{"gene":"SARAF","run_date":"2026-06-10T07:46:29","timeline":{"discoveries":[{"year":2012,"finding":"SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM1 to facilitate slow Ca2+-dependent inactivation (SCDI) of store-operated calcium entry (SOCE), acting as a negative regulator to prevent Ca2+ overfilling of intracellular stores.","method":"Heterologous expression, RNAi-mediated silencing, site-directed mutagenesis, electrophysiology, Co-immunoprecipitation, and Ca2+ imaging","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (electrophysiology, biochemistry, imaging, mutagenesis, RNAi) in a single rigorous study, independently replicated by multiple subsequent labs","pmids":["22464749"],"is_preprint":false},{"year":2013,"finding":"STIM1 contains a C-terminal inhibitory domain (CTID, aa 448-530) that regulates SCDI by controlling SARAF's access to and interaction with the STIM1 Orai1 activation region (SOAR); CTID has two lobes with distinct roles: the STIM1(448-490) lobe restricts SARAF access to SOAR while the STIM1(490-530) lobe directs SARAF to SOAR.","method":"Homology modeling, deletion mutagenesis, Co-immunoprecipitation, electrophysiology, Ca2+ imaging, fluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis combined with functional electrophysiology and biochemical interaction assays in a single study","pmids":["23816623"],"is_preprint":false},{"year":2016,"finding":"SARAF dynamically interacts with both STIM1 and Orai1 in a temporally regulated manner: store depletion causes SARAF to dissociate from STIM1 (~30 s after thapsigargin) and associate with Orai1, followed by re-association with STIM1 and dissociation from Orai1. SARAF interacts with the C-terminus of Orai1 (C-terminal deletion mutants abolish SARAF-Orai1 interaction). A STIM1-independent interaction of SARAF with Orai1 leads to channel activation.","method":"Co-immunoprecipitation, RNAi silencing, overexpression, Orai1 N- and C-terminal deletion mutants, C-terminus blocking peptides, Ca2+ imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal Co-IP with mutant analysis and functional assays, single lab, multiple orthogonal approaches","pmids":["27068144"],"is_preprint":false},{"year":2016,"finding":"In addition to its ER localization, SARAF is constitutively expressed in the plasma membrane where it interacts with plasma membrane-resident Orai1 subunits of arachidonate-regulated Ca2+ (ARC) channels and negatively regulates store-independent Ca2+ entry through ARC channels. Arachidonic acid increases the association of PM-resident SARAF with Orai1.","method":"siRNA silencing, overexpression, subcellular fractionation, Co-immunoprecipitation, Ca2+ imaging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with functional Ca2+ measurements and RNAi in single lab, multiple methods","pmids":["26817842"],"is_preprint":false},{"year":2016,"finding":"SARAF interacts with TRPC1 in a STIM1-independent manner and negatively regulates TRPC1-mediated Ca2+ entry; agonist stimulation (ATP) enhances SARAF-TRPC1 interaction. In contrast, SARAF-TRPC6 interaction is constitutive and SARAF silencing has no effect on TRPC6-mediated Ca2+ entry.","method":"Co-immunoprecipitation in STIM1-deficient NG115-401L cells and STIM1-expressing SH-SY5Y cells, siRNA silencing, Ca2+ imaging","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP combined with functional RNAi knockdown assays in two cell lines, single lab","pmids":["27506849"],"is_preprint":false},{"year":2016,"finding":"The surface expression/plasma membrane localization of SARAF is dependent on the expression of STIM1 in the plasma membrane; transfection with surface-translocating pHluorin-STIM1 enhances plasma membrane location of SARAF compared to non-surface-translocating YFP-STIM1.","method":"Transfection of STIM1 variants with different PM-targeting abilities in STIM1-deficient NG115-401L cells, fluorescence microscopy","journal":"Channels (Austin, Tex.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single localization experiment in one cell line, single lab, single method","pmids":["27414851"],"is_preprint":false},{"year":2018,"finding":"EFHB (EF-hand domain family member B/CFAP21), a cytosolic Ca2+ sensor, interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. EFHB silencing abolishes the dissociation of SARAF from STIM1, indicating EFHB modulates the STIM1-SARAF dynamic interaction required for Orai1 activation and subsequent SCDI.","method":"Co-immunoprecipitation, RNAi silencing, overexpression, Ca2+ imaging, confocal microscopy (NFAT translocation assay)","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with functional RNAi and overexpression and multiple readouts, single lab","pmids":["30481768"],"is_preprint":false},{"year":2019,"finding":"X-ray crystal structure of the SARAF luminal domain (SARAFL) reveals a novel 10-stranded β-sandwich fold ('SARAF-fold') with three conserved disulfide bonds. The structure forms a domain-swapped dimer via exchange of the last two β-strands (β9 and β10), termed the 'SARAF luminal switch'. FRET experiments validate domain-swapped dimerization in cells, showing it is reversible. A variant lacking the luminal switch loses dimerization and shows impaired function (accelerated SOCE inactivation), demonstrating the dimer is essential for SARAF function.","method":"X-ray crystallography, FRET with full-length SARAF, designed domain-swap variant (structure-guided mutagenesis), functional SOCE assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with FRET validation in cells and mutagenesis showing functional consequence, single lab but multiple orthogonal methods","pmids":["31082439"],"is_preprint":false},{"year":2019,"finding":"Phosphorylation of STIM1 at Y316 modulates its interaction with SARAF: the Y316F mutation alters the STIM1-SARAF interaction pattern under resting and store-depleted conditions, enhances SCDI of Orai1 in a SARAF-dependent manner (effect abolished by SARAF knockdown), and reduces STIM1-Orai1 colocalization.","method":"Site-directed mutagenesis (Y316F), Co-immunoprecipitation, siRNA knockdown of SARAF, electrophysiology (ICRAC), confocal colocalization, Ca2+ imaging in HEK293, NG115-401L, and MEG-01 cells","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with Co-IP, electrophysiology, and SARAF knockdown rescue, single lab","pmids":["30975919"],"is_preprint":false},{"year":2019,"finding":"In acinar cells, STIM1 interacts stably with SARAF following physiological levels of carbachol stimulation but only transiently following pathological levels, leading to excessive Ca2+ influx. SARAF knockout mice develop more severe acute pancreatitis with increased Ca2+ influx, while SARAF overexpression in acini reduces Ca2+ influx and severity of pancreatitis. SARAF protein levels initially increase then decrease during pancreatitis-inducing stimulation via degradation.","method":"CRISPR/Cas9-generated SARAF-HA knock-in mice, Saraf-/- and Saraf-overexpressing mice, FRET microscopy, immunoprecipitation, Ca2+ imaging, in vivo pancreatitis models (caerulein and L-arginine injection)","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO and OE mouse models with FRET, Co-IP, and in vivo functional readouts across multiple experimental approaches","pmids":["31493399"],"is_preprint":false},{"year":2020,"finding":"The penta-EF-hand Ca2+-binding protein ALG-2 interacts with the cytosolic domain (CytD) of SARAF via an ALG-2-binding motif (ABM-2). ALG-2 binding interferes with SARAF ubiquitination by NEDD4 family E3 ubiquitin ligases (which target PPXY motifs in SARAF CytD), thereby stabilizing SARAF protein. A ubiquitination-defective SARAF mutant (Lys-to-Arg in CytD) shows slower degradation, and ALG-2 promotes Ca2+-dependent CytD-to-CytD interactions of SARAF.","method":"Semi-quantitative in vitro pulldown assay, GFP pulldown, Co-immunoprecipitation, site-directed mutagenesis (F228S, Lys-to-Arg), half-life analysis, ubiquitination assay in HEK293 cells","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple biochemical assays with mutagenesis confirming mechanism, single lab","pmids":["32878247"],"is_preprint":false},{"year":2020,"finding":"SARAF activates mTORC1 and increases protein synthesis in cardiomyocytes; mTORC1 inhibition blunts SARAF-dependent cell growth. SARAF-deficient cardiomyocytes do not show hypertrophic growth after neurohumoral stimulation, while SARAF overexpression causes cell growth associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content.","method":"Saraf knockout mice, cardiomyocyte-specific overexpression in vivo (angiotensin II model), mTORC1 inhibitor treatment (rapamycin), protein synthesis assays, echocardiography, Ca2+ measurements","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and OE with pharmacological inhibition identifying mTORC1 as downstream effector, single lab","pmids":["32173353"],"is_preprint":false},{"year":2021,"finding":"SARAF has a dual regulatory role in CRAC channel function: (1) following ER Ca2+ depletion, SARAF facilitates a conformational change in STIM1 that relieves an activation constraint imposed by the STIM1 inactivation domain (ID, aa 475-483), promoting initial STIM1 activation, translocation to ER-PM junctions, and Orai1 coupling; (2) following intracellular Ca2+ rise, SARAF cooperates with STIM1 ID to control CRAC channel SCDI. In T lymphocytes, SARAF is required for proper TCR-evoked transcription.","method":"SARAF loss-of-function, STIM1 inactivation domain mutagenesis, FRET, electrophysiology (CRAC current), confocal imaging, T cell Ca2+ signaling and gene expression assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (FRET, electrophysiology, mutagenesis, gene expression) establishing dual mechanistic role, single rigorous study","pmids":["34705029"],"is_preprint":false},{"year":2021,"finding":"SARAF interacts with Orai1 in human umbilical vein endothelial cells (HUVECs), as demonstrated by proximity ligation assay and immunostaining. SARAF and Orai1 knockdown impairs VEGF-mediated Ca2+ increase, HUVEC tube formation, proliferation, and migration.","method":"siRNA knockdown, in situ proximity ligation assay, immunostaining, Ca2+ imaging, tube formation assay, proliferation and migration assays","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — PLA interaction assay combined with functional RNAi phenotypes in primary endothelial cells, single lab","pmids":["33748129"],"is_preprint":false},{"year":2023,"finding":"In neonatal platelets, SARAF is overexpressed, leading to increased STIM1/SARAF interaction even under resting conditions, which impairs thrombin-induced Ca2+ influx and contributes to deficient platelet aggregation. Higher SARAF/PDCD61(ALG-2) interaction reduces SARAF ubiquitination and prolongs its half-life. These effects are reproduced by SARAF overexpression in MEG01 and DAMI cells.","method":"Co-immunoprecipitation, Western blotting, siRNA overexpression in megakaryocyte cell lines, Ca2+ mobilization assays, ubiquitination assay","journal":"British journal of haematology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with cell line overexpression reproducing primary platelet findings, single lab","pmids":["38062782"],"is_preprint":false},{"year":2023,"finding":"Overexpression of the C-terminal cytoplasmic region of SARAF reduces SOCE and decreases proliferation, migration, and invasion of triple-negative breast cancer cells both in vitro and in vivo.","method":"Overexpression of C-terminal SARAF fragment, Ca2+ imaging, proliferation/migration/invasion assays, in vivo mouse xenograft model","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — domain-specific overexpression with functional in vitro and in vivo readouts, single lab","pmids":["36982380"],"is_preprint":false}],"current_model":"SARAF is an endoplasmic reticulum (and plasma membrane) single-pass membrane protein whose luminal domain forms a domain-swapped β-sandwich dimer essential for its function; it associates with STIM1 at rest to prevent spontaneous SOCE activation, then—following ER Ca2+ depletion—facilitates a conformational change in STIM1 that relieves the STIM1 inactivation domain (ID, aa 475-483) constraint to promote initial STIM1 activation and Orai1 coupling, and subsequently cooperates with the STIM1 CTID domain (aa 448-530) to control slow Ca2+-dependent inactivation (SCDI) of Orai1 channels as cytosolic Ca2+ rises; SARAF also negatively regulates ARC channels and TRPC1 (but not TRPC6) in a STIM1-independent manner through direct plasma membrane interactions; its stability is regulated by ALG-2-dependent interference with NEDD4-family E3 ubiquitin ligase-mediated ubiquitination of PPXY motifs in its cytosolic domain; and in cardiomyocytes SARAF links sarcoplasmic reticulum Ca2+ homeostasis to mTORC1 activation and protein synthesis."},"narrative":{"mechanistic_narrative":"SARAF is an endoplasmic reticulum (and plasma membrane) single-pass membrane protein that functions as a negative regulator of store-operated calcium entry (SOCE), preventing Ca2+ overfilling of intracellular stores by associating with STIM1 to drive slow Ca2+-dependent inactivation (SCDI) of the CRAC channel [PMID:22464749]. SARAF acts through a dual mechanism: after ER Ca2+ depletion it facilitates a conformational change in STIM1 that relieves the constraint imposed by the STIM1 inactivation domain (aa 475-483), promoting initial STIM1 activation and Orai1 coupling, and after cytosolic Ca2+ rises it cooperates with the STIM1 inactivation/CTID region (aa 448-530) to enforce SCDI [PMID:34705029, PMID:23816623]. This interaction is temporally choreographed — store depletion transiently dissociates SARAF from STIM1 and shifts it onto the Orai1 C-terminus before re-association — and is gated by the cytosolic Ca2+ sensor EFHB and by STIM1 Y316 phosphorylation [PMID:27068144, PMID:30481768, PMID:30975919]. Its luminal domain adopts a novel 10-stranded β-sandwich fold that forms a reversible domain-swapped dimer essential for proper SOCE inactivation [PMID:31082439]. SARAF additionally restrains STIM1-independent Ca2+ entry through direct plasma membrane interactions with the Orai1 subunits of ARC channels and with TRPC1 (but not TRPC6) [PMID:26817842, PMID:27506849]. SARAF protein stability is set by ALG-2, which binds its cytosolic domain and shields PPXY motifs from NEDD4-family E3 ligase-mediated ubiquitination [PMID:32878247]. Physiologically, SARAF tunes Ca2+ signaling in acinar cells (where its loss worsens acute pancreatitis), T lymphocytes, platelets, endothelial cells, and cardiomyocytes, where it links sarcoplasmic reticulum Ca2+ homeostasis to mTORC1-driven protein synthesis and hypertrophic growth [PMID:31493399, PMID:34705029, PMID:38062782, PMID:33748129, PMID:32173353].","teleology":[{"year":2012,"claim":"Established SARAF as the molecular component responsible for limiting SOCE, answering how cells prevent Ca2+ overfilling of stores via STIM1.","evidence":"Heterologous expression, RNAi, mutagenesis, electrophysiology, Co-IP and Ca2+ imaging defining SARAF-STIM1-mediated SCDI","pmids":["22464749"],"confidence":"High","gaps":["Did not define the STIM1 region mediating SARAF access","Structural basis of SARAF function unresolved","Did not address non-STIM1 channel targets"]},{"year":2013,"claim":"Mapped the STIM1 C-terminal inhibitory domain (CTID, aa 448-530) that gates SARAF access to the SOAR region, explaining how STIM1 itself controls when SARAF can act.","evidence":"Homology modeling, deletion mutagenesis, Co-IP, electrophysiology and Ca2+ imaging","pmids":["23816623"],"confidence":"High","gaps":["Atomic structure of the STIM1-SARAF interface not solved","Did not establish the temporal order of SARAF engagement"]},{"year":2016,"claim":"Showed SARAF interactions are temporally dynamic and extend beyond STIM1 to Orai1, ARC channels, and TRPC1, revealing STIM1-independent regulatory roles at the plasma membrane.","evidence":"Reciprocal Co-IP with Orai1 deletion mutants and blocking peptides, subcellular fractionation, RNAi, and Ca2+ imaging across multiple cell lines","pmids":["27068144","26817842","27506849","27414851"],"confidence":"Medium","gaps":["Binding interfaces on Orai1, ARC channels, and TRPC1 not mapped at residue level","STIM1-dependence of PM trafficking based on a single localization experiment","Whether these interactions are direct vs. complex-mediated unresolved"]},{"year":2018,"claim":"Identified EFHB as a cytosolic Ca2+ sensor required for the STIM1-SARAF dynamic interaction, providing an upstream Ca2+-dependent switch controlling SARAF dissociation.","evidence":"Co-IP, RNAi silencing, overexpression, Ca2+ imaging and NFAT translocation assays","pmids":["30481768"],"confidence":"Medium","gaps":["Whether EFHB binds SARAF directly is unknown","Structural/mechanistic basis of EFHB-driven dissociation unresolved"]},{"year":2019,"claim":"Determined the SARAF luminal domain structure as a domain-swapped β-sandwich dimer and showed dimerization is functionally essential, linking molecular architecture to SOCE control.","evidence":"X-ray crystallography, in-cell FRET, and structure-guided domain-swap mutant with functional SOCE assays","pmids":["31082439"],"confidence":"High","gaps":["Structure of the full-length protein or STIM1 complex not solved","How luminal dimerization is transmitted to cytosolic regulatory output unknown"]},{"year":2019,"claim":"Connected SARAF function to physiological Ca2+ signaling in vivo, showing it protects acinar cells from Ca2+ overload and that its loss exacerbates acute pancreatitis.","evidence":"CRISPR knock-in, knockout and overexpression mice, FRET, Co-IP, Ca2+ imaging, and in vivo pancreatitis models","pmids":["31493399"],"confidence":"High","gaps":["Mechanism distinguishing stable vs. transient STIM1-SARAF binding across stimulus intensity not fully resolved","Degradation pathway during pancreatitis not molecularly defined here"]},{"year":2019,"claim":"Showed STIM1 Y316 phosphorylation modulates SARAF engagement, adding a post-translational layer to SCDI control.","evidence":"Y316F mutagenesis, Co-IP, SARAF knockdown rescue, ICRAC electrophysiology and colocalization across three cell lines","pmids":["30975919"],"confidence":"Medium","gaps":["Kinase responsible for Y316 phosphorylation not identified","Whether phosphorylation directly alters the SARAF interface unknown"]},{"year":2020,"claim":"Defined how SARAF abundance is controlled, showing ALG-2 binds its cytosolic domain to block NEDD4-family ubiquitination of PPXY motifs and stabilize the protein.","evidence":"In vitro pulldown, GFP pulldown, Co-IP, mutagenesis, half-life and ubiquitination assays in HEK293 cells","pmids":["32878247"],"confidence":"Medium","gaps":["Specific NEDD4-family ligase(s) acting in vivo not pinned down","Physiological triggers that shift the ALG-2/ubiquitination balance unclear"]},{"year":2020,"claim":"Linked SARAF-controlled SR Ca2+ homeostasis to mTORC1 activation and cardiomyocyte hypertrophic growth, extending SARAF function to downstream growth signaling.","evidence":"Knockout and cardiomyocyte-specific overexpression mice, rapamycin treatment, protein synthesis assays, echocardiography and Ca2+ measurements","pmids":["32173353"],"confidence":"Medium","gaps":["Molecular link between Ca2+ signaling and mTORC1 not defined","Single lab; tissue-specificity of the growth axis not broadly tested"]},{"year":2021,"claim":"Resolved the dual mechanistic role of SARAF in CRAC channel function — both promoting initial STIM1 activation and enforcing later SCDI via the STIM1 inactivation domain — and showed it is required for TCR-evoked transcription.","evidence":"SARAF loss-of-function, STIM1 ID mutagenesis, FRET, CRAC electrophysiology, confocal imaging and T-cell gene expression assays","pmids":["34705029"],"confidence":"High","gaps":["Conformational details of the STIM1 change SARAF induces not structurally resolved","How the same protein switches between activating and inactivating roles mechanistically unresolved"]},{"year":2021,"claim":"Extended SARAF-Orai1 function to endothelial biology, implicating it in VEGF-driven Ca2+ signaling and angiogenic behavior.","evidence":"siRNA knockdown, proximity ligation assay, immunostaining, Ca2+ imaging, and tube formation/proliferation/migration assays in HUVECs","pmids":["33748129"],"confidence":"Medium","gaps":["Whether the SARAF-Orai1 PLA signal reflects direct binding unresolved","Downstream angiogenic signaling pathway not mapped"]},{"year":2023,"claim":"Demonstrated that altered SARAF abundance reshapes Ca2+ signaling in disease-relevant contexts — impairing platelet aggregation when overexpressed and restraining tumor cell aggressiveness via its cytoplasmic region.","evidence":"Co-IP, ubiquitination assays, and overexpression in megakaryocyte lines for platelets; C-terminal SARAF overexpression with in vitro/in vivo assays in triple-negative breast cancer","pmids":["38062782","36982380"],"confidence":"Medium","gaps":["Causal contribution of SARAF dysregulation to disease versus correlation not fully established","Cytoplasmic fragment mechanism of SOCE suppression not defined at residue level"]},{"year":null,"claim":"How SARAF physically toggles between promoting STIM1 activation and enforcing channel inactivation, and the atomic structure of the SARAF-STIM1-Orai1 complex, remain unresolved.","evidence":"No structural model of the assembled complex exists in the available corpus","pmids":[],"confidence":"High","gaps":["No structure of the cytosolic domain or its STIM1 contacts","Mechanism coupling luminal dimerization to cytosolic regulatory output unknown","Determinants of the dual activating/inactivating switch unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,12]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[0,3,4]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,4,5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,12]}],"complexes":[],"partners":["STIM1","ORAI1","TRPC1","EFHB","PDCD6","NEDD4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96BY9","full_name":"Store-operated calcium entry-associated regulatory factor","aliases":["HBV X-transactivated gene 3 protein","HBV XAg-transactivated protein 3","Protein FOAP-7","Transmembrane protein 66"],"length_aa":339,"mass_kda":37.0,"function":"Negative regulator of store-operated Ca(2+) entry (SOCE) involved in protecting cells from Ca(2+) overfilling. In response to cytosolic Ca(2+) elevation after endoplasmic reticulum Ca(2+) refilling, promotes a slow inactivation of STIM (STIM1 or STIM2)-dependent SOCE activity: possibly act by facilitating the deoligomerization of STIM to efficiently turn off ORAI when the endoplasmic reticulum lumen is filled with the appropriate Ca(2+) levels, and thus preventing the overload of the cell with excessive Ca(2+) ions","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q96BY9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SARAF","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SARAF","total_profiled":1310},"omim":[{"mim_id":"617958","title":"INTERACTOR OF LITTLE ELONGATION COMPLEX ELL SUBUNIT 1; ICE1","url":"https://www.omim.org/entry/617958"},{"mim_id":"614768","title":"TRANSMEMBRANE PROTEIN 66; TMEM66","url":"https://www.omim.org/entry/614768"},{"mim_id":"609544","title":"CENTRIOLAR COILED-COIL PROTEIN, 110-KD; CCP110","url":"https://www.omim.org/entry/609544"},{"mim_id":"605652","title":"F-BOX AND LEUCINE-RICH REPEAT PROTEIN 2; FBXL2","url":"https://www.omim.org/entry/605652"},{"mim_id":"601728","title":"PHOSPHATASE AND TENSIN HOMOLOG; PTEN","url":"https://www.omim.org/entry/601728"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Rods & Rings","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SARAF"},"hgnc":{"alias_symbol":["MGC8721"],"prev_symbol":["TMEM66"]},"alphafold":{"accession":"Q96BY9","domains":[{"cath_id":"-","chopping":"35-142_327-337","consensus_level":"high","plddt":95.7446,"start":35,"end":337}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BY9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BY9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BY9-F1-predicted_aligned_error_v6.png","plddt_mean":68.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SARAF","jax_strain_url":"https://www.jax.org/strain/search?query=SARAF"},"sequence":{"accession":"Q96BY9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96BY9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96BY9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BY9"}},"corpus_meta":[{"pmid":"22464749","id":"PMC_22464749","title":"SARAF inactivates the store operated calcium entry machinery to prevent excess calcium refilling.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/22464749","citation_count":232,"is_preprint":false},{"pmid":"23816623","id":"PMC_23816623","title":"The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function.","date":"2013","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23816623","citation_count":108,"is_preprint":false},{"pmid":"27068144","id":"PMC_27068144","title":"Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27068144","citation_count":63,"is_preprint":false},{"pmid":"31493399","id":"PMC_31493399","title":"Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis.","date":"2019","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/31493399","citation_count":44,"is_preprint":false},{"pmid":"30481768","id":"PMC_30481768","title":"EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/30481768","citation_count":32,"is_preprint":false},{"pmid":"30975919","id":"PMC_30975919","title":"STIM1 phosphorylation at Y316 modulates its interaction with SARAF and the activation of SOCE and ICRAC.","date":"2019","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/30975919","citation_count":30,"is_preprint":false},{"pmid":"26817842","id":"PMC_26817842","title":"Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26817842","citation_count":30,"is_preprint":false},{"pmid":"33748129","id":"PMC_33748129","title":"SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33748129","citation_count":25,"is_preprint":false},{"pmid":"34705029","id":"PMC_34705029","title":"Bidirectional regulation of calcium release-activated calcium (CRAC) channel by SARAF.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34705029","citation_count":25,"is_preprint":false},{"pmid":"27506849","id":"PMC_27506849","title":"SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner.","date":"2016","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/27506849","citation_count":25,"is_preprint":false},{"pmid":"29223474","id":"PMC_29223474","title":"Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF.","date":"2017","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/29223474","citation_count":24,"is_preprint":false},{"pmid":"29807359","id":"PMC_29807359","title":"Overexpression of SARAF Ameliorates Pressure Overload-Induced Cardiac Hypertrophy Through Suppressing STIM1-Orai1 in Mice.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29807359","citation_count":23,"is_preprint":false},{"pmid":"27414851","id":"PMC_27414851","title":"Role of STIM1 in the surface expression of SARAF.","date":"2016","source":"Channels (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/27414851","citation_count":20,"is_preprint":false},{"pmid":"32569806","id":"PMC_32569806","title":"Modulation of Cerebral Store-operated Calcium Entry-regulatory Factor (SARAF) and Peripheral Orai1 Following Focal Cerebral Ischemia and Preconditioning in Mice.","date":"2020","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/32569806","citation_count":16,"is_preprint":false},{"pmid":"34439314","id":"PMC_34439314","title":"SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34439314","citation_count":13,"is_preprint":false},{"pmid":"34440656","id":"PMC_34440656","title":"Regulation of Store-Operated Ca2+ Entry by SARAF.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/34440656","citation_count":12,"is_preprint":false},{"pmid":"32173353","id":"PMC_32173353","title":"Saraf-dependent activation of mTORC1 regulates cardiac growth.","date":"2020","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/32173353","citation_count":12,"is_preprint":false},{"pmid":"31082439","id":"PMC_31082439","title":"SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation.","date":"2019","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/31082439","citation_count":12,"is_preprint":false},{"pmid":"32878247","id":"PMC_32878247","title":"The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32878247","citation_count":9,"is_preprint":false},{"pmid":"25469256","id":"PMC_25469256","title":"Differential upregulation of the hypothetical transmembrane protein 66 (TMEM66) in multiple sclerosis patients with potential inflammatory response.","date":"2014","source":"Biomedical reports","url":"https://pubmed.ncbi.nlm.nih.gov/25469256","citation_count":9,"is_preprint":false},{"pmid":"38062782","id":"PMC_38062782","title":"SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets.","date":"2023","source":"British journal of haematology","url":"https://pubmed.ncbi.nlm.nih.gov/38062782","citation_count":4,"is_preprint":false},{"pmid":"37372417","id":"PMC_37372417","title":"Cholecalciferol Supplementation Induced Up-Regulation of SARAF Gene and Down-Regulated miR-155-5p Expression in Slovenian Patients with Multiple Sclerosis.","date":"2023","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/37372417","citation_count":3,"is_preprint":false},{"pmid":"36982380","id":"PMC_36982380","title":"The Cytoplasmic Region of SARAF Reduces Triple-Negative Breast Cancer Metastasis through the Regulation of Store-Operated Calcium Entry.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36982380","citation_count":2,"is_preprint":false},{"pmid":"33959619","id":"PMC_33959619","title":"Corrigendum: SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33959619","citation_count":1,"is_preprint":false},{"pmid":"38802035","id":"PMC_38802035","title":"MiR-30a-5p isoform -1|1 promotes the progression of gastric cancer by inhibiting TMEM66 and reducing intratumoral cytotoxic T cells.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/38802035","citation_count":0,"is_preprint":false},{"pmid":"40429665","id":"PMC_40429665","title":"Progressive Elevation of Store-Operated Calcium Entry-Associated Regulatory Factor (SARAF) and Calcium Pathway Dysregulation in Multiple Sclerosis.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40429665","citation_count":0,"is_preprint":false},{"pmid":"38073055","id":"PMC_38073055","title":"Reduced Ca2+ mobilization in neonatal human platelets involves SARAF and pannexin-1.","date":"2023","source":"British journal of haematology","url":"https://pubmed.ncbi.nlm.nih.gov/38073055","citation_count":0,"is_preprint":false},{"pmid":"40362663","id":"PMC_40362663","title":"Calcium Homeostasis Disrupted-How Store-Operated Calcium Entry Factor SARAF Silencing Impacts HepG2 Liver Cancer Cells.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40362663","citation_count":0,"is_preprint":false},{"pmid":"41821656","id":"PMC_41821656","title":"Ca2 + influx channel inhibitor SARAF protects rats from severe acute pancreatitis induced colonitis.","date":"2026","source":"3 Biotech","url":"https://pubmed.ncbi.nlm.nih.gov/41821656","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16068,"output_tokens":4578,"usd":0.058437,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12586,"output_tokens":4248,"usd":0.084565,"stage2_stop_reason":"end_turn"},"total_usd":0.143002,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM1 to facilitate slow Ca2+-dependent inactivation (SCDI) of store-operated calcium entry (SOCE), acting as a negative regulator to prevent Ca2+ overfilling of intracellular stores.\",\n      \"method\": \"Heterologous expression, RNAi-mediated silencing, site-directed mutagenesis, electrophysiology, Co-immunoprecipitation, and Ca2+ imaging\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (electrophysiology, biochemistry, imaging, mutagenesis, RNAi) in a single rigorous study, independently replicated by multiple subsequent labs\",\n      \"pmids\": [\"22464749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"STIM1 contains a C-terminal inhibitory domain (CTID, aa 448-530) that regulates SCDI by controlling SARAF's access to and interaction with the STIM1 Orai1 activation region (SOAR); CTID has two lobes with distinct roles: the STIM1(448-490) lobe restricts SARAF access to SOAR while the STIM1(490-530) lobe directs SARAF to SOAR.\",\n      \"method\": \"Homology modeling, deletion mutagenesis, Co-immunoprecipitation, electrophysiology, Ca2+ imaging, fluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis combined with functional electrophysiology and biochemical interaction assays in a single study\",\n      \"pmids\": [\"23816623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SARAF dynamically interacts with both STIM1 and Orai1 in a temporally regulated manner: store depletion causes SARAF to dissociate from STIM1 (~30 s after thapsigargin) and associate with Orai1, followed by re-association with STIM1 and dissociation from Orai1. SARAF interacts with the C-terminus of Orai1 (C-terminal deletion mutants abolish SARAF-Orai1 interaction). A STIM1-independent interaction of SARAF with Orai1 leads to channel activation.\",\n      \"method\": \"Co-immunoprecipitation, RNAi silencing, overexpression, Orai1 N- and C-terminal deletion mutants, C-terminus blocking peptides, Ca2+ imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal Co-IP with mutant analysis and functional assays, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"27068144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In addition to its ER localization, SARAF is constitutively expressed in the plasma membrane where it interacts with plasma membrane-resident Orai1 subunits of arachidonate-regulated Ca2+ (ARC) channels and negatively regulates store-independent Ca2+ entry through ARC channels. Arachidonic acid increases the association of PM-resident SARAF with Orai1.\",\n      \"method\": \"siRNA silencing, overexpression, subcellular fractionation, Co-immunoprecipitation, Ca2+ imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with functional Ca2+ measurements and RNAi in single lab, multiple methods\",\n      \"pmids\": [\"26817842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SARAF interacts with TRPC1 in a STIM1-independent manner and negatively regulates TRPC1-mediated Ca2+ entry; agonist stimulation (ATP) enhances SARAF-TRPC1 interaction. In contrast, SARAF-TRPC6 interaction is constitutive and SARAF silencing has no effect on TRPC6-mediated Ca2+ entry.\",\n      \"method\": \"Co-immunoprecipitation in STIM1-deficient NG115-401L cells and STIM1-expressing SH-SY5Y cells, siRNA silencing, Ca2+ imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP combined with functional RNAi knockdown assays in two cell lines, single lab\",\n      \"pmids\": [\"27506849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The surface expression/plasma membrane localization of SARAF is dependent on the expression of STIM1 in the plasma membrane; transfection with surface-translocating pHluorin-STIM1 enhances plasma membrane location of SARAF compared to non-surface-translocating YFP-STIM1.\",\n      \"method\": \"Transfection of STIM1 variants with different PM-targeting abilities in STIM1-deficient NG115-401L cells, fluorescence microscopy\",\n      \"journal\": \"Channels (Austin, Tex.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single localization experiment in one cell line, single lab, single method\",\n      \"pmids\": [\"27414851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EFHB (EF-hand domain family member B/CFAP21), a cytosolic Ca2+ sensor, interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. EFHB silencing abolishes the dissociation of SARAF from STIM1, indicating EFHB modulates the STIM1-SARAF dynamic interaction required for Orai1 activation and subsequent SCDI.\",\n      \"method\": \"Co-immunoprecipitation, RNAi silencing, overexpression, Ca2+ imaging, confocal microscopy (NFAT translocation assay)\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with functional RNAi and overexpression and multiple readouts, single lab\",\n      \"pmids\": [\"30481768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"X-ray crystal structure of the SARAF luminal domain (SARAFL) reveals a novel 10-stranded β-sandwich fold ('SARAF-fold') with three conserved disulfide bonds. The structure forms a domain-swapped dimer via exchange of the last two β-strands (β9 and β10), termed the 'SARAF luminal switch'. FRET experiments validate domain-swapped dimerization in cells, showing it is reversible. A variant lacking the luminal switch loses dimerization and shows impaired function (accelerated SOCE inactivation), demonstrating the dimer is essential for SARAF function.\",\n      \"method\": \"X-ray crystallography, FRET with full-length SARAF, designed domain-swap variant (structure-guided mutagenesis), functional SOCE assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with FRET validation in cells and mutagenesis showing functional consequence, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"31082439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Phosphorylation of STIM1 at Y316 modulates its interaction with SARAF: the Y316F mutation alters the STIM1-SARAF interaction pattern under resting and store-depleted conditions, enhances SCDI of Orai1 in a SARAF-dependent manner (effect abolished by SARAF knockdown), and reduces STIM1-Orai1 colocalization.\",\n      \"method\": \"Site-directed mutagenesis (Y316F), Co-immunoprecipitation, siRNA knockdown of SARAF, electrophysiology (ICRAC), confocal colocalization, Ca2+ imaging in HEK293, NG115-401L, and MEG-01 cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with Co-IP, electrophysiology, and SARAF knockdown rescue, single lab\",\n      \"pmids\": [\"30975919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In acinar cells, STIM1 interacts stably with SARAF following physiological levels of carbachol stimulation but only transiently following pathological levels, leading to excessive Ca2+ influx. SARAF knockout mice develop more severe acute pancreatitis with increased Ca2+ influx, while SARAF overexpression in acini reduces Ca2+ influx and severity of pancreatitis. SARAF protein levels initially increase then decrease during pancreatitis-inducing stimulation via degradation.\",\n      \"method\": \"CRISPR/Cas9-generated SARAF-HA knock-in mice, Saraf-/- and Saraf-overexpressing mice, FRET microscopy, immunoprecipitation, Ca2+ imaging, in vivo pancreatitis models (caerulein and L-arginine injection)\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO and OE mouse models with FRET, Co-IP, and in vivo functional readouts across multiple experimental approaches\",\n      \"pmids\": [\"31493399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The penta-EF-hand Ca2+-binding protein ALG-2 interacts with the cytosolic domain (CytD) of SARAF via an ALG-2-binding motif (ABM-2). ALG-2 binding interferes with SARAF ubiquitination by NEDD4 family E3 ubiquitin ligases (which target PPXY motifs in SARAF CytD), thereby stabilizing SARAF protein. A ubiquitination-defective SARAF mutant (Lys-to-Arg in CytD) shows slower degradation, and ALG-2 promotes Ca2+-dependent CytD-to-CytD interactions of SARAF.\",\n      \"method\": \"Semi-quantitative in vitro pulldown assay, GFP pulldown, Co-immunoprecipitation, site-directed mutagenesis (F228S, Lys-to-Arg), half-life analysis, ubiquitination assay in HEK293 cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple biochemical assays with mutagenesis confirming mechanism, single lab\",\n      \"pmids\": [\"32878247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SARAF activates mTORC1 and increases protein synthesis in cardiomyocytes; mTORC1 inhibition blunts SARAF-dependent cell growth. SARAF-deficient cardiomyocytes do not show hypertrophic growth after neurohumoral stimulation, while SARAF overexpression causes cell growth associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content.\",\n      \"method\": \"Saraf knockout mice, cardiomyocyte-specific overexpression in vivo (angiotensin II model), mTORC1 inhibitor treatment (rapamycin), protein synthesis assays, echocardiography, Ca2+ measurements\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and OE with pharmacological inhibition identifying mTORC1 as downstream effector, single lab\",\n      \"pmids\": [\"32173353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SARAF has a dual regulatory role in CRAC channel function: (1) following ER Ca2+ depletion, SARAF facilitates a conformational change in STIM1 that relieves an activation constraint imposed by the STIM1 inactivation domain (ID, aa 475-483), promoting initial STIM1 activation, translocation to ER-PM junctions, and Orai1 coupling; (2) following intracellular Ca2+ rise, SARAF cooperates with STIM1 ID to control CRAC channel SCDI. In T lymphocytes, SARAF is required for proper TCR-evoked transcription.\",\n      \"method\": \"SARAF loss-of-function, STIM1 inactivation domain mutagenesis, FRET, electrophysiology (CRAC current), confocal imaging, T cell Ca2+ signaling and gene expression assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (FRET, electrophysiology, mutagenesis, gene expression) establishing dual mechanistic role, single rigorous study\",\n      \"pmids\": [\"34705029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SARAF interacts with Orai1 in human umbilical vein endothelial cells (HUVECs), as demonstrated by proximity ligation assay and immunostaining. SARAF and Orai1 knockdown impairs VEGF-mediated Ca2+ increase, HUVEC tube formation, proliferation, and migration.\",\n      \"method\": \"siRNA knockdown, in situ proximity ligation assay, immunostaining, Ca2+ imaging, tube formation assay, proliferation and migration assays\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — PLA interaction assay combined with functional RNAi phenotypes in primary endothelial cells, single lab\",\n      \"pmids\": [\"33748129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In neonatal platelets, SARAF is overexpressed, leading to increased STIM1/SARAF interaction even under resting conditions, which impairs thrombin-induced Ca2+ influx and contributes to deficient platelet aggregation. Higher SARAF/PDCD61(ALG-2) interaction reduces SARAF ubiquitination and prolongs its half-life. These effects are reproduced by SARAF overexpression in MEG01 and DAMI cells.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, siRNA overexpression in megakaryocyte cell lines, Ca2+ mobilization assays, ubiquitination assay\",\n      \"journal\": \"British journal of haematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with cell line overexpression reproducing primary platelet findings, single lab\",\n      \"pmids\": [\"38062782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Overexpression of the C-terminal cytoplasmic region of SARAF reduces SOCE and decreases proliferation, migration, and invasion of triple-negative breast cancer cells both in vitro and in vivo.\",\n      \"method\": \"Overexpression of C-terminal SARAF fragment, Ca2+ imaging, proliferation/migration/invasion assays, in vivo mouse xenograft model\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — domain-specific overexpression with functional in vitro and in vivo readouts, single lab\",\n      \"pmids\": [\"36982380\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SARAF is an endoplasmic reticulum (and plasma membrane) single-pass membrane protein whose luminal domain forms a domain-swapped β-sandwich dimer essential for its function; it associates with STIM1 at rest to prevent spontaneous SOCE activation, then—following ER Ca2+ depletion—facilitates a conformational change in STIM1 that relieves the STIM1 inactivation domain (ID, aa 475-483) constraint to promote initial STIM1 activation and Orai1 coupling, and subsequently cooperates with the STIM1 CTID domain (aa 448-530) to control slow Ca2+-dependent inactivation (SCDI) of Orai1 channels as cytosolic Ca2+ rises; SARAF also negatively regulates ARC channels and TRPC1 (but not TRPC6) in a STIM1-independent manner through direct plasma membrane interactions; its stability is regulated by ALG-2-dependent interference with NEDD4-family E3 ubiquitin ligase-mediated ubiquitination of PPXY motifs in its cytosolic domain; and in cardiomyocytes SARAF links sarcoplasmic reticulum Ca2+ homeostasis to mTORC1 activation and protein synthesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SARAF is an endoplasmic reticulum (and plasma membrane) single-pass membrane protein that functions as a negative regulator of store-operated calcium entry (SOCE), preventing Ca2+ overfilling of intracellular stores by associating with STIM1 to drive slow Ca2+-dependent inactivation (SCDI) of the CRAC channel [#0]. SARAF acts through a dual mechanism: after ER Ca2+ depletion it facilitates a conformational change in STIM1 that relieves the constraint imposed by the STIM1 inactivation domain (aa 475-483), promoting initial STIM1 activation and Orai1 coupling, and after cytosolic Ca2+ rises it cooperates with the STIM1 inactivation/CTID region (aa 448-530) to enforce SCDI [#12, #1]. This interaction is temporally choreographed — store depletion transiently dissociates SARAF from STIM1 and shifts it onto the Orai1 C-terminus before re-association — and is gated by the cytosolic Ca2+ sensor EFHB and by STIM1 Y316 phosphorylation [#2, #6, #8]. Its luminal domain adopts a novel 10-stranded β-sandwich fold that forms a reversible domain-swapped dimer essential for proper SOCE inactivation [#7]. SARAF additionally restrains STIM1-independent Ca2+ entry through direct plasma membrane interactions with the Orai1 subunits of ARC channels and with TRPC1 (but not TRPC6) [#3, #4]. SARAF protein stability is set by ALG-2, which binds its cytosolic domain and shields PPXY motifs from NEDD4-family E3 ligase-mediated ubiquitination [#10]. Physiologically, SARAF tunes Ca2+ signaling in acinar cells (where its loss worsens acute pancreatitis), T lymphocytes, platelets, endothelial cells, and cardiomyocytes, where it links sarcoplasmic reticulum Ca2+ homeostasis to mTORC1-driven protein synthesis and hypertrophic growth [#9, #12, #14, #13, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established SARAF as the molecular component responsible for limiting SOCE, answering how cells prevent Ca2+ overfilling of stores via STIM1.\",\n      \"evidence\": \"Heterologous expression, RNAi, mutagenesis, electrophysiology, Co-IP and Ca2+ imaging defining SARAF-STIM1-mediated SCDI\",\n      \"pmids\": [\"22464749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the STIM1 region mediating SARAF access\", \"Structural basis of SARAF function unresolved\", \"Did not address non-STIM1 channel targets\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped the STIM1 C-terminal inhibitory domain (CTID, aa 448-530) that gates SARAF access to the SOAR region, explaining how STIM1 itself controls when SARAF can act.\",\n      \"evidence\": \"Homology modeling, deletion mutagenesis, Co-IP, electrophysiology and Ca2+ imaging\",\n      \"pmids\": [\"23816623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the STIM1-SARAF interface not solved\", \"Did not establish the temporal order of SARAF engagement\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed SARAF interactions are temporally dynamic and extend beyond STIM1 to Orai1, ARC channels, and TRPC1, revealing STIM1-independent regulatory roles at the plasma membrane.\",\n      \"evidence\": \"Reciprocal Co-IP with Orai1 deletion mutants and blocking peptides, subcellular fractionation, RNAi, and Ca2+ imaging across multiple cell lines\",\n      \"pmids\": [\"27068144\", \"26817842\", \"27506849\", \"27414851\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interfaces on Orai1, ARC channels, and TRPC1 not mapped at residue level\", \"STIM1-dependence of PM trafficking based on a single localization experiment\", \"Whether these interactions are direct vs. complex-mediated unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified EFHB as a cytosolic Ca2+ sensor required for the STIM1-SARAF dynamic interaction, providing an upstream Ca2+-dependent switch controlling SARAF dissociation.\",\n      \"evidence\": \"Co-IP, RNAi silencing, overexpression, Ca2+ imaging and NFAT translocation assays\",\n      \"pmids\": [\"30481768\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EFHB binds SARAF directly is unknown\", \"Structural/mechanistic basis of EFHB-driven dissociation unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Determined the SARAF luminal domain structure as a domain-swapped β-sandwich dimer and showed dimerization is functionally essential, linking molecular architecture to SOCE control.\",\n      \"evidence\": \"X-ray crystallography, in-cell FRET, and structure-guided domain-swap mutant with functional SOCE assays\",\n      \"pmids\": [\"31082439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full-length protein or STIM1 complex not solved\", \"How luminal dimerization is transmitted to cytosolic regulatory output unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected SARAF function to physiological Ca2+ signaling in vivo, showing it protects acinar cells from Ca2+ overload and that its loss exacerbates acute pancreatitis.\",\n      \"evidence\": \"CRISPR knock-in, knockout and overexpression mice, FRET, Co-IP, Ca2+ imaging, and in vivo pancreatitis models\",\n      \"pmids\": [\"31493399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism distinguishing stable vs. transient STIM1-SARAF binding across stimulus intensity not fully resolved\", \"Degradation pathway during pancreatitis not molecularly defined here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed STIM1 Y316 phosphorylation modulates SARAF engagement, adding a post-translational layer to SCDI control.\",\n      \"evidence\": \"Y316F mutagenesis, Co-IP, SARAF knockdown rescue, ICRAC electrophysiology and colocalization across three cell lines\",\n      \"pmids\": [\"30975919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for Y316 phosphorylation not identified\", \"Whether phosphorylation directly alters the SARAF interface unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined how SARAF abundance is controlled, showing ALG-2 binds its cytosolic domain to block NEDD4-family ubiquitination of PPXY motifs and stabilize the protein.\",\n      \"evidence\": \"In vitro pulldown, GFP pulldown, Co-IP, mutagenesis, half-life and ubiquitination assays in HEK293 cells\",\n      \"pmids\": [\"32878247\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific NEDD4-family ligase(s) acting in vivo not pinned down\", \"Physiological triggers that shift the ALG-2/ubiquitination balance unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked SARAF-controlled SR Ca2+ homeostasis to mTORC1 activation and cardiomyocyte hypertrophic growth, extending SARAF function to downstream growth signaling.\",\n      \"evidence\": \"Knockout and cardiomyocyte-specific overexpression mice, rapamycin treatment, protein synthesis assays, echocardiography and Ca2+ measurements\",\n      \"pmids\": [\"32173353\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between Ca2+ signaling and mTORC1 not defined\", \"Single lab; tissue-specificity of the growth axis not broadly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the dual mechanistic role of SARAF in CRAC channel function — both promoting initial STIM1 activation and enforcing later SCDI via the STIM1 inactivation domain — and showed it is required for TCR-evoked transcription.\",\n      \"evidence\": \"SARAF loss-of-function, STIM1 ID mutagenesis, FRET, CRAC electrophysiology, confocal imaging and T-cell gene expression assays\",\n      \"pmids\": [\"34705029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational details of the STIM1 change SARAF induces not structurally resolved\", \"How the same protein switches between activating and inactivating roles mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended SARAF-Orai1 function to endothelial biology, implicating it in VEGF-driven Ca2+ signaling and angiogenic behavior.\",\n      \"evidence\": \"siRNA knockdown, proximity ligation assay, immunostaining, Ca2+ imaging, and tube formation/proliferation/migration assays in HUVECs\",\n      \"pmids\": [\"33748129\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the SARAF-Orai1 PLA signal reflects direct binding unresolved\", \"Downstream angiogenic signaling pathway not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that altered SARAF abundance reshapes Ca2+ signaling in disease-relevant contexts — impairing platelet aggregation when overexpressed and restraining tumor cell aggressiveness via its cytoplasmic region.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, and overexpression in megakaryocyte lines for platelets; C-terminal SARAF overexpression with in vitro/in vivo assays in triple-negative breast cancer\",\n      \"pmids\": [\"38062782\", \"36982380\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal contribution of SARAF dysregulation to disease versus correlation not fully established\", \"Cytoplasmic fragment mechanism of SOCE suppression not defined at residue level\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SARAF physically toggles between promoting STIM1 activation and enforcing channel inactivation, and the atomic structure of the SARAF-STIM1-Orai1 complex, remain unresolved.\",\n      \"evidence\": \"No structural model of the assembled complex exists in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the cytosolic domain or its STIM1 contacts\", \"Mechanism coupling luminal dimerization to cytosolic regulatory output unknown\", \"Determinants of the dual activating/inactivating switch unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 12]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STIM1\", \"ORAI1\", \"TRPC1\", \"EFHB\", \"PDCD6\", \"NEDD4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}