evidence":"Reciprocal co-IP, in vitro Sac1 phosphatase assay, siRNA depletion with PI(4)P and cargo secretion readouts, super-resolution microscopy","pmids":["30659099"],"confidence":"High","gaps":["Cargo-selectivity determinants of the gatekeeper function unresolved","How carrier-formation and PI(4)P-consumption roles are coordinated unclear"]},{"year":null,"claim":"How FAPP1 reconciles its membrane-tubulating carrier-formation activity with its Sac1-mediated PI(4)P-consuming gatekeeper role, and what determines which cargoes it gates, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No model integrating tubulation and PI(4)P-consumption at the TGN","Physiological regulators switching between the two functions unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,3,4,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,8]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[0,8]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,2,8]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,8]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,8]}],"complexes":[],"partners":["ARF1","SACM1L"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9HB20","full_name":"Pleckstrin homology domain-containing family A member 3","aliases":["Phosphatidylinositol-four-phosphate adapter protein 1","FAPP-1","Phosphoinositol 4-phosphate adapter protein 1"],"length_aa":300,"mass_kda":33.9,"function":"Plays a role in regulation of vesicular cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PubMed:15107860). Regulates Golgi phosphatidylinositol 4-phosphate (PtdIns(4)P) levels and activates the PtdIns(4)P phosphatase activity of SACM1L when it binds PtdIns(4)P in 'trans' configuration (PubMed:30659099). Binds preferentially to PtdIns(4)P (PubMed:11001876, PubMed:15107860). Negatively regulates APOB secretion from hepatocytes (PubMed:30659099)","subcellular_location":"Golgi apparatus, trans-Golgi network membrane","url":"https://www.uniprot.org/uniprotkb/Q9HB20/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLEKHA3","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ECPAS","stoichiometry":0.2},{"gene":"OSBP","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PLEKHA3","total_profiled":1310},"omim":[{"mim_id":"607774","title":"PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING PROTEIN, FAMILY A, MEMBER 3; PLEKHA3","url":"https://www.omim.org/entry/607774"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Golgi apparatus","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PLEKHA3"},"hgnc":{"alias_symbol":["FAPP1"],"prev_symbol":[]},"alphafold":{"accession":"Q9HB20","domains":[{"cath_id":"2.30.29.30","chopping":"1-76","consensus_level":"high","plddt":94.3772,"start":1,"end":76},{"cath_id":"1.20.58","chopping":"102-180","consensus_level":"high","plddt":87.4723,"start":102,"end":180}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HB20","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HB20-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HB20-F1-predicted_aligned_error_v6.png","plddt_mean":71.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLEKHA3","jax_strain_url":"https://www.jax.org/strain/search?query=PLEKHA3"},"sequence":{"accession":"Q9HB20","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HB20.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HB20/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HB20"}},"corpus_meta":[{"pmid":"11001876","id":"PMC_11001876","title":"Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11001876","citation_count":480,"is_preprint":false},{"pmid":"15107860","id":"PMC_15107860","title":"FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P.","date":"2004","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15107860","citation_count":451,"is_preprint":false},{"pmid":"16710455","id":"PMC_16710455","title":"Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole.","date":"2006","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/16710455","citation_count":245,"is_preprint":false},{"pmid":"15635101","id":"PMC_15635101","title":"A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/15635101","citation_count":220,"is_preprint":false},{"pmid":"18785997","id":"PMC_18785997","title":"Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.","date":"2008","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18785997","citation_count":142,"is_preprint":false},{"pmid":"16103222","id":"PMC_16103222","title":"FAPP2 is involved in the transport of apical cargo in polarized MDCK cells.","date":"2005","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16103222","citation_count":83,"is_preprint":false},{"pmid":"30659099","id":"PMC_30659099","title":"The activity of Sac1 across ER-TGN contact sites requires the four-phosphate-adaptor-protein-1.","date":"2019","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30659099","citation_count":70,"is_preprint":false},{"pmid":"21454700","id":"PMC_21454700","title":"Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21454700","citation_count":67,"is_preprint":false},{"pmid":"20300118","id":"PMC_20300118","title":"Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domains.","date":"2010","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/20300118","citation_count":65,"is_preprint":false},{"pmid":"25394204","id":"PMC_25394204","title":"Discovery of novel membrane binding structures and functions.","date":"2014","source":"Biochemistry and cell biology = Biochimie et biologie cellulaire","url":"https://pubmed.ncbi.nlm.nih.gov/25394204","citation_count":45,"is_preprint":false},{"pmid":"16053445","id":"PMC_16053445","title":"High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/16053445","citation_count":35,"is_preprint":false},{"pmid":"24462251","id":"PMC_24462251","title":"Interaction of Fapp1 with Arf1 and PI4P at a membrane surface: an example of coincidence detection.","date":"2014","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/24462251","citation_count":33,"is_preprint":false},{"pmid":"25579996","id":"PMC_25579996","title":"Structural basis of dynamic membrane recognition by trans-Golgi network specific FAPP proteins.","date":"2015","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25579996","citation_count":30,"is_preprint":false},{"pmid":"25300501","id":"PMC_25300501","title":"Copy number variants in short children born small for gestational age.","date":"2014","source":"Hormone research in paediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/25300501","citation_count":29,"is_preprint":false},{"pmid":"21119004","id":"PMC_21119004","title":"Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.","date":"2010","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21119004","citation_count":23,"is_preprint":false},{"pmid":"24220498","id":"PMC_24220498","title":"Membranes and mammalian glycolipid transferring proteins.","date":"2013","source":"Chemistry and physics of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/24220498","citation_count":20,"is_preprint":false},{"pmid":"16837555","id":"PMC_16837555","title":"Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta stimulate extracellular signal-regulated kinase 1/2 signaling by accelerating recycling through the endocytic recycling compartment.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16837555","citation_count":19,"is_preprint":false},{"pmid":"30728282","id":"PMC_30728282","title":"Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells.","date":"2019","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/30728282","citation_count":18,"is_preprint":false},{"pmid":"29627573","id":"PMC_29627573","title":"Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/29627573","citation_count":11,"is_preprint":false},{"pmid":"31396242","id":"PMC_31396242","title":"Tethering of Multi-Vesicular Bodies and the Tonoplast to the Plasma Membrane in Plants.","date":"2019","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/31396242","citation_count":10,"is_preprint":false},{"pmid":"31231579","id":"PMC_31231579","title":"Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane.","date":"2019","source":"Animal cells and systems","url":"https://pubmed.ncbi.nlm.nih.gov/31231579","citation_count":7,"is_preprint":false},{"pmid":"35399503","id":"PMC_35399503","title":"Origin and Evolution of the Multifaceted Adherens Junction Component Plekha7.","date":"2022","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/35399503","citation_count":6,"is_preprint":false},{"pmid":"22035800","id":"PMC_22035800","title":"Metabolically stabilized derivatives of phosphatidylinositol 4-phosphate: synthesis and applications.","date":"2011","source":"Chemistry & biology","url":"https://pubmed.ncbi.nlm.nih.gov/22035800","citation_count":6,"is_preprint":false},{"pmid":"15881666","id":"PMC_15881666","title":"P5644 interacts with phosphatidylinositol-4-phosphate adaptor protein-1 associated protein-1.","date":"2005","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15881666","citation_count":6,"is_preprint":false},{"pmid":"37705287","id":"PMC_37705287","title":"Genome-wide association study identifies variants associated with semen volume in white-feathered broilers.","date":"2023","source":"Animal genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37705287","citation_count":6,"is_preprint":false},{"pmid":"27553233","id":"PMC_27553233","title":"NMR of Membrane Proteins: Beyond Crystals.","date":"2016","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/27553233","citation_count":4,"is_preprint":false},{"pmid":"40653076","id":"PMC_40653076","title":"Ursodeoxycholic acid alleviates multiple sclerosis via TGR5-dependent microglial regulation in mice.","date":"2025","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40653076","citation_count":2,"is_preprint":false},{"pmid":"40022259","id":"PMC_40022259","title":"Genetic insights into cardiac conduction disorders from genome-wide association studies.","date":"2025","source":"Human genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40022259","citation_count":1,"is_preprint":false},{"pmid":"21298564","id":"PMC_21298564","title":"Secondary structure and 1H, 13C, 15N resonance assignments of the Golgi-specific PH domain of FAPP1.","date":"2011","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/21298564","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16275,"output_tokens":2720,"usd":0.044812,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10002,"output_tokens":3260,"usd":0.065755,"stage2_stop_reason":"end_turn"},"total_usd":0.110567,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"The PH domain of FAPP1 (PLEKHA3) specifically binds phosphatidylinositol-4-phosphate (PtdIns(4)P) in vitro, with distinct selectivity from PtdIns(3,4,5)P3 and other phosphoinositides.\",\n      \"method\": \"In vitro phosphoinositide-binding assay using recombinant PH domains\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro binding assay with specificity determination, foundational result replicated extensively in subsequent studies across multiple labs\",\n      \"pmids\": [\"11001876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FAPP1 localizes to the trans-Golgi network (TGN) on nascent carriers and interacts with both PtdIns(4)P and the small GTPase ARF through its PH domain; knockdown or displacement of FAPP1 inhibits cargo transfer to the plasma membrane, and overexpression of FAPP-PH impairs carrier fission, establishing FAPP1 as an essential component of a PtdIns(4)P- and ARF-regulated machinery controlling post-Golgi carrier generation.\",\n      \"method\": \"Subcellular localization (fluorescence microscopy), siRNA knockdown, overexpression with cargo transport assays, co-immunoprecipitation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (localization, knockdown, overexpression, binding), replicated across subsequent studies\",\n      \"pmids\": [\"15107860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The FAPP1 PH domain detects PI(4)P in distinct cellular compartments including the Golgi and plasma membrane; PI4KIIalpha and PI4KIIIbeta regulate Golgi PI(4)P pools sensed by FAPP1-PH, whereas plasma membrane PI(4)P pool detected by FAPP1-PH is specifically regulated by PI4KIIIalpha.\",\n      \"method\": \"GFP-fusion live-cell imaging, siRNA knockdown of individual PI4 kinases, pharmacological inhibition (wortmannin, PAO)\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with genetic and pharmacological perturbations, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"15635101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The FAPP1 PH domain contains a prominent hydrophobic wedge that independently tubulates Golgi membranes by penetrating into the lipid leaflet; the mechanism involves electrostatic approach, phosphoinositide sampling, and perpendicular bilayer penetration.\",\n      \"method\": \"Solution NMR structure of micelle-bound FAPP1-PH domain, mutagenesis of hydrophobic wedge residues, membrane tubulation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis and functional membrane tubulation assay in a single study\",\n      \"pmids\": [\"20300118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the FAPP1 PH domain at 1.9 Å resolution reveals a seven-stranded β-barrel with a lipid-binding pocket; PtdIns(4)P binding is enhanced in acidic environment and required for membrane penetration and tubulation; ARF1 binds to the outer side of the β-barrel at a site distinct from the lipid-binding pocket, allowing simultaneous and independent binding of both ligands.\",\n      \"method\": \"X-ray crystallography (1.9 Å), NMR resonance perturbation analysis, site-directed mutagenesis, binding affinity measurements, membrane tubulation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with NMR, mutagenesis, and functional assays in a single rigorous study\",\n      \"pmids\": [\"21454700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Metabolically stabilized analogs of PtdIns(4)P (methylenephosphonate and phosphorothioate derivatives) are recognized by the FAPP1 PH domain and stimulate membrane insertion and tubulation activity of the PH domain, demonstrating that the head group recognition drives membrane remodeling.\",\n      \"method\": \"Chemical synthesis of PtdIns(4)P analogs, binding assays, membrane tubulation assay\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with synthetic analogs, single lab, single study\",\n      \"pmids\": [\"22035800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"At a model membrane surface, yeast Arf1 (yArf1) interacts with the FAPP1-PH domain primarily through contacts between switch I residues of Arf1 and the C-terminal extension of the PH domain; the Arf1 binding site is distinct from the PI4P binding site, supporting coincidence detection of activated ARF and PI4P as the membrane recruitment mechanism.\",\n      \"method\": \"NMR chemical shift perturbation analysis at model membrane surface, structural modeling\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR-based structural mapping at membrane surface, single lab, consistent with crystal structure data from independent study\",\n      \"pmids\": [\"24462251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The FAPP1-PH domain preferentially targets PI4P-containing liquid disordered membranes over liquid ordered membranes; membrane penetration is mediated by an exposed conserved hydrophobic wedge adjacent to the PI4P recognition site, providing selectivity for dynamic TGN membrane zones.\",\n      \"method\": \"Liposome sedimentation assays, membrane partitioning assays, NMR spectroscopy with PI4P-containing bicelles\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods (sedimentation, NMR) in single lab, consistent with prior structural studies\",\n      \"pmids\": [\"25579996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FAPP1 localizes at ER-TGN contact sites (ERTGoCS), physically interacts with the ER phosphatase Sac1, and promotes Sac1's in-trans dephosphorylation of PI4P at the TGN in vitro; depletion of FAPP1 increases TGN PI4P levels and increases secretion of selected cargoes (e.g., ApoB100), establishing FAPP1 as a PI4P detector/adaptor that positions Sac1 to consume TGN PI4P and thereby acts as a gatekeeper of Golgi exit.\",\n      \"method\": \"Co-immunoprecipitation (FAPP1–Sac1 interaction), in vitro phosphatase assay (Sac1 activity with/without FAPP1), FAPP1 depletion (siRNA) with PI4P level measurement and cargo secretion assay, super-resolution microscopy for ERTGoCS localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, in vitro reconstitution of phosphatase activity, genetic depletion with defined biochemical and secretory phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"30659099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"FAPP1 depletion (siRNA) had no effect on apical or basolateral transport in polarized MDCK cells, in contrast to FAPP2 depletion which specifically reduced apical transport.\",\n      \"method\": \"RNA interference, adenovirus-mediated siRNA, transport assays in polarized MDCK cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — negative finding from clean siRNA knockdown in defined transport assay; single lab but rigorous transport readout\",\n      \"pmids\": [\"16103222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A protein named FASP1 (FAPP1-associated protein-1) was identified as a binding partner of pp5644; FASP1 is described as being associated with FAPP1, and co-localizes with pp5644 in the cytoplasm of HeLa cells.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, co-localization by fluorescence microscopy\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — interaction identified for FASP1 (a FAPP1-associated protein), not directly establishing new mechanism for FAPP1 itself; single lab, limited follow-up\",\n      \"pmids\": [\"15881666\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FAPP1/PLEKHA3 is a PH domain-containing adaptor protein that is recruited to the trans-Golgi network through coincidence detection of PI(4)P and GTP-bound ARF1 via distinct sites on its PH domain; once at the TGN, FAPP1 promotes post-Golgi carrier formation through membrane tubulation via a hydrophobic wedge, and at ER-TGN contact sites it positions the ER phosphatase Sac1 to dephosphorylate TGN PI(4)P in trans, thereby acting as a gatekeeper that regulates PI(4)P levels and the rate of selective cargo secretion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLEKHA3 (FAPP1) is a pleckstrin-homology (PH) domain adaptor that controls PI(4)P homeostasis and selective cargo exit at the trans-Golgi network (TGN) [#1, #8]. Its PH domain is recruited to the TGN by coincidence detection of two ligands engaged at structurally distinct sites: PtdIns(4)P bound in a lipid-binding pocket and GTP-bound ARF engaged on the outer face of the β-barrel, allowing simultaneous and independent binding of both [#0, #4, #6]. Membrane recognition is sharpened by preferential targeting of PI(4)P-containing liquid-disordered bilayers and by a conserved hydrophobic wedge adjacent to the lipid pocket that penetrates the leaflet and can independently tubulate Golgi membranes, linking head-group recognition to membrane remodeling [#3, #5, #7]. Functionally, FAPP1 is required for post-Golgi carrier generation, as its displacement blocks cargo transfer to the plasma membrane and PH-domain overexpression impairs carrier fission [#1]. At ER-TGN contact sites FAPP1 binds the ER phosphatase Sac1 and positions it to dephosphorylate TGN PI(4)P in trans; FAPP1 depletion raises TGN PI(4)P and increases secretion of selected cargoes, defining FAPP1 as a gatekeeper of Golgi exit [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the molecular ligand of FAPP1 by showing its PH domain is a specific PtdIns(4)P reader, distinguishing it from PIP3-binding PH domains and providing the lipid-recognition basis for its function.\",\n      \"evidence\": \"In vitro phosphoinositide-binding assay with recombinant PH domains\",\n      \"pmids\": [\"11001876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address subcellular localization or cellular function\", \"No structural basis for the selectivity\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Placed FAPP1 in a cellular pathway by showing it localizes to TGN carriers, binds both PI(4)P and ARF, and is required for post-Golgi cargo transport and carrier fission.\",\n      \"evidence\": \"Fluorescence localization, siRNA knockdown, PH-domain overexpression with cargo transport assays, co-IP\",\n      \"pmids\": [\"15107860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how PI(4)P and ARF are simultaneously engaged\", \"Molecular mechanism of carrier fission unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined which kinases generate the PI(4)P pools FAPP1 senses, distinguishing Golgi pools (PI4KIIalpha/PI4KIIIbeta) from a plasma-membrane pool (PI4KIIIalpha).\",\n      \"evidence\": \"GFP-fusion live-cell imaging with PI4-kinase siRNA and pharmacological inhibition\",\n      \"pmids\": [\"15635101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of plasma-membrane PI(4)P sensing not established\", \"Single-lab imaging-based assignment\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Clarified FAPP1's transport specificity by showing, in contrast to FAPP2, that FAPP1 depletion does not affect apical/basolateral transport in polarized cells.\",\n      \"evidence\": \"Adenovirus-mediated siRNA with transport assays in polarized MDCK cells\",\n      \"pmids\": [\"16103222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result; does not exclude roles in non-polarized secretion\", \"Cargo-specific dependencies not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed how lipid recognition is coupled to membrane remodeling, showing a hydrophobic wedge in the PH domain that penetrates the bilayer and independently tubulates Golgi membranes.\",\n      \"evidence\": \"Solution NMR of micelle-bound PH domain, wedge mutagenesis, membrane tubulation assay\",\n      \"pmids\": [\"20300118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tubulation shown in vitro; in-cell contribution to fission not directly quantified\", \"Role of ARF in wedge insertion not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided the high-resolution structural basis for coincidence detection, showing PI(4)P binds a defined pocket while ARF1 binds a distinct outer β-barrel site, permitting independent simultaneous engagement.\",\n      \"evidence\": \"1.9 Å crystal structure with NMR perturbation, mutagenesis, affinity measurements, tubulation assay\",\n      \"pmids\": [\"21454700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane-bound ternary geometry not directly visualized\", \"Regulation of binding by ARF nucleotide state in cells not shown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that head-group recognition itself drives remodeling, since stabilized PtdIns(4)P analogs are recognized and stimulate PH-domain membrane insertion and tubulation.\",\n      \"evidence\": \"Synthetic PI(4)P analogs with binding and membrane tubulation assays\",\n      \"pmids\": [\"22035800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro analog system; physiological relevance of analogs limited\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped the ARF-binding interface at a membrane surface, showing Arf1 switch I contacts the PH domain C-terminal extension at a site distinct from the PI(4)P pocket, confirming the coincidence-detection recruitment model.\",\n      \"evidence\": \"NMR chemical-shift perturbation at model membrane surface with structural modeling\",\n      \"pmids\": [\"24462251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Used yeast Arf1; human ARF1 contacts inferred\", \"Affinity and stoichiometry on cellular membranes not measured\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Refined membrane targeting selectivity, showing preference for PI(4)P in liquid-disordered membranes via a hydrophobic wedge adjacent to the recognition site, tuning FAPP1 to dynamic TGN zones.\",\n      \"evidence\": \"Liposome sedimentation, membrane partitioning, NMR with PI(4)P bicelles\",\n      \"pmids\": [\"25579996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lipid-phase preference shown in reconstituted membranes\", \"Functional impact on cargo selection not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Redefined FAPP1 as a PI(4)P-consuming gatekeeper rather than only a carrier-forming factor, showing it operates at ER-TGN contacts to position Sac1 for in-trans PI(4)P dephosphorylation and to limit secretion of selected cargoes.\",\n      \"evidence\": \"Reciprocal co-IP, in vitro Sac1 phosphatase assay, siRNA depletion with PI(4)P and cargo secretion readouts, super-resolution microscopy\",\n      \"pmids\": [\"30659099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cargo-selectivity determinants of the gatekeeper function unresolved\", \"How carrier-formation and PI(4)P-consumption roles are coordinated unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FAPP1 reconciles its membrane-tubulating carrier-formation activity with its Sac1-mediated PI(4)P-consuming gatekeeper role, and what determines which cargoes it gates, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No model integrating tubulation and PI(4)P-consumption at the TGN\", \"Physiological regulators switching between the two functions unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 3, 4, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ARF1\",\n      \"SACM1L\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}