{"gene":"TMEM165","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2012,"finding":"TMEM165 deficiency causes Golgi glycosylation defects (CDG-II), identified via siRNA knockdown in HEK cells showing abnormal serum-transferrin isoelectric focusing patterns; TMEM165 encodes a putative transmembrane protein whose loss disrupts Golgi N-glycosylation.","method":"siRNA knockdown in HEK cells, transferrin isoelectric focusing, autozygosity mapping","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function with defined cellular phenotype, replicated across multiple patients and cell types","pmids":["22683087"],"is_preprint":false},{"year":2013,"finding":"Wild-type TMEM165 localizes to the Golgi compartment, plasma membrane, and late endosomes/lysosomes; disease-causing mutations alter its subcellular localization; the YNRL motif is critical for proper TMEM165 subcellular localization; mutations associated with mild phenotype can complement yeast gdt1Δ whereas truncation mutations cannot.","method":"Fluorescence microscopy of tagged TMEM165 variants, yeast complementation assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2/3 — direct localization experiment with functional consequence via yeast complementation, single lab","pmids":["23575229"],"is_preprint":false},{"year":2016,"finding":"Golgi glycosylation defects in TMEM165/Gdt1p-deficient cells result from Golgi manganese (Mn2+) homeostasis defect; Mn2+ supplementation rescues normal glycosylation in both yeast gdt1Δ and mammalian TMEM165-depleted cells; GPP130 Mn2+ sensitivity is altered in TMEM165-depleted cells.","method":"Genetic knockdown/knockout, Mn2+ supplementation rescue, GPP130 sensitivity assay in yeast and mammalian cells","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated in two model systems (yeast and mammalian cells)","pmids":["27008884"],"is_preprint":false},{"year":2016,"finding":"Yeast Gdt1p (TMEM165 ortholog) has Ca2+ transport activity demonstrated by heterologous expression in Lactococcus lactis; Ca2+ uptake is pH-dependent, indicating Gdt1p acts as a Ca2+/H+ antiporter; Gdt1p controls cellular calcium stores and is required for glycosylation of carboxypeptidase Y and Gas1p under high external calcium, which is rescued by Mn2+ supplementation.","method":"Heterologous expression in Lactococcus lactis with Ca2+ uptake assay, yeast glycosylation assays, calcium signaling measurements","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — direct transport activity demonstrated by reconstitution in bacterial cells, supported by in vivo yeast assays","pmids":["27075443"],"is_preprint":false},{"year":2017,"finding":"TMEM165 is a novel Mn2+-sensitive Golgi protein that undergoes lysosomal degradation upon high Mn2+ exposure; the glutamic acid at position E108 within the cytosolic ELGDK motif is crucial for Mn2+-induced degradation of TMEM165; the CDG patient variant E108G renders TMEM165 insensitive to Mn2+-induced degradation.","method":"Mn2+ exposure assays, lysosomal degradation pathway analysis, site-directed mutagenesis of E108G variant in mammalian cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — mutational analysis with defined molecular phenotype, identifies specific motif responsible for Mn2+-induced degradation","pmids":["28270545"],"is_preprint":false},{"year":2017,"finding":"TMEM165-deficient cells show severe hypogalactosylation and GalNAc transfer defects in N-linked glycans and glycolipids; these defects are rescued by Mn2+ and also by galactose supplementation; oral D-galactose supplementation in TMEM165-CDG patients improves glycosylation parameters.","method":"Mass spectrometry of N-linked glycans and glycolipids in TMEM165 KO HEK293 cells, Mn2+ and galactose rescue experiments, clinical intervention in patients","journal":"The Journal of clinical endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods in cells and patients, identifies specific glycosylation substrates","pmids":["28323990"],"is_preprint":false},{"year":2018,"finding":"Mn2+ uptake rescuing N-glycosylation in TMEM165 KO cells does not rely on endocytosis but occurs via plasma membrane transporters; the rescue of LAMP2 glycosylation defects involves thapsigargin- and cyclopiazonic acid-sensitive pumps (SERCA pumps); overexpression of SERCA2b partially rescues LAMP2 glycosylation defects in TMEM165 KO cells.","method":"Endocytosis inhibition assays, thapsigargin/CPA inhibitor experiments, SERCA2b overexpression in TMEM165 KO HEK293 cells, western blot of LAMP2 glycosylation","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological and genetic rescue experiments in knockout cells, single lab","pmids":["30307768"],"is_preprint":false},{"year":2019,"finding":"Human TMEM165 directly transports both Ca2+ and Mn2+ when heterologously expressed in yeast devoid of Golgi Ca2+/Mn2+ transporters and in Lactococcus lactis loaded with Fura-2 fluorescent probe; expression abrogates Ca2+- and Mn2+-induced growth defects and glycosylation defects in yeast; the E108G disease-causing mutation significantly reduces TMEM165 transport activity.","method":"Heterologous expression in S. cerevisiae and L. lactis, Fura-2 fluorescent Ca2+/Mn2+ influx assay, yeast growth and glycosylation rescue assays, mutagenesis of E108G","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical transport activity demonstrated in two heterologous systems with mutagenesis validation","pmids":["32047108"],"is_preprint":false},{"year":2019,"finding":"TMEM165 expression and abundance are functionally linked to SPCA1 (the Golgi Ca2+/Mn2+ P-type ATPase); TMEM165 is constitutively degraded in lysosomes in the absence of SPCA1; SPCA1's Mn2+-pumping capacity (but not Ca2+ pumping, as shown by Q747A mutant favoring Mn2+) rescues TMEM165 abundance and Golgi localization; SERCA2b overexpression also rescues TMEM165 expression.","method":"SPCA1-deficient Hap1 cells, SPCA1 mutant complementation, TMEM165 immunoblot and localization, lysosomal degradation tracking","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — reciprocal functional link established with mutagenesis separating Mn2+ and Ca2+ transport roles, multiple orthogonal approaches","pmids":["31652305"],"is_preprint":false},{"year":2019,"finding":"TMEM165 is required for normal milk biosynthesis in the lactating mammary gland; conditional knockout mice show reduced lactose biosynthesis, altered milk composition (elevated fat, protein, iron, zinc; lower calcium and manganese), and impaired nursing pup growth; TMEM165 supplies Ca2+ and Mn2+ to the Golgi in exchange for H+ to sustain lactose synthase and glycosyltransferases.","method":"Conditional tissue-specific knockout mice, milk composition biochemical assays, immunostaining, lactation phenotype measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — in vivo conditional KO with defined molecular and physiological phenotypes, mechanistic interpretation supported by biochemistry","pmids":["30622138"],"is_preprint":false},{"year":2019,"finding":"The two conserved UPF0016 consensus motifs (E-φ-G-D-[KR]-[TS]) in TMEM165 are crucial for its function in Golgi glycosylation and its Mn2+ sensitivity; specific amino acids within these motifs contribute differentially to glycosylation function versus Mn2+-induced degradation sensitivity.","method":"Site-directed mutagenesis of UPF0016 motif residues, glycosylation rescue assays, Mn2+ sensitivity assays in mammalian cells","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with defined functional readout, single lab","pmids":["31351090"],"is_preprint":false},{"year":2021,"finding":"TMEM165 deficiency impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan (GAG) chains of proteoglycans; the blockage is not due to defective Golgi elongating enzymes but to Mn2+ cofactor insufficiency; Mn2+ supplementation rescues GAG chain elongation; TMEM165 loss impairs TGFβ and BMP signaling in chondrocytes and accelerates Ihh expression promoting early chondrocyte hypertrophy.","method":"TMEM165 KO cells, GAG chain length analysis, enzyme activity assays, Mn2+ supplementation rescue, signaling pathway analysis, chondrogenic differentiation assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function with multiple defined molecular phenotypes and rescue, single lab","pmids":["34930890"],"is_preprint":false},{"year":2023,"finding":"Yeast Gdt1p (TMEM165 ortholog) mediates H+ transport in exchange for Ca2+ and Mn2+ across the Golgi membrane; the direction of H+ flow is reversible depending on physiological ion concentration gradients; Gdt1p influences Golgi pH measured by genetically encoded pH probes in vivo.","method":"Heterologous expression in L. lactis with extracellular/intracellular pH recording, in vivo cytosolic and Golgi pH measurements with genetically encoded probes in S. cerevisiae","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct transport mechanism demonstrated by electrophysiology/pH recording across biological membrane plus in vivo pH measurement","pmids":["36963491"],"is_preprint":false},{"year":2023,"finding":"TMEM165 plays a crucial role in cellular Mn2+ homeostasis: Mn2+ supplementation fully rescues Mn2+ content in the secretory pathway of TMEM165-depleted cells and restores glycosylation; TMEM165 and SPCA1 together regulate cellular Mn2+ homeostasis; TMEM165's Mn2+-induced degradation is linked to cytosolic Mn2+ detoxification mediated by SPCA1.","method":"ICP-MS measurement of subcellular Mn2+ content, GPP130 as Golgi Mn2+ sensor, TMEM165/SPCA1 double-depletion experiments","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 — direct elemental measurement of Mn2+ in subcellular compartments, functional link between TMEM165 and SPCA1, single lab","pmids":["37062452"],"is_preprint":false},{"year":2023,"finding":"AlphaFold 2-based structural model of TMEM165 (validated by molecular dynamics simulation) reveals a two-fold repeat of three transmembrane helices where the conserved E-φ-G-D-[KR]-[TS] consensus motifs face each other forming a putative acidic cation-binding site at the cytosolic side; this model explains the impact of CDG patient mutations including G304R on transporter function.","method":"AlphaFold 2 structural prediction, molecular dynamics simulation with membrane lipids and water, functional validation using patient mutation data","journal":"Computational and structural biotechnology journal","confidence":"Medium","confidence_rationale":"Tier 1/4 — structural model with MD refinement explains mutation pathogenicity, but not experimentally solved structure; functional validation indirect","pmids":["37416081"],"is_preprint":false},{"year":2017,"finding":"TMEM165 splice transcript variants (Short-Form, 129 aa; Long-Form, 259 aa) exist in human tissues, particularly in brain; both isoforms localize to the endoplasmic reticulum (distinct from wild-type Golgi localization) and have different effects on glycosylation compared to the full-length protein; the Short-Form forms homodimers.","method":"RT-PCR and RT-qPCR from human brain tissues, overexpression of isoforms with fluorescence microscopy, glycosylation assays","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 3 — direct localization and functional experiments with defined isoforms, single lab","pmids":["28088503"],"is_preprint":false},{"year":2020,"finding":"SPCA1 deficiency in Hailey-Hailey disease fibroblasts and keratinocytes renders TMEM165 more sensitive to Mn2+-induced degradation due to cytosolic Mn2+ accumulation; this links SPCA1 function to TMEM165 stability in a pathological context.","method":"Hailey-Hailey disease patient fibroblasts/keratinocytes, ICP-MS, GPP130 Golgi Mn2+ sensor, Mn2+ exposure assays","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 — functional link in patient-derived cells confirmed by direct Mn2+ measurement, single lab","pmids":["32335229"],"is_preprint":false},{"year":2025,"finding":"A fraction of TMEM165 localizes on the lysosomal limiting membrane where it imports Ca2+ into the lysosomal lumen and mediates Ca2+-induced lysosomal proton leakage; this lysosomal TMEM165 activity accelerates recovery from cytosolic Ca2+ overload, enhances cell survival, and causes cytosolic acidification.","method":"Genetic depletion and overexpression, electrophysiology, fluorescent visualization of subcellular ion concentrations and fluxes across lysosomal membrane","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — direct electrophysiological measurement of ion transport across lysosomal membrane combined with genetic depletion/overexpression and live imaging of ion fluxes","pmids":["40473625"],"is_preprint":false},{"year":2015,"finding":"Morpholino knockdown of tmem165 in zebrafish causes craniofacial abnormalities with fewer chondrocytes, altered N-glycan processing (mirroring human patients), and decreased expression of cartilage and bone development markers, establishing that TMEM165 deficiency impairs chondrocyte and osteoblast differentiation in vivo.","method":"Morpholino knockdown in zebrafish embryos, glycomic analysis, craniofacial phenotype quantification, marker gene expression","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo loss-of-function with defined cellular and glycomic phenotypes in a vertebrate model system","pmids":["25609749"],"is_preprint":false},{"year":2020,"finding":"TMEM165 knockout in human MDA-MB-231 breast cancer cells results in significant reduction of cell migration, tumor growth, and tumor vascularization in vivo; TMEM165 loss alters glycosylation of cancer cells and affects expression of EMT-related glycoproteins including E-cadherin.","method":"CRISPR/Cas9 KO, migration assays, in vivo tumor growth assays, glycoproteomic analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotypes in vitro and in vivo, mechanistic link to glycosylation changes, single lab","pmids":["32733646"],"is_preprint":false}],"current_model":"TMEM165 is a Golgi (and lysosomal) membrane protein belonging to the UPF0016/CaCA2 family that functions as a Ca2+/Mn2+:H+ antiporter, pumping Ca2+ and Mn2+ into the Golgi lumen in exchange for H+ to maintain Golgi cation homeostasis required for glycosyltransferase activity; at the lysosome it imports Ca2+ and mediates Ca2+-induced proton leakage to regulate intracellular ion homeostasis and cell survival; TMEM165 abundance is regulated by cytosolic Mn2+ concentration through lysosomal degradation involving the ELGDK motif (E108), and is functionally coupled to the SPCA1 Mn2+/Ca2+ ATPase pump."},"narrative":{"teleology":[{"year":2012,"claim":"The molecular basis of a CDG-II subtype was unknown; identification of TMEM165 mutations in patients and replication of glycosylation defects by siRNA knockdown established TMEM165 as a Golgi-resident protein required for N-glycosylation.","evidence":"Autozygosity mapping in CDG-II families, siRNA knockdown in HEK cells, transferrin isoelectric focusing","pmids":["22683087"],"confidence":"High","gaps":["Transport mechanism unknown","Substrate ion not identified","No structure available"]},{"year":2013,"claim":"It was unclear where TMEM165 resides and how disease mutations affect its trafficking; fluorescence microscopy and yeast complementation showed wild-type TMEM165 localizes to Golgi, plasma membrane, and late endosomes/lysosomes, with the YNRL motif governing its subcellular targeting.","evidence":"Fluorescence microscopy of tagged TMEM165 variants, yeast gdt1Δ complementation","pmids":["23575229"],"confidence":"Medium","gaps":["Mechanism of YNRL-mediated sorting not defined","Relative functional contribution of each compartment not established"]},{"year":2016,"claim":"The ion species responsible for glycosylation failure was unknown; Mn²⁺ supplementation rescued glycosylation defects in both yeast gdt1Δ and mammalian TMEM165-depleted cells, identifying Golgi Mn²⁺ homeostasis as the critical function, while direct Ca²⁺/H⁺ antiport activity was demonstrated for Gdt1p in Lactococcus lactis.","evidence":"Mn²⁺ supplementation rescue in yeast and mammalian cells, heterologous Ca²⁺ uptake assays in L. lactis, GPP130 Mn²⁺ sensitivity","pmids":["27008884","27075443"],"confidence":"High","gaps":["Direct Mn²⁺ transport by human TMEM165 not yet demonstrated","Stoichiometry of Ca²⁺/Mn²⁺:H⁺ exchange unknown"]},{"year":2017,"claim":"How TMEM165 protein levels are controlled was unclear; high cytosolic Mn²⁺ triggers lysosomal degradation of TMEM165 via the E108 residue within the ELGDK motif, and the CDG patient mutation E108G abolishes this regulation, separating transport function from abundance control.","evidence":"Site-directed mutagenesis, Mn²⁺ exposure and lysosomal degradation assays in mammalian cells","pmids":["28270545","31351090"],"confidence":"High","gaps":["Identity of the Mn²⁺ sensor that recognizes E108 is unknown","Whether degradation is ubiquitin-dependent not tested"]},{"year":2017,"claim":"The specific glycan structures affected and potential therapeutic rescue were undefined; mass spectrometry revealed hypogalactosylation and GalNAc transfer defects in N-glycans and glycolipids, both rescued by Mn²⁺ and galactose supplementation, with oral galactose improving CDG patient glycosylation.","evidence":"Mass spectrometry of glycans in TMEM165 KO HEK293 cells, Mn²⁺/galactose rescue, clinical galactose supplementation","pmids":["28323990"],"confidence":"High","gaps":["Long-term clinical efficacy of galactose therapy not established","Mechanism by which galactose bypasses Mn²⁺ deficiency not fully resolved"]},{"year":2019,"claim":"Whether human TMEM165 itself directly transports Mn²⁺ (not just Ca²⁺) was unresolved; heterologous expression in yeast and L. lactis with Fura-2 fluorescence demonstrated direct Ca²⁺ and Mn²⁺ transport by human TMEM165, with the E108G mutation reducing transport activity.","evidence":"Fura-2 fluorescent influx assay in L. lactis, yeast growth/glycosylation rescue, E108G mutagenesis","pmids":["32047108"],"confidence":"High","gaps":["No reconstitution in proteoliposomes for kinetic characterization","H⁺ counterflux not directly measured for the human protein at this stage"]},{"year":2019,"claim":"The relationship between TMEM165 and the SPCA1 Golgi pump was unknown; SPCA1 deficiency causes constitutive lysosomal degradation of TMEM165, and SPCA1's Mn²⁺-pumping capacity (not Ca²⁺) is required to stabilize TMEM165, revealing a coupled regulatory axis for Golgi Mn²⁺ supply.","evidence":"SPCA1-deficient Hap1 cells, Q747A Mn²⁺-selective SPCA1 mutant complementation, immunoblot and localization","pmids":["31652305"],"confidence":"High","gaps":["Whether TMEM165 and SPCA1 physically interact is unknown","Mechanism by which SPCA1 Mn²⁺ transport prevents TMEM165 degradation not defined"]},{"year":2019,"claim":"The physiological relevance of TMEM165 in intact tissues was unclear; conditional knockout in mouse mammary gland showed reduced lactose biosynthesis, lower milk Ca²⁺ and Mn²⁺, and impaired pup growth, demonstrating that TMEM165 supplies Golgi Ca²⁺/Mn²⁺ required for lactose synthase in vivo.","evidence":"Conditional tissue-specific knockout mice, milk composition analysis, immunostaining","pmids":["30622138"],"confidence":"High","gaps":["Systemic phenotype of whole-body knockout not reported","Contribution to bone/cartilage phenotype in mice not tested"]},{"year":2021,"claim":"The molecular basis of the skeletal phenotype in TMEM165-CDG was poorly understood; TMEM165 loss impairs glycosaminoglycan chain elongation due to Mn²⁺ cofactor insufficiency, disrupting TGFβ/BMP signaling and promoting premature chondrocyte hypertrophy.","evidence":"TMEM165 KO cells, GAG chain analysis, Mn²⁺ supplementation rescue, chondrogenic signaling assays","pmids":["34930890"],"confidence":"Medium","gaps":["In vivo cartilage-specific knockout not performed","Whether all glycosyltransferases are equally Mn²⁺-dependent through TMEM165 is unclear"]},{"year":2023,"claim":"Whether H⁺ flow through Gdt1p/TMEM165 is unidirectional was unresolved; direct pH recording in L. lactis and in vivo Golgi pH probes showed that H⁺ transport is reversible depending on the prevailing ion gradient, establishing Gdt1p as a bidirectional Ca²⁺(Mn²⁺)/H⁺ exchanger that influences Golgi pH.","evidence":"Extracellular/intracellular pH recording in L. lactis, genetically encoded Golgi pH probes in S. cerevisiae","pmids":["36963491"],"confidence":"High","gaps":["Bidirectionality not yet confirmed for human TMEM165 directly","Impact on Golgi pH in mammalian cells not measured"]},{"year":2023,"claim":"A structural framework for understanding TMEM165 transport and CDG mutations was lacking; an AlphaFold 2-based model refined by molecular dynamics revealed a pseudo-symmetric architecture with the two conserved acidic motifs facing each other to form the cation-binding site, rationalizing the impact of patient mutations including G304R.","evidence":"AlphaFold 2 prediction, molecular dynamics simulation, correlation with patient mutation data","pmids":["37416081"],"confidence":"Medium","gaps":["No experimental structure (cryo-EM, X-ray) available","Ion-binding site not validated by mutagenesis of predicted coordinating residues beyond known motifs"]},{"year":2025,"claim":"Whether the lysosomal pool of TMEM165 has a distinct function beyond degradation was unknown; electrophysiology and live imaging demonstrated that lysosomal TMEM165 imports Ca²⁺ into the lysosomal lumen and mediates Ca²⁺-induced proton leakage, accelerating recovery from Ca²⁺ overload and promoting cell survival.","evidence":"Lysosomal electrophysiology, fluorescent ion imaging, genetic depletion and overexpression in mammalian cells","pmids":["40473625"],"confidence":"High","gaps":["Relative contribution of lysosomal versus Golgi TMEM165 to whole-cell Ca²⁺/H⁺ homeostasis not quantified","Whether lysosomal function is relevant to CDG pathology not determined"]},{"year":null,"claim":"An experimentally determined atomic structure of TMEM165 is needed to define the ion translocation pathway, binding-site stoichiometry, and gating mechanism; how the Mn²⁺-sensing/degradation machinery recognizes the E108 residue and the identity of the receptor/ubiquitin ligase remain unknown.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimental structure","Mn²⁺ sensor/E3 ligase for TMEM165 degradation unidentified","Relative physiological importance of Ca²⁺ versus Mn²⁺ transport in different tissues not resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[3,7,12,17]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,1,2,4,7,9]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,4,17]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5,11]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,7,12,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,5]}],"complexes":[],"partners":["ATP2C1"],"other_free_text":[]},"mechanistic_narrative":"TMEM165 is a Golgi- and lysosome-resident Ca²⁺/Mn²⁺:H⁺ antiporter of the UPF0016/CaCA2 family that maintains luminal cation homeostasis essential for glycosyltransferase and lactose synthase activity. Direct transport assays in Lactococcus lactis and yeast demonstrate that TMEM165 imports both Ca²⁺ and Mn²⁺ into the Golgi lumen in exchange for H⁺, with reversible H⁺ flow direction depending on ion gradients; its loss causes Mn²⁺ depletion in the secretory pathway, leading to hypogalactosylation, defective glycosaminoglycan elongation, and impaired chondrocyte differentiation [PMID:27075443, PMID:32047108, PMID:36963491, PMID:34930890]. TMEM165 abundance is regulated by cytosolic Mn²⁺ through lysosomal degradation controlled by the conserved E-ϕ-G-D-[KR]-[TS] motif (specifically E108), and this regulatory axis is functionally coupled to the SPCA1 Golgi Mn²⁺/Ca²⁺ pump [PMID:28270545, PMID:31652305]. Biallelic loss-of-function mutations in TMEM165 cause a congenital disorder of glycosylation (CDG-II) characterized by skeletal and multisystemic abnormalities [PMID:22683087]."},"prefetch_data":{"uniprot":{"accession":"Q9HC07","full_name":"Putative divalent cation/proton antiporter TMEM165","aliases":["Transmembrane protein 165","Transmembrane protein PT27","Transmembrane protein TPARL"],"length_aa":324,"mass_kda":34.9,"function":"Putative divalent cation:proton antiporter that exchanges calcium or manganese ions for protons across the Golgi membrane. Mediates the reversible transport of calcium or manganese to the Golgi lumen driven by the proton gradient and possibly the membrane potential generated by V-ATPase. Provides calcium or manganese cofactors to resident Golgi enzymes and contributes to the maintenance of an acidic luminal Golgi pH required for proper functioning of the secretory pathway (By similarity) (PubMed:22683087, PubMed:23569283, PubMed:27008884, PubMed:32047108). Promotes Ca(2+) storage within the Golgi lumen of the mammary epithelial cells to be then secreted into milk (By similarity). The transport mechanism and stoichiometry remains to be elucidated","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q9HC07/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TMEM165","classification":"Not Classified","n_dependent_lines":162,"n_total_lines":1208,"dependency_fraction":0.13410596026490065},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SLC35E1","stoichiometry":4.0},{"gene":"GORASP2","stoichiometry":0.2},{"gene":"GPR107","stoichiometry":0.2},{"gene":"STX5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TMEM165","total_profiled":1310},"omim":[{"mim_id":"614727","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE IIk; CDG2K","url":"https://www.omim.org/entry/614727"},{"mim_id":"614726","title":"TRANSMEMBRANE PROTEIN 165; TMEM165","url":"https://www.omim.org/entry/614726"}],"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/TMEM165"},"hgnc":{"alias_symbol":["TMPT27","TPARL","GDT1","SLC64A1"],"prev_symbol":[]},"alphafold":{"accession":"Q9HC07","domains":[{"cath_id":"-","chopping":"87-210_233-324","consensus_level":"medium","plddt":90.4784,"start":87,"end":324}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HC07","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HC07-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HC07-F1-predicted_aligned_error_v6.png","plddt_mean":76.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TMEM165","jax_strain_url":"https://www.jax.org/strain/search?query=TMEM165"},"sequence":{"accession":"Q9HC07","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HC07.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HC07/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HC07"}},"corpus_meta":[{"pmid":"22683087","id":"PMC_22683087","title":"TMEM165 deficiency causes a congenital disorder of glycosylation.","date":"2012","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22683087","citation_count":172,"is_preprint":false},{"pmid":"27008884","id":"PMC_27008884","title":"Glycosylation abnormalities in Gdt1p/TMEM165 deficient cells result from a defect in Golgi manganese homeostasis.","date":"2016","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27008884","citation_count":97,"is_preprint":false},{"pmid":"28323990","id":"PMC_28323990","title":"Galactose Supplementation in Patients With TMEM165-CDG Rescues the Glycosylation Defects.","date":"2017","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/28323990","citation_count":72,"is_preprint":false},{"pmid":"27075443","id":"PMC_27075443","title":"Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27075443","citation_count":52,"is_preprint":false},{"pmid":"28270545","id":"PMC_28270545","title":"Manganese-induced turnover of TMEM165.","date":"2017","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/28270545","citation_count":48,"is_preprint":false},{"pmid":"23430531","id":"PMC_23430531","title":"Bone Dysplasia as a Key Feature in Three Patients with a Novel Congenital Disorder of Glycosylation (CDG) Type II Due to a Deep Intronic Splice Mutation in TMEM165.","date":"2012","source":"JIMD reports","url":"https://pubmed.ncbi.nlm.nih.gov/23430531","citation_count":44,"is_preprint":false},{"pmid":"23575229","id":"PMC_23575229","title":"Impact of disease-causing mutations on TMEM165 subcellular localization, a recently identified protein involved in CDG-II.","date":"2013","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23575229","citation_count":40,"is_preprint":false},{"pmid":"27401145","id":"PMC_27401145","title":"TMEM165 deficiencies in Congenital Disorders of Glycosylation type II (CDG-II): Clues and evidences for roles of the protein in Golgi functions and ion homeostasis.","date":"2016","source":"Tissue & cell","url":"https://pubmed.ncbi.nlm.nih.gov/27401145","citation_count":40,"is_preprint":false},{"pmid":"32047108","id":"PMC_32047108","title":"The human Golgi protein TMEM165 transports calcium and manganese in yeast and bacterial cells.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32047108","citation_count":36,"is_preprint":false},{"pmid":"25609749","id":"PMC_25609749","title":"Abnormal cartilage development and altered N-glycosylation in Tmem165-deficient zebrafish mirrors the phenotypes associated with TMEM165-CDG.","date":"2015","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/25609749","citation_count":32,"is_preprint":false},{"pmid":"30307768","id":"PMC_30307768","title":"Involvement of thapsigargin- and cyclopiazonic acid-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2.","date":"2018","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/30307768","citation_count":28,"is_preprint":false},{"pmid":"31351090","id":"PMC_31351090","title":"Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity.","date":"2019","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/31351090","citation_count":26,"is_preprint":false},{"pmid":"26238249","id":"PMC_26238249","title":"TMEM165 Deficiency: Postnatal Changes in Glycosylation.","date":"2015","source":"JIMD 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/31415112","citation_count":17,"is_preprint":false},{"pmid":"34930890","id":"PMC_34930890","title":"TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/34930890","citation_count":17,"is_preprint":false},{"pmid":"32743000","id":"PMC_32743000","title":"From the Uncharacterized Protein Family 0016 to the GDT1 family: Molecular insights into a newly-characterized family of cation secondary transporters.","date":"2020","source":"Microbial cell (Graz, Austria)","url":"https://pubmed.ncbi.nlm.nih.gov/32743000","citation_count":16,"is_preprint":false},{"pmid":"31652305","id":"PMC_31652305","title":"Investigating the functional link between TMEM165 and SPCA1.","date":"2019","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31652305","citation_count":15,"is_preprint":false},{"pmid":"24720419","id":"PMC_24720419","title":"Antisense-mediated therapeutic pseudoexon skipping in TMEM165-CDG.","date":"2014","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24720419","citation_count":13,"is_preprint":false},{"pmid":"39002685","id":"PMC_39002685","title":"Insights into molecular and cellular functions of the Golgi calcium/manganese-proton antiporter TMEM165.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39002685","citation_count":9,"is_preprint":false},{"pmid":"37062452","id":"PMC_37062452","title":"Insights into the regulation of cellular Mn2+ homeostasis via TMEM165.","date":"2023","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/37062452","citation_count":9,"is_preprint":false},{"pmid":"28088503","id":"PMC_28088503","title":"Evidence for splice transcript variants of TMEM165, a gene involved in CDG.","date":"2017","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/28088503","citation_count":9,"is_preprint":false},{"pmid":"32335229","id":"PMC_32335229","title":"SPCA1 governs the stability of TMEM165 in Hailey-Hailey disease.","date":"2020","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/32335229","citation_count":8,"is_preprint":false},{"pmid":"36963491","id":"PMC_36963491","title":"The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36963491","citation_count":7,"is_preprint":false},{"pmid":"37416081","id":"PMC_37416081","title":"New insights into the pathogenicity of TMEM165 variants using structural modeling based on AlphaFold 2 predictions.","date":"2023","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/37416081","citation_count":6,"is_preprint":false},{"pmid":"38013006","id":"PMC_38013006","title":"Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.","date":"2023","source":"Translational research : the journal of laboratory and clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38013006","citation_count":6,"is_preprint":false},{"pmid":"11270120","id":"PMC_11270120","title":"Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium.","date":"2001","source":"Differentiation; research in biological diversity","url":"https://pubmed.ncbi.nlm.nih.gov/11270120","citation_count":6,"is_preprint":false},{"pmid":"40473625","id":"PMC_40473625","title":"Lysosomal TMEM165 controls cellular ion homeostasis and survival by mediating lysosomal Ca2+ import and H+ efflux.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40473625","citation_count":3,"is_preprint":false},{"pmid":"34703799","id":"PMC_34703799","title":"The Relationship between rs534654 Polymorphism in TMEM165 Gene and Increased Risk of Bipolar Disorder Type 1.","date":"2021","source":"International journal of molecular and cellular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34703799","citation_count":1,"is_preprint":false},{"pmid":"41308767","id":"PMC_41308767","title":"TMEM165 promotes glioblastoma progression through epithelial-mesenchymal transition-mediated invasion and temozolomide chemoresistance.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/41308767","citation_count":0,"is_preprint":false},{"pmid":"41583011","id":"PMC_41583011","title":"Putative role of TMEM165 in congenital cardiomyopathies.","date":"2026","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/41583011","citation_count":0,"is_preprint":false},{"pmid":"38816278","id":"PMC_38816278","title":"Sharing is caring: TMEM165 a Golgi calcium importer used by the lysosome.","date":"2024","source":"Trends in biochemical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38816278","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.05.09.593345","title":"TMEM165 replenishes lysosomal Ca<sup>2+</sup>stores, protects cells against Ca<sup>2+</sup>overload, and mediates Ca<sup>2+</sup>-induced lysosomal H<sup>+</sup>leakage","date":"2024-05-13","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.09.593345","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18479,"output_tokens":4984,"usd":0.065099},"stage2":{"model":"claude-opus-4-6","input_tokens":8485,"output_tokens":3395,"usd":0.19095},"total_usd":0.256049,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"TMEM165 deficiency causes Golgi glycosylation defects (CDG-II), identified via siRNA knockdown in HEK cells showing abnormal serum-transferrin isoelectric focusing patterns; TMEM165 encodes a putative transmembrane protein whose loss disrupts Golgi N-glycosylation.\",\n      \"method\": \"siRNA knockdown in HEK cells, transferrin isoelectric focusing, autozygosity mapping\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined cellular phenotype, replicated across multiple patients and cell types\",\n      \"pmids\": [\"22683087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Wild-type TMEM165 localizes to the Golgi compartment, plasma membrane, and late endosomes/lysosomes; disease-causing mutations alter its subcellular localization; the YNRL motif is critical for proper TMEM165 subcellular localization; mutations associated with mild phenotype can complement yeast gdt1Δ whereas truncation mutations cannot.\",\n      \"method\": \"Fluorescence microscopy of tagged TMEM165 variants, yeast complementation assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — direct localization experiment with functional consequence via yeast complementation, single lab\",\n      \"pmids\": [\"23575229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Golgi glycosylation defects in TMEM165/Gdt1p-deficient cells result from Golgi manganese (Mn2+) homeostasis defect; Mn2+ supplementation rescues normal glycosylation in both yeast gdt1Δ and mammalian TMEM165-depleted cells; GPP130 Mn2+ sensitivity is altered in TMEM165-depleted cells.\",\n      \"method\": \"Genetic knockdown/knockout, Mn2+ supplementation rescue, GPP130 sensitivity assay in yeast and mammalian cells\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated in two model systems (yeast and mammalian cells)\",\n      \"pmids\": [\"27008884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Yeast Gdt1p (TMEM165 ortholog) has Ca2+ transport activity demonstrated by heterologous expression in Lactococcus lactis; Ca2+ uptake is pH-dependent, indicating Gdt1p acts as a Ca2+/H+ antiporter; Gdt1p controls cellular calcium stores and is required for glycosylation of carboxypeptidase Y and Gas1p under high external calcium, which is rescued by Mn2+ supplementation.\",\n      \"method\": \"Heterologous expression in Lactococcus lactis with Ca2+ uptake assay, yeast glycosylation assays, calcium signaling measurements\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct transport activity demonstrated by reconstitution in bacterial cells, supported by in vivo yeast assays\",\n      \"pmids\": [\"27075443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM165 is a novel Mn2+-sensitive Golgi protein that undergoes lysosomal degradation upon high Mn2+ exposure; the glutamic acid at position E108 within the cytosolic ELGDK motif is crucial for Mn2+-induced degradation of TMEM165; the CDG patient variant E108G renders TMEM165 insensitive to Mn2+-induced degradation.\",\n      \"method\": \"Mn2+ exposure assays, lysosomal degradation pathway analysis, site-directed mutagenesis of E108G variant in mammalian cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutational analysis with defined molecular phenotype, identifies specific motif responsible for Mn2+-induced degradation\",\n      \"pmids\": [\"28270545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM165-deficient cells show severe hypogalactosylation and GalNAc transfer defects in N-linked glycans and glycolipids; these defects are rescued by Mn2+ and also by galactose supplementation; oral D-galactose supplementation in TMEM165-CDG patients improves glycosylation parameters.\",\n      \"method\": \"Mass spectrometry of N-linked glycans and glycolipids in TMEM165 KO HEK293 cells, Mn2+ and galactose rescue experiments, clinical intervention in patients\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in cells and patients, identifies specific glycosylation substrates\",\n      \"pmids\": [\"28323990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mn2+ uptake rescuing N-glycosylation in TMEM165 KO cells does not rely on endocytosis but occurs via plasma membrane transporters; the rescue of LAMP2 glycosylation defects involves thapsigargin- and cyclopiazonic acid-sensitive pumps (SERCA pumps); overexpression of SERCA2b partially rescues LAMP2 glycosylation defects in TMEM165 KO cells.\",\n      \"method\": \"Endocytosis inhibition assays, thapsigargin/CPA inhibitor experiments, SERCA2b overexpression in TMEM165 KO HEK293 cells, western blot of LAMP2 glycosylation\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological and genetic rescue experiments in knockout cells, single lab\",\n      \"pmids\": [\"30307768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human TMEM165 directly transports both Ca2+ and Mn2+ when heterologously expressed in yeast devoid of Golgi Ca2+/Mn2+ transporters and in Lactococcus lactis loaded with Fura-2 fluorescent probe; expression abrogates Ca2+- and Mn2+-induced growth defects and glycosylation defects in yeast; the E108G disease-causing mutation significantly reduces TMEM165 transport activity.\",\n      \"method\": \"Heterologous expression in S. cerevisiae and L. lactis, Fura-2 fluorescent Ca2+/Mn2+ influx assay, yeast growth and glycosylation rescue assays, mutagenesis of E108G\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical transport activity demonstrated in two heterologous systems with mutagenesis validation\",\n      \"pmids\": [\"32047108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM165 expression and abundance are functionally linked to SPCA1 (the Golgi Ca2+/Mn2+ P-type ATPase); TMEM165 is constitutively degraded in lysosomes in the absence of SPCA1; SPCA1's Mn2+-pumping capacity (but not Ca2+ pumping, as shown by Q747A mutant favoring Mn2+) rescues TMEM165 abundance and Golgi localization; SERCA2b overexpression also rescues TMEM165 expression.\",\n      \"method\": \"SPCA1-deficient Hap1 cells, SPCA1 mutant complementation, TMEM165 immunoblot and localization, lysosomal degradation tracking\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional link established with mutagenesis separating Mn2+ and Ca2+ transport roles, multiple orthogonal approaches\",\n      \"pmids\": [\"31652305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM165 is required for normal milk biosynthesis in the lactating mammary gland; conditional knockout mice show reduced lactose biosynthesis, altered milk composition (elevated fat, protein, iron, zinc; lower calcium and manganese), and impaired nursing pup growth; TMEM165 supplies Ca2+ and Mn2+ to the Golgi in exchange for H+ to sustain lactose synthase and glycosyltransferases.\",\n      \"method\": \"Conditional tissue-specific knockout mice, milk composition biochemical assays, immunostaining, lactation phenotype measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo conditional KO with defined molecular and physiological phenotypes, mechanistic interpretation supported by biochemistry\",\n      \"pmids\": [\"30622138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The two conserved UPF0016 consensus motifs (E-φ-G-D-[KR]-[TS]) in TMEM165 are crucial for its function in Golgi glycosylation and its Mn2+ sensitivity; specific amino acids within these motifs contribute differentially to glycosylation function versus Mn2+-induced degradation sensitivity.\",\n      \"method\": \"Site-directed mutagenesis of UPF0016 motif residues, glycosylation rescue assays, Mn2+ sensitivity assays in mammalian cells\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with defined functional readout, single lab\",\n      \"pmids\": [\"31351090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM165 deficiency impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan (GAG) chains of proteoglycans; the blockage is not due to defective Golgi elongating enzymes but to Mn2+ cofactor insufficiency; Mn2+ supplementation rescues GAG chain elongation; TMEM165 loss impairs TGFβ and BMP signaling in chondrocytes and accelerates Ihh expression promoting early chondrocyte hypertrophy.\",\n      \"method\": \"TMEM165 KO cells, GAG chain length analysis, enzyme activity assays, Mn2+ supplementation rescue, signaling pathway analysis, chondrogenic differentiation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with multiple defined molecular phenotypes and rescue, single lab\",\n      \"pmids\": [\"34930890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Yeast Gdt1p (TMEM165 ortholog) mediates H+ transport in exchange for Ca2+ and Mn2+ across the Golgi membrane; the direction of H+ flow is reversible depending on physiological ion concentration gradients; Gdt1p influences Golgi pH measured by genetically encoded pH probes in vivo.\",\n      \"method\": \"Heterologous expression in L. lactis with extracellular/intracellular pH recording, in vivo cytosolic and Golgi pH measurements with genetically encoded probes in S. cerevisiae\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct transport mechanism demonstrated by electrophysiology/pH recording across biological membrane plus in vivo pH measurement\",\n      \"pmids\": [\"36963491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TMEM165 plays a crucial role in cellular Mn2+ homeostasis: Mn2+ supplementation fully rescues Mn2+ content in the secretory pathway of TMEM165-depleted cells and restores glycosylation; TMEM165 and SPCA1 together regulate cellular Mn2+ homeostasis; TMEM165's Mn2+-induced degradation is linked to cytosolic Mn2+ detoxification mediated by SPCA1.\",\n      \"method\": \"ICP-MS measurement of subcellular Mn2+ content, GPP130 as Golgi Mn2+ sensor, TMEM165/SPCA1 double-depletion experiments\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct elemental measurement of Mn2+ in subcellular compartments, functional link between TMEM165 and SPCA1, single lab\",\n      \"pmids\": [\"37062452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AlphaFold 2-based structural model of TMEM165 (validated by molecular dynamics simulation) reveals a two-fold repeat of three transmembrane helices where the conserved E-φ-G-D-[KR]-[TS] consensus motifs face each other forming a putative acidic cation-binding site at the cytosolic side; this model explains the impact of CDG patient mutations including G304R on transporter function.\",\n      \"method\": \"AlphaFold 2 structural prediction, molecular dynamics simulation with membrane lipids and water, functional validation using patient mutation data\",\n      \"journal\": \"Computational and structural biotechnology journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/4 — structural model with MD refinement explains mutation pathogenicity, but not experimentally solved structure; functional validation indirect\",\n      \"pmids\": [\"37416081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM165 splice transcript variants (Short-Form, 129 aa; Long-Form, 259 aa) exist in human tissues, particularly in brain; both isoforms localize to the endoplasmic reticulum (distinct from wild-type Golgi localization) and have different effects on glycosylation compared to the full-length protein; the Short-Form forms homodimers.\",\n      \"method\": \"RT-PCR and RT-qPCR from human brain tissues, overexpression of isoforms with fluorescence microscopy, glycosylation assays\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct localization and functional experiments with defined isoforms, single lab\",\n      \"pmids\": [\"28088503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPCA1 deficiency in Hailey-Hailey disease fibroblasts and keratinocytes renders TMEM165 more sensitive to Mn2+-induced degradation due to cytosolic Mn2+ accumulation; this links SPCA1 function to TMEM165 stability in a pathological context.\",\n      \"method\": \"Hailey-Hailey disease patient fibroblasts/keratinocytes, ICP-MS, GPP130 Golgi Mn2+ sensor, Mn2+ exposure assays\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional link in patient-derived cells confirmed by direct Mn2+ measurement, single lab\",\n      \"pmids\": [\"32335229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A fraction of TMEM165 localizes on the lysosomal limiting membrane where it imports Ca2+ into the lysosomal lumen and mediates Ca2+-induced lysosomal proton leakage; this lysosomal TMEM165 activity accelerates recovery from cytosolic Ca2+ overload, enhances cell survival, and causes cytosolic acidification.\",\n      \"method\": \"Genetic depletion and overexpression, electrophysiology, fluorescent visualization of subcellular ion concentrations and fluxes across lysosomal membrane\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct electrophysiological measurement of ion transport across lysosomal membrane combined with genetic depletion/overexpression and live imaging of ion fluxes\",\n      \"pmids\": [\"40473625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Morpholino knockdown of tmem165 in zebrafish causes craniofacial abnormalities with fewer chondrocytes, altered N-glycan processing (mirroring human patients), and decreased expression of cartilage and bone development markers, establishing that TMEM165 deficiency impairs chondrocyte and osteoblast differentiation in vivo.\",\n      \"method\": \"Morpholino knockdown in zebrafish embryos, glycomic analysis, craniofacial phenotype quantification, marker gene expression\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss-of-function with defined cellular and glycomic phenotypes in a vertebrate model system\",\n      \"pmids\": [\"25609749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM165 knockout in human MDA-MB-231 breast cancer cells results in significant reduction of cell migration, tumor growth, and tumor vascularization in vivo; TMEM165 loss alters glycosylation of cancer cells and affects expression of EMT-related glycoproteins including E-cadherin.\",\n      \"method\": \"CRISPR/Cas9 KO, migration assays, in vivo tumor growth assays, glycoproteomic analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotypes in vitro and in vivo, mechanistic link to glycosylation changes, single lab\",\n      \"pmids\": [\"32733646\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TMEM165 is a Golgi (and lysosomal) membrane protein belonging to the UPF0016/CaCA2 family that functions as a Ca2+/Mn2+:H+ antiporter, pumping Ca2+ and Mn2+ into the Golgi lumen in exchange for H+ to maintain Golgi cation homeostasis required for glycosyltransferase activity; at the lysosome it imports Ca2+ and mediates Ca2+-induced proton leakage to regulate intracellular ion homeostasis and cell survival; TMEM165 abundance is regulated by cytosolic Mn2+ concentration through lysosomal degradation involving the ELGDK motif (E108), and is functionally coupled to the SPCA1 Mn2+/Ca2+ ATPase pump.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TMEM165 is a Golgi- and lysosome-resident Ca²⁺/Mn²⁺:H⁺ antiporter of the UPF0016/CaCA2 family that maintains luminal cation homeostasis essential for glycosyltransferase and lactose synthase activity. Direct transport assays in Lactococcus lactis and yeast demonstrate that TMEM165 imports both Ca²⁺ and Mn²⁺ into the Golgi lumen in exchange for H⁺, with reversible H⁺ flow direction depending on ion gradients; its loss causes Mn²⁺ depletion in the secretory pathway, leading to hypogalactosylation, defective glycosaminoglycan elongation, and impaired chondrocyte differentiation [PMID:27075443, PMID:32047108, PMID:36963491, PMID:34930890]. TMEM165 abundance is regulated by cytosolic Mn²⁺ through lysosomal degradation controlled by the conserved E-ϕ-G-D-[KR]-[TS] motif (specifically E108), and this regulatory axis is functionally coupled to the SPCA1 Golgi Mn²⁺/Ca²⁺ pump [PMID:28270545, PMID:31652305]. Biallelic loss-of-function mutations in TMEM165 cause a congenital disorder of glycosylation (CDG-II) characterized by skeletal and multisystemic abnormalities [PMID:22683087].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"The molecular basis of a CDG-II subtype was unknown; identification of TMEM165 mutations in patients and replication of glycosylation defects by siRNA knockdown established TMEM165 as a Golgi-resident protein required for N-glycosylation.\",\n      \"evidence\": \"Autozygosity mapping in CDG-II families, siRNA knockdown in HEK cells, transferrin isoelectric focusing\",\n      \"pmids\": [\"22683087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transport mechanism unknown\", \"Substrate ion not identified\", \"No structure available\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"It was unclear where TMEM165 resides and how disease mutations affect its trafficking; fluorescence microscopy and yeast complementation showed wild-type TMEM165 localizes to Golgi, plasma membrane, and late endosomes/lysosomes, with the YNRL motif governing its subcellular targeting.\",\n      \"evidence\": \"Fluorescence microscopy of tagged TMEM165 variants, yeast gdt1Δ complementation\",\n      \"pmids\": [\"23575229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of YNRL-mediated sorting not defined\", \"Relative functional contribution of each compartment not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The ion species responsible for glycosylation failure was unknown; Mn²⁺ supplementation rescued glycosylation defects in both yeast gdt1Δ and mammalian TMEM165-depleted cells, identifying Golgi Mn²⁺ homeostasis as the critical function, while direct Ca²⁺/H⁺ antiport activity was demonstrated for Gdt1p in Lactococcus lactis.\",\n      \"evidence\": \"Mn²⁺ supplementation rescue in yeast and mammalian cells, heterologous Ca²⁺ uptake assays in L. lactis, GPP130 Mn²⁺ sensitivity\",\n      \"pmids\": [\"27008884\", \"27075443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Mn²⁺ transport by human TMEM165 not yet demonstrated\", \"Stoichiometry of Ca²⁺/Mn²⁺:H⁺ exchange unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"How TMEM165 protein levels are controlled was unclear; high cytosolic Mn²⁺ triggers lysosomal degradation of TMEM165 via the E108 residue within the ELGDK motif, and the CDG patient mutation E108G abolishes this regulation, separating transport function from abundance control.\",\n      \"evidence\": \"Site-directed mutagenesis, Mn²⁺ exposure and lysosomal degradation assays in mammalian cells\",\n      \"pmids\": [\"28270545\", \"31351090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Mn²⁺ sensor that recognizes E108 is unknown\", \"Whether degradation is ubiquitin-dependent not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The specific glycan structures affected and potential therapeutic rescue were undefined; mass spectrometry revealed hypogalactosylation and GalNAc transfer defects in N-glycans and glycolipids, both rescued by Mn²⁺ and galactose supplementation, with oral galactose improving CDG patient glycosylation.\",\n      \"evidence\": \"Mass spectrometry of glycans in TMEM165 KO HEK293 cells, Mn²⁺/galactose rescue, clinical galactose supplementation\",\n      \"pmids\": [\"28323990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Long-term clinical efficacy of galactose therapy not established\", \"Mechanism by which galactose bypasses Mn²⁺ deficiency not fully resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether human TMEM165 itself directly transports Mn²⁺ (not just Ca²⁺) was unresolved; heterologous expression in yeast and L. lactis with Fura-2 fluorescence demonstrated direct Ca²⁺ and Mn²⁺ transport by human TMEM165, with the E108G mutation reducing transport activity.\",\n      \"evidence\": \"Fura-2 fluorescent influx assay in L. lactis, yeast growth/glycosylation rescue, E108G mutagenesis\",\n      \"pmids\": [\"32047108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No reconstitution in proteoliposomes for kinetic characterization\", \"H⁺ counterflux not directly measured for the human protein at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The relationship between TMEM165 and the SPCA1 Golgi pump was unknown; SPCA1 deficiency causes constitutive lysosomal degradation of TMEM165, and SPCA1's Mn²⁺-pumping capacity (not Ca²⁺) is required to stabilize TMEM165, revealing a coupled regulatory axis for Golgi Mn²⁺ supply.\",\n      \"evidence\": \"SPCA1-deficient Hap1 cells, Q747A Mn²⁺-selective SPCA1 mutant complementation, immunoblot and localization\",\n      \"pmids\": [\"31652305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TMEM165 and SPCA1 physically interact is unknown\", \"Mechanism by which SPCA1 Mn²⁺ transport prevents TMEM165 degradation not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The physiological relevance of TMEM165 in intact tissues was unclear; conditional knockout in mouse mammary gland showed reduced lactose biosynthesis, lower milk Ca²⁺ and Mn²⁺, and impaired pup growth, demonstrating that TMEM165 supplies Golgi Ca²⁺/Mn²⁺ required for lactose synthase in vivo.\",\n      \"evidence\": \"Conditional tissue-specific knockout mice, milk composition analysis, immunostaining\",\n      \"pmids\": [\"30622138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Systemic phenotype of whole-body knockout not reported\", \"Contribution to bone/cartilage phenotype in mice not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The molecular basis of the skeletal phenotype in TMEM165-CDG was poorly understood; TMEM165 loss impairs glycosaminoglycan chain elongation due to Mn²⁺ cofactor insufficiency, disrupting TGFβ/BMP signaling and promoting premature chondrocyte hypertrophy.\",\n      \"evidence\": \"TMEM165 KO cells, GAG chain analysis, Mn²⁺ supplementation rescue, chondrogenic signaling assays\",\n      \"pmids\": [\"34930890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo cartilage-specific knockout not performed\", \"Whether all glycosyltransferases are equally Mn²⁺-dependent through TMEM165 is unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Whether H⁺ flow through Gdt1p/TMEM165 is unidirectional was unresolved; direct pH recording in L. lactis and in vivo Golgi pH probes showed that H⁺ transport is reversible depending on the prevailing ion gradient, establishing Gdt1p as a bidirectional Ca²⁺(Mn²⁺)/H⁺ exchanger that influences Golgi pH.\",\n      \"evidence\": \"Extracellular/intracellular pH recording in L. lactis, genetically encoded Golgi pH probes in S. cerevisiae\",\n      \"pmids\": [\"36963491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Bidirectionality not yet confirmed for human TMEM165 directly\", \"Impact on Golgi pH in mammalian cells not measured\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A structural framework for understanding TMEM165 transport and CDG mutations was lacking; an AlphaFold 2-based model refined by molecular dynamics revealed a pseudo-symmetric architecture with the two conserved acidic motifs facing each other to form the cation-binding site, rationalizing the impact of patient mutations including G304R.\",\n      \"evidence\": \"AlphaFold 2 prediction, molecular dynamics simulation, correlation with patient mutation data\",\n      \"pmids\": [\"37416081\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure (cryo-EM, X-ray) available\", \"Ion-binding site not validated by mutagenesis of predicted coordinating residues beyond known motifs\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Whether the lysosomal pool of TMEM165 has a distinct function beyond degradation was unknown; electrophysiology and live imaging demonstrated that lysosomal TMEM165 imports Ca²⁺ into the lysosomal lumen and mediates Ca²⁺-induced proton leakage, accelerating recovery from Ca²⁺ overload and promoting cell survival.\",\n      \"evidence\": \"Lysosomal electrophysiology, fluorescent ion imaging, genetic depletion and overexpression in mammalian cells\",\n      \"pmids\": [\"40473625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of lysosomal versus Golgi TMEM165 to whole-cell Ca²⁺/H⁺ homeostasis not quantified\", \"Whether lysosomal function is relevant to CDG pathology not determined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"An experimentally determined atomic structure of TMEM165 is needed to define the ion translocation pathway, binding-site stoichiometry, and gating mechanism; how the Mn²⁺-sensing/degradation machinery recognizes the E108 residue and the identity of the receptor/ubiquitin ligase remain unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental structure\", \"Mn²⁺ sensor/E3 ligase for TMEM165 degradation unidentified\", \"Relative physiological importance of Ca²⁺ versus Mn²⁺ transport in different tissues not resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [3, 7, 12, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 1, 2, 4, 7, 9]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 4, 17]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5, 11]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 7, 12, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ATP2C1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}