{"gene":"TMEM165","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2012,"finding":"TMEM165 is required for Golgi glycosylation; siRNA-mediated knockdown of TMEM165 in HEK cells causes Golgi glycosylation defects, identifying it as a protein involved in congenital disorders of glycosylation.","method":"siRNA knockdown in HEK cells with glycosylation readout","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with defined cellular phenotype, independently replicated across multiple labs","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 subcellular localization differentially depending on the mutation. The YNRL motif is critical for TMEM165 subcellular localization.","method":"Fluorescence microscopy of wild-type and mutant TMEM165 constructs; yeast complementation assay with gdt1Δ strain","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional consequence via yeast complementation, single lab, two orthogonal methods","pmids":["23575229"],"is_preprint":false},{"year":2016,"finding":"Glycosylation defects in Gdt1p/TMEM165-deficient cells result from a defect in Golgi manganese (Mn2+) homeostasis; Mn2+ supplementation restores normal glycosylation in both yeast gdt1Δ mutants and TMEM165-depleted mammalian cells. GPP130 Mn2+ sensitivity is altered in TMEM165-depleted cells.","method":"Yeast and mammalian cell knockout/depletion with Mn2+ supplementation rescue; GPP130 as Golgi Mn2+ sensor","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast genetics, mammalian knockdown, Mn2+ rescue, Golgi sensor), replicated across organisms","pmids":["27008884"],"is_preprint":false},{"year":2016,"finding":"Yeast Gdt1p (TMEM165 ortholog) has direct Ca2+ transport activity functioning as a Ca2+/H+ antiporter, demonstrated by heterologous expression in Lactococcus lactis; Gdt1p controls cellular calcium stores and is required for glycosylation at high external calcium concentrations, with glycosylation restored by Mn2+ supplementation.","method":"Heterologous expression in Lactococcus lactis with Ca2+ uptake assay; yeast genetic analysis; glycosylation assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical transport assay in reconstituted bacterial system plus in vivo yeast genetics, single lab but multiple orthogonal methods","pmids":["27075443"],"is_preprint":false},{"year":2017,"finding":"High Mn2+ concentrations cause lysosomal degradation of TMEM165. The glutamic acid at position E108 within the cytosolic ELGDK motif is crucial for Mn2+-induced degradation; the E108G variant is insensitive to Mn2+-induced degradation but does not abolish TMEM165 function in Golgi glycosylation.","method":"Western blot of TMEM165 levels upon MnCl2 exposure in cell lines expressing wild-type or mutant TMEM165; lysosomal inhibitor experiments","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis with functional readout, single lab, multiple mutants tested","pmids":["28270545"],"is_preprint":false},{"year":2017,"finding":"TMEM165 deficiency causes severe hypogalactosylation and GalNAc transfer defects; these N-glycosylation defects are corrected by Mn2+ supplementation and also rescued by galactose supplementation in cells and in patients.","method":"Mass spectrometry glycan analysis in TMEM165 KO HEK293 cells; patient treatment with oral galactose; transferrin isoelectrofocusing","journal":"The Journal of clinical endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (MS glycomics, patient biochemistry, cell rescue), replicated in both cell model and patients","pmids":["28323990"],"is_preprint":false},{"year":2017,"finding":"TMEM165 exists as splice-transcript isoforms (Short Form 129 aa and Long Form 259 aa in addition to full-length 324 aa); both isoforms localize to the endoplasmic reticulum (not Golgi), the Short Form forms homodimers and is expressed at low levels broadly but enriched in brain, and the Long Form is expressed only in the temporal lobe. These isoforms have different effects on glycosylation compared to wild-type protein.","method":"RT-PCR from human brain tissue, RT-Q-PCR, fluorescence microscopy of expressed isoforms, western blot for glycosylation readout","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by imaging and gel mobility glycosylation assay, single lab, multiple methods","pmids":["28088503"],"is_preprint":false},{"year":2018,"finding":"The rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ is mediated by thapsigargin- and cyclopiazonic acid-sensitive pumps (SERCA pumps), not by endocytosis or SPCA1. Overexpression of SERCA2b partially rescues LAMP2 glycosylation defect in TMEM165 KO cells.","method":"Pharmacological inhibition with thapsigargin/CPA in TMEM165 KO HEK293 cells; SERCA2b overexpression; glycosylation readout by western blot","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological dissection and rescue by overexpression, single lab, two orthogonal approaches","pmids":["30307768"],"is_preprint":false},{"year":2019,"finding":"TMEM165 directly mediates Ca2+ and Mn2+ transport when expressed heterologously in yeast and Lactococcus lactis; expression in a yeast strain lacking Golgi Ca2+/Mn2+ transporters abrogates Mn2+-induced growth defects, excessive Mn2+ accumulation, and glycosylation defects. The E108G disease-causing mutation significantly reduces TMEM165 transport activity.","method":"Heterologous expression in S. cerevisiae and L. lactis; Fura-2 fluorescent probe Ca2+ influx assay in bacteria; yeast growth assays; glycosylation assays; site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical transport assay reconstituted in two independent heterologous systems, mutagenesis of disease-causing variants, multiple orthogonal methods","pmids":["32047108"],"is_preprint":false},{"year":2019,"finding":"TMEM165 abundance is directly dependent on SPCA1 function—specifically SPCA1's capacity to pump Mn2+ into the Golgi lumen. In SPCA1-deficient cells, TMEM165 is constitutively degraded in lysosomes; the SPCA1 Q747A mutant that favors Mn2+ pumping rescues TMEM165 abundance and Golgi localization. SERCA2b overexpression also rescues TMEM165 expression.","method":"SPCA1-deficient Hap1 cells; complementation with SPCA1 mutants differentially impairing Mn2+/Ca2+ transport; western blot; fluorescence microscopy; lysosomal inhibitors","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical approach with structure-function mutants and multiple readouts, single lab but multiple orthogonal methods","pmids":["31652305"],"is_preprint":false},{"year":2019,"finding":"TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and affects milk calcium and manganese levels; conditional knockout mice show decreased lactose biosynthesis and only calcium and manganese levels are significantly lower in milk (normalized to protein), consistent with TMEM165 supplying Ca2+ and Mn2+ to the Golgi in exchange for H+.","method":"Conditional tissue-specific knockout mice; biochemical assays of milk composition; immunostaining","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO in mammals with defined biochemical phenotype, multiple readouts, in vivo model","pmids":["30622138"],"is_preprint":false},{"year":2019,"finding":"The conserved UPF0016 consensus motifs E-φ-G-D-[KR]-[TS] in TMEM165 are crucial for both Golgi glycosylation function and Mn2+-induced sensitivity; specific amino acids within these motifs differentially contribute to these two activities.","method":"Site-directed mutagenesis of conserved motifs; glycosylation assays; western blot for Mn2+-induced degradation","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with functional readout, single lab, two orthogonal functional assays","pmids":["31351090"],"is_preprint":false},{"year":2020,"finding":"TMEM165 depletion in SPCA1-deficient context (Hailey-Hailey disease fibroblasts and keratinocytes) leads to increased sensitivity to Mn2+-induced degradation, linked to cytosolic Mn2+ accumulation as measured by ICP-MS and GPP130 as Golgi Mn2+ sensor.","method":"HHD patient fibroblasts and keratinocytes; western blot for TMEM165 upon MnCl2; ICP-MS for Mn2+ levels; GPP130 immunofluorescence as Golgi Mn2+ sensor","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient cells with multiple orthogonal readouts, single lab","pmids":["32335229"],"is_preprint":false},{"year":2021,"finding":"TMEM165 deficiency impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans (producing shorter GAG chains) not by defects in Golgi elongating enzymes but by reduced Mn2+ cofactor availability; Mn2+ supplementation rescues the elongation defect. TMEM165 deficiency also impairs TGFβ and BMP signaling in chondrocytes and accelerates chondrocyte maturation/hypertrophy.","method":"TMEM165 KO cells; GAG chain analysis; Mn2+ rescue; TGFβ/BMP signaling assays; chondrocyte differentiation assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined biochemical and pathway phenotypes, Mn2+ rescue mechanistically informative, single lab","pmids":["34930890"],"is_preprint":false},{"year":2023,"finding":"Yeast Gdt1p transports H+ ions in exchange for Ca2+ and Mn2+ cations (antiporter mechanism); the direction of H+ transport can be reversed depending on physiological concentration gradients. Direct transport measurements were made by expressing Gdt1p in L. lactis and recording extracellular/intracellular pH during application of Ca2+, Mn2+ or H+ gradients; in vivo cytosolic and Golgi pH measurements confirmed Gdt1p influences Golgi pH.","method":"Heterologous expression in L. lactis with pH recording; genetically encoded pH probes in S. cerevisiae targeting cytosol and Golgi","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical transport assays in reconstituted system plus in vivo pH measurements, multiple orthogonal methods","pmids":["36963491"],"is_preprint":false},{"year":2023,"finding":"Mn2+ supplementation fully rescues the Mn2+ content in secretory pathway organelles of TMEM165-depleted cells and restores glycosylation. Both TMEM165 and SPCA1 are crucial for cellular Mn2+ homeostasis; cytosolic Mn2+ accumulation in TMEM165- and SPCA1-depleted cells is demonstrated by ICP-MS upon increasing Mn2+ concentrations. The Mn2+-detoxifying capacity through SPCA1 relies on the Mn2+-induced degradation mechanism of TMEM165.","method":"TMEM165 siRNA depletion; SPCA1 siRNA depletion; ICP-MS for organellar and cytosolic Mn2+; glycosylation assays","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ion measurement by ICP-MS with functional glycosylation readout, single lab, two orthogonal methods","pmids":["37062452"],"is_preprint":false},{"year":2023,"finding":"AlphaFold2 structural modeling of TMEM165, refined by molecular dynamics simulation with membrane lipids, reveals a two-fold repeat of three transmembrane helices where the conserved consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side. This model explains the functional impact of patient mutations including G304R, which is distant from the consensus motifs in sequence.","method":"AlphaFold2 structural prediction refined by molecular dynamics simulation; functional validation of mutations by expression in cells","journal":"Computational and structural biotechnology journal","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational structural model with limited experimental validation of specific structural predictions","pmids":["37416081"],"is_preprint":false},{"year":2025,"finding":"A fraction of TMEM165 localizes to the lysosomal limiting membrane (in addition to its Golgi localization) where it imports calcium into the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. This lysosomal TMEM165 activity accelerates cellular recovery from cytosolic calcium overload, enhancing cell survival, and causes significant cytosolic acidification.","method":"Genetic depletion and overexpression; electrophysiology (patch-clamp of lysosomes); visualization of subcellular ion concentrations and fluxes across lysosomal membrane; cell survival assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct electrophysiology plus ion flux visualization and functional survival assay, multiple orthogonal methods in single rigorous study","pmids":["40473625"],"is_preprint":false}],"current_model":"TMEM165 is a multi-pass transmembrane protein localizing to the medial/trans-Golgi network and the lysosomal membrane that functions as a Ca2+/Mn2+:H+ antiporter—pumping Ca2+ and Mn2+ into the Golgi (and lysosomal) lumen in exchange for H+ efflux—thereby maintaining Golgi Mn2+ homeostasis required as a cofactor for glycosyltransferases and glycosidases; loss of TMEM165 causes Golgi N- and O-glycosylation defects, impaired proteoglycan GAG chain elongation, and altered lipid glycosylation, all rescuable by Mn2+ supplementation; TMEM165 abundance is regulated by Mn2+-induced lysosomal degradation dependent on the cytosolic ELGDK motif (E108) and on SPCA1-mediated Golgi Mn2+ pumping; at the lysosome, TMEM165 imports Ca2+ and mediates proton leakage to regulate intracellular ion homeostasis and cell survival."},"narrative":{"mechanistic_narrative":"TMEM165 is a multi-pass transmembrane cation/H+ antiporter that supplies the secretory pathway with the Mn2+ and Ca2+ required for Golgi glycosylation reactions [PMID:22683087, PMID:27008884, PMID:32047108]. Direct transport assays in reconstituted Lactococcus lactis and yeast systems established that TMEM165 (and its yeast ortholog Gdt1p) imports Ca2+ and Mn2+ in exchange for H+, with the direction of H+ flux set by the prevailing ion gradients, and that this activity controls Golgi pH [PMID:27075443, PMID:32047108, PMID:36963491]. Its glycosylation function reflects delivery of luminal Mn2+ as a cofactor for glycosyltransferases and glycosidases: TMEM165 loss produces N- and O-glycosylation defects, severe hypogalactosylation and GalNAc-transfer failures, and shortened chondroitin/heparan-sulfate GAG chains, all corrected by Mn2+ (and, for galactosylation, galactose) supplementation [PMID:27008884, PMID:28323990, PMID:34930890]. TMEM165 abundance is itself governed by Mn2+: high Golgi Mn2+ pumped by the P-type ATPase SPCA1 triggers lysosomal degradation of TMEM165 via the cytosolic ELGDK motif (E108), coupling the two transporters into a system that detoxifies cytosolic Mn2+ and maintains organellar Mn2+ homeostasis [PMID:28270545, PMID:31652305, PMID:37062452]. The conserved UPF0016 E-φ-G-D-[KR]-[TS] motifs form a cytosolic acidic cation-binding site and are required for both transport/glycosylation and Mn2+-induced degradation [PMID:31351090, PMID:37416081]. Beyond the Golgi, a lysosomal pool of TMEM165 imports Ca2+ into the lysosomal lumen and mediates Ca2+-induced proton leakage, accelerating recovery from cytosolic Ca2+ overload and promoting cell survival [PMID:40473625]. TMEM165 deficiency causes a congenital disorder of glycosylation, and in lactating mammary gland it is required for lactose biosynthesis and normal milk Ca2+/Mn2+ content [PMID:22683087, PMID:30622138].","teleology":[{"year":2012,"claim":"Established that TMEM165 is functionally required for Golgi glycosylation, defining it as a candidate for congenital disorders of glycosylation rather than an orphan membrane protein.","evidence":"siRNA knockdown in HEK cells with glycosylation readout","pmids":["22683087"],"confidence":"High","gaps":["Did not identify the molecular activity underlying the glycosylation requirement","No localization or transport mechanism defined"]},{"year":2013,"claim":"Mapped TMEM165 to Golgi, plasma membrane, and late endosome/lysosome compartments and showed disease mutations mislocalize the protein, identifying the YNRL motif as a localization determinant.","evidence":"Fluorescence microscopy of WT/mutant constructs plus yeast gdt1Δ complementation","pmids":["23575229"],"confidence":"Medium","gaps":["Did not establish the biochemical activity","Functional significance of the non-Golgi pools left unresolved"]},{"year":2016,"claim":"Identified Golgi Mn2+ homeostasis as the mechanistic basis of the glycosylation defect, since Mn2+ supplementation rescues both yeast and mammalian loss-of-function.","evidence":"Yeast/mammalian depletion with Mn2+ rescue and GPP130 Golgi Mn2+ sensor","pmids":["27008884"],"confidence":"High","gaps":["Did not demonstrate direct Mn2+ transport by TMEM165","Stoichiometry and coupling ion unknown at this stage"]},{"year":2016,"claim":"Demonstrated that the ortholog Gdt1p directly transports Ca2+ as a Ca2+/H+ antiporter, providing the first direct biochemical evidence for transport activity and linking it to cellular calcium stores.","evidence":"Heterologous expression in L. lactis with Ca2+ uptake assay plus yeast genetics","pmids":["27075443"],"confidence":"High","gaps":["Direct Mn2+ transport by the human protein not yet shown","Physiological relevance of Ca2+ vs Mn2+ selectivity unclear"]},{"year":2017,"claim":"Showed TMEM165 abundance is controlled by Mn2+-induced lysosomal degradation through the cytosolic ELGDK motif (E108), revealing a regulatory feedback layer distinct from its transport function.","evidence":"MnCl2 exposure with WT/mutant western blots and lysosomal inhibitors","pmids":["28270545"],"confidence":"Medium","gaps":["Degradation machinery and Mn2+-sensing step not identified","E108G separates degradation from glycosylation function but its transport effect was not tested here"]},{"year":2017,"claim":"Defined the specific glycan lesions (hypogalactosylation, GalNAc-transfer defects) and showed both Mn2+ and galactose correct them in cells and patients, translating the Mn2+ model toward therapy.","evidence":"MS glycomics in KO HEK293, patient galactose treatment, transferrin IEF","pmids":["28323990"],"confidence":"High","gaps":["Mechanism by which galactose bypasses the Mn2+ deficiency not fully resolved","Long-term clinical efficacy not assessed"]},{"year":2017,"claim":"Characterized ER-localized splice isoforms with distinct tissue expression and altered glycosylation effects, indicating isoform-specific regulation of the protein.","evidence":"RT-PCR/RT-qPCR from brain tissue, isoform imaging, glycosylation western blots","pmids":["28088503"],"confidence":"Medium","gaps":["Physiological role of ER-retained isoforms unknown","Whether isoforms transport ions untested"]},{"year":2018,"claim":"Dissected the route of extracellular Mn2+ rescue, showing SERCA pumps rather than endocytosis or SPCA1 deliver rescuing Mn2+ in TMEM165 KO cells.","evidence":"Thapsigargin/CPA inhibition and SERCA2b overexpression in KO HEK293 cells","pmids":["30307768"],"confidence":"Medium","gaps":["Quantitative contribution of SERCA vs other pumps unresolved","Relationship between SERCA-delivered ER Mn2+ and Golgi Mn2+ not detailed"]},{"year":2019,"claim":"Provided direct biochemical proof that human TMEM165 transports both Ca2+ and Mn2+ and that the disease variant E108G reduces transport, unifying the transport and disease mechanisms.","evidence":"Reconstitution in S. cerevisiae and L. lactis, Fura-2 Ca2+ assay, growth/glycosylation assays, mutagenesis","pmids":["32047108"],"confidence":"High","gaps":["Transport stoichiometry not quantified","High-resolution structure of the transport site absent"]},{"year":2019,"claim":"Established a reciprocal regulatory circuit in which SPCA1-mediated Golgi Mn2+ pumping sustains TMEM165 by preventing constitutive lysosomal degradation.","evidence":"SPCA1-deficient Hap1 cells, SPCA1 transport-selective mutants, western blot, microscopy","pmids":["31652305"],"confidence":"High","gaps":["Molecular sensor coupling Golgi Mn2+ to TMEM165 degradation unidentified","Whether SPCA1 and TMEM165 physically interact untested"]},{"year":2019,"claim":"Demonstrated a physiological requirement in vivo: mammary TMEM165 supports lactose biosynthesis and normal milk Ca2+/Mn2+, consistent with Ca2+/Mn2+-for-H+ exchange into the Golgi.","evidence":"Tissue-specific conditional knockout mice with milk composition and immunostaining","pmids":["30622138"],"confidence":"High","gaps":["Cell-type contributions within mammary tissue not separated","Systemic vs local Mn2+ effects not dissected"]},{"year":2019,"claim":"Showed the conserved UPF0016 motifs underlie both glycosylation and Mn2+-sensitivity, with individual residues differentially partitioning between the two activities.","evidence":"Site-directed mutagenesis of conserved motifs with glycosylation and degradation readouts","pmids":["31351090"],"confidence":"Medium","gaps":["Structural basis of motif function not resolved here","Coupling between ion binding and degradation signal unclear"]},{"year":2020,"claim":"Extended the SPCA1–TMEM165 link to disease cells, showing SPCA1 loss (Hailey-Hailey) sensitizes TMEM165 to Mn2+-induced degradation via cytosolic Mn2+ accumulation.","evidence":"HHD patient fibroblasts/keratinocytes, MnCl2 western blot, ICP-MS, GPP130 sensor","pmids":["32335229"],"confidence":"Medium","gaps":["Causal contribution of TMEM165 loss to HHD phenotype unproven","Patient-derived single-lab data"]},{"year":2021,"claim":"Showed Mn2+ limitation from TMEM165 loss impairs proteoglycan GAG chain elongation and disrupts TGFβ/BMP signaling and chondrocyte maturation, broadening the functional consequences beyond N/O-glycans.","evidence":"KO cells, GAG chain analysis, Mn2+ rescue, TGFβ/BMP and chondrocyte differentiation assays","pmids":["34930890"],"confidence":"Medium","gaps":["Direct link between GAG defects and signaling changes not fully traced","In vivo skeletal phenotype not established here"]},{"year":2023,"claim":"Defined the antiporter mechanism precisely, showing Gdt1p exchanges H+ for Ca2+/Mn2+ with reversible H+ directionality set by gradients and that it shapes Golgi pH.","evidence":"L. lactis pH recording with ion gradients plus genetically encoded cytosolic/Golgi pH probes in yeast","pmids":["36963491"],"confidence":"High","gaps":["Whether human TMEM165 reverses H+ direction in vivo not directly shown","Quantitative coupling ratio unresolved"]},{"year":2023,"claim":"Quantified the cooperative role of TMEM165 and SPCA1 in organellar Mn2+ filling and cytosolic Mn2+ detoxification, tying the degradation mechanism to Mn2+ handling.","evidence":"siRNA depletion of TMEM165/SPCA1, ICP-MS of organellar and cytosolic Mn2+, glycosylation assays","pmids":["37062452"],"confidence":"Medium","gaps":["Single-lab dataset","Dynamic kinetics of Mn2+ partitioning not modeled"]},{"year":2023,"claim":"Proposed a structural model in which the duplicated three-helix repeats place the conserved motifs face-to-face to form a cytosolic acidic cation-binding site, rationalizing distant patient mutations.","evidence":"AlphaFold2 model refined by molecular dynamics with cellular mutation validation","pmids":["37416081"],"confidence":"Low","gaps":["Computational model lacks experimental structure","Predicted binding-site residues not biochemically confirmed"]},{"year":2025,"claim":"Revealed a distinct lysosomal function: a lysosomal pool of TMEM165 imports Ca2+ and drives Ca2+-induced proton leakage, accelerating recovery from cytosolic Ca2+ overload and enhancing survival.","evidence":"Lysosomal patch-clamp electrophysiology, ion-flux imaging, depletion/overexpression, survival assays","pmids":["40473625"],"confidence":"High","gaps":["Relationship between lysosomal and Golgi pools' regulation unresolved","Physiological contexts triggering lysosomal Ca2+ handling not defined"]},{"year":null,"claim":"How TMEM165 is partitioned and independently regulated between Golgi and lysosomal membranes, and whether its lysosomal Ca2+/H+ activity intersects with Golgi Mn2+ homeostasis, remain open.","evidence":"","pmids":[],"confidence":"Low","gaps":["No experimental structure of the human transporter","Mechanism sensing Golgi Mn2+ to trigger lysosomal degradation unidentified","Determinants of dual Golgi/lysosomal targeting unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[3,8,14,17]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[3,8,14]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,2,9]},{"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":[6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,8,14,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,5,13]}],"complexes":[],"partners":["ATP2C1"],"other_free_text":[]}},"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":53,"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":"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":"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":"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 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activity.","date":"2018","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/30015898","citation_count":19,"is_preprint":false},{"pmid":"30622138","id":"PMC_30622138","title":"Milk biosynthesis requires the Golgi cation exchanger TMEM165.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30622138","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":"32733646","id":"PMC_32733646","title":"Novel role for the Golgi membrane protein TMEM165 in control of migration and invasion for breast carcinoma.","date":"2020","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/32733646","citation_count":17,"is_preprint":false},{"pmid":"31415112","id":"PMC_31415112","title":"Fetal bovine serum impacts the observed N-glycosylation defects in TMEM165 KO HEK cells.","date":"2019","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/31415112","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 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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":"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":"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":"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":4,"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18818,"output_tokens":4657,"usd":0.063155,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12797,"output_tokens":5351,"usd":0.09888,"stage2_stop_reason":"end_turn"},"total_usd":0.162035,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"TMEM165 is required for Golgi glycosylation; siRNA-mediated knockdown of TMEM165 in HEK cells causes Golgi glycosylation defects, identifying it as a protein involved in congenital disorders of glycosylation.\",\n      \"method\": \"siRNA knockdown in HEK cells with glycosylation readout\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with defined cellular phenotype, independently replicated across multiple labs\",\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 subcellular localization differentially depending on the mutation. The YNRL motif is critical for TMEM165 subcellular localization.\",\n      \"method\": \"Fluorescence microscopy of wild-type and mutant TMEM165 constructs; yeast complementation assay with gdt1Δ strain\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional consequence via yeast complementation, single lab, two orthogonal methods\",\n      \"pmids\": [\"23575229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Glycosylation defects in Gdt1p/TMEM165-deficient cells result from a defect in Golgi manganese (Mn2+) homeostasis; Mn2+ supplementation restores normal glycosylation in both yeast gdt1Δ mutants and TMEM165-depleted mammalian cells. GPP130 Mn2+ sensitivity is altered in TMEM165-depleted cells.\",\n      \"method\": \"Yeast and mammalian cell knockout/depletion with Mn2+ supplementation rescue; GPP130 as Golgi Mn2+ sensor\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast genetics, mammalian knockdown, Mn2+ rescue, Golgi sensor), replicated across organisms\",\n      \"pmids\": [\"27008884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Yeast Gdt1p (TMEM165 ortholog) has direct Ca2+ transport activity functioning as a Ca2+/H+ antiporter, demonstrated by heterologous expression in Lactococcus lactis; Gdt1p controls cellular calcium stores and is required for glycosylation at high external calcium concentrations, with glycosylation restored by Mn2+ supplementation.\",\n      \"method\": \"Heterologous expression in Lactococcus lactis with Ca2+ uptake assay; yeast genetic analysis; glycosylation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical transport assay in reconstituted bacterial system plus in vivo yeast genetics, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"27075443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"High Mn2+ concentrations cause lysosomal degradation of TMEM165. The glutamic acid at position E108 within the cytosolic ELGDK motif is crucial for Mn2+-induced degradation; the E108G variant is insensitive to Mn2+-induced degradation but does not abolish TMEM165 function in Golgi glycosylation.\",\n      \"method\": \"Western blot of TMEM165 levels upon MnCl2 exposure in cell lines expressing wild-type or mutant TMEM165; lysosomal inhibitor experiments\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis with functional readout, single lab, multiple mutants tested\",\n      \"pmids\": [\"28270545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM165 deficiency causes severe hypogalactosylation and GalNAc transfer defects; these N-glycosylation defects are corrected by Mn2+ supplementation and also rescued by galactose supplementation in cells and in patients.\",\n      \"method\": \"Mass spectrometry glycan analysis in TMEM165 KO HEK293 cells; patient treatment with oral galactose; transferrin isoelectrofocusing\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (MS glycomics, patient biochemistry, cell rescue), replicated in both cell model and patients\",\n      \"pmids\": [\"28323990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM165 exists as splice-transcript isoforms (Short Form 129 aa and Long Form 259 aa in addition to full-length 324 aa); both isoforms localize to the endoplasmic reticulum (not Golgi), the Short Form forms homodimers and is expressed at low levels broadly but enriched in brain, and the Long Form is expressed only in the temporal lobe. These isoforms have different effects on glycosylation compared to wild-type protein.\",\n      \"method\": \"RT-PCR from human brain tissue, RT-Q-PCR, fluorescence microscopy of expressed isoforms, western blot for glycosylation readout\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by imaging and gel mobility glycosylation assay, single lab, multiple methods\",\n      \"pmids\": [\"28088503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ is mediated by thapsigargin- and cyclopiazonic acid-sensitive pumps (SERCA pumps), not by endocytosis or SPCA1. Overexpression of SERCA2b partially rescues LAMP2 glycosylation defect in TMEM165 KO cells.\",\n      \"method\": \"Pharmacological inhibition with thapsigargin/CPA in TMEM165 KO HEK293 cells; SERCA2b overexpression; glycosylation readout by western blot\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological dissection and rescue by overexpression, single lab, two orthogonal approaches\",\n      \"pmids\": [\"30307768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM165 directly mediates Ca2+ and Mn2+ transport when expressed heterologously in yeast and Lactococcus lactis; expression in a yeast strain lacking Golgi Ca2+/Mn2+ transporters abrogates Mn2+-induced growth defects, excessive Mn2+ accumulation, and glycosylation defects. The E108G disease-causing mutation significantly reduces TMEM165 transport activity.\",\n      \"method\": \"Heterologous expression in S. cerevisiae and L. lactis; Fura-2 fluorescent probe Ca2+ influx assay in bacteria; yeast growth assays; glycosylation assays; site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical transport assay reconstituted in two independent heterologous systems, mutagenesis of disease-causing variants, multiple orthogonal methods\",\n      \"pmids\": [\"32047108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM165 abundance is directly dependent on SPCA1 function—specifically SPCA1's capacity to pump Mn2+ into the Golgi lumen. In SPCA1-deficient cells, TMEM165 is constitutively degraded in lysosomes; the SPCA1 Q747A mutant that favors Mn2+ pumping rescues TMEM165 abundance and Golgi localization. SERCA2b overexpression also rescues TMEM165 expression.\",\n      \"method\": \"SPCA1-deficient Hap1 cells; complementation with SPCA1 mutants differentially impairing Mn2+/Ca2+ transport; western blot; fluorescence microscopy; lysosomal inhibitors\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical approach with structure-function mutants and multiple readouts, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"31652305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and affects milk calcium and manganese levels; conditional knockout mice show decreased lactose biosynthesis and only calcium and manganese levels are significantly lower in milk (normalized to protein), consistent with TMEM165 supplying Ca2+ and Mn2+ to the Golgi in exchange for H+.\",\n      \"method\": \"Conditional tissue-specific knockout mice; biochemical assays of milk composition; immunostaining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO in mammals with defined biochemical phenotype, multiple readouts, in vivo model\",\n      \"pmids\": [\"30622138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The conserved UPF0016 consensus motifs E-φ-G-D-[KR]-[TS] in TMEM165 are crucial for both Golgi glycosylation function and Mn2+-induced sensitivity; specific amino acids within these motifs differentially contribute to these two activities.\",\n      \"method\": \"Site-directed mutagenesis of conserved motifs; glycosylation assays; western blot for Mn2+-induced degradation\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with functional readout, single lab, two orthogonal functional assays\",\n      \"pmids\": [\"31351090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM165 depletion in SPCA1-deficient context (Hailey-Hailey disease fibroblasts and keratinocytes) leads to increased sensitivity to Mn2+-induced degradation, linked to cytosolic Mn2+ accumulation as measured by ICP-MS and GPP130 as Golgi Mn2+ sensor.\",\n      \"method\": \"HHD patient fibroblasts and keratinocytes; western blot for TMEM165 upon MnCl2; ICP-MS for Mn2+ levels; GPP130 immunofluorescence as Golgi Mn2+ sensor\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient cells with multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"32335229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM165 deficiency impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans (producing shorter GAG chains) not by defects in Golgi elongating enzymes but by reduced Mn2+ cofactor availability; Mn2+ supplementation rescues the elongation defect. TMEM165 deficiency also impairs TGFβ and BMP signaling in chondrocytes and accelerates chondrocyte maturation/hypertrophy.\",\n      \"method\": \"TMEM165 KO cells; GAG chain analysis; Mn2+ rescue; TGFβ/BMP signaling assays; chondrocyte differentiation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined biochemical and pathway phenotypes, Mn2+ rescue mechanistically informative, single lab\",\n      \"pmids\": [\"34930890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Yeast Gdt1p transports H+ ions in exchange for Ca2+ and Mn2+ cations (antiporter mechanism); the direction of H+ transport can be reversed depending on physiological concentration gradients. Direct transport measurements were made by expressing Gdt1p in L. lactis and recording extracellular/intracellular pH during application of Ca2+, Mn2+ or H+ gradients; in vivo cytosolic and Golgi pH measurements confirmed Gdt1p influences Golgi pH.\",\n      \"method\": \"Heterologous expression in L. lactis with pH recording; genetically encoded pH probes in S. cerevisiae targeting cytosol and Golgi\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical transport assays in reconstituted system plus in vivo pH measurements, multiple orthogonal methods\",\n      \"pmids\": [\"36963491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Mn2+ supplementation fully rescues the Mn2+ content in secretory pathway organelles of TMEM165-depleted cells and restores glycosylation. Both TMEM165 and SPCA1 are crucial for cellular Mn2+ homeostasis; cytosolic Mn2+ accumulation in TMEM165- and SPCA1-depleted cells is demonstrated by ICP-MS upon increasing Mn2+ concentrations. The Mn2+-detoxifying capacity through SPCA1 relies on the Mn2+-induced degradation mechanism of TMEM165.\",\n      \"method\": \"TMEM165 siRNA depletion; SPCA1 siRNA depletion; ICP-MS for organellar and cytosolic Mn2+; glycosylation assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ion measurement by ICP-MS with functional glycosylation readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"37062452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AlphaFold2 structural modeling of TMEM165, refined by molecular dynamics simulation with membrane lipids, reveals a two-fold repeat of three transmembrane helices where the conserved consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side. This model explains the functional impact of patient mutations including G304R, which is distant from the consensus motifs in sequence.\",\n      \"method\": \"AlphaFold2 structural prediction refined by molecular dynamics simulation; functional validation of mutations by expression in cells\",\n      \"journal\": \"Computational and structural biotechnology journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational structural model with limited experimental validation of specific structural predictions\",\n      \"pmids\": [\"37416081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A fraction of TMEM165 localizes to the lysosomal limiting membrane (in addition to its Golgi localization) where it imports calcium into the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. This lysosomal TMEM165 activity accelerates cellular recovery from cytosolic calcium overload, enhancing cell survival, and causes significant cytosolic acidification.\",\n      \"method\": \"Genetic depletion and overexpression; electrophysiology (patch-clamp of lysosomes); visualization of subcellular ion concentrations and fluxes across lysosomal membrane; cell survival assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct electrophysiology plus ion flux visualization and functional survival assay, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"40473625\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TMEM165 is a multi-pass transmembrane protein localizing to the medial/trans-Golgi network and the lysosomal membrane that functions as a Ca2+/Mn2+:H+ antiporter—pumping Ca2+ and Mn2+ into the Golgi (and lysosomal) lumen in exchange for H+ efflux—thereby maintaining Golgi Mn2+ homeostasis required as a cofactor for glycosyltransferases and glycosidases; loss of TMEM165 causes Golgi N- and O-glycosylation defects, impaired proteoglycan GAG chain elongation, and altered lipid glycosylation, all rescuable by Mn2+ supplementation; TMEM165 abundance is regulated by Mn2+-induced lysosomal degradation dependent on the cytosolic ELGDK motif (E108) and on SPCA1-mediated Golgi Mn2+ pumping; at the lysosome, TMEM165 imports Ca2+ and mediates proton leakage to regulate intracellular ion homeostasis and cell survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TMEM165 is a multi-pass transmembrane cation/H+ antiporter that supplies the secretory pathway with the Mn2+ and Ca2+ required for Golgi glycosylation reactions [#0, #2, #8]. Direct transport assays in reconstituted Lactococcus lactis and yeast systems established that TMEM165 (and its yeast ortholog Gdt1p) imports Ca2+ and Mn2+ in exchange for H+, with the direction of H+ flux set by the prevailing ion gradients, and that this activity controls Golgi pH [#3, #8, #14]. Its glycosylation function reflects delivery of luminal Mn2+ as a cofactor for glycosyltransferases and glycosidases: TMEM165 loss produces N- and O-glycosylation defects, severe hypogalactosylation and GalNAc-transfer failures, and shortened chondroitin/heparan-sulfate GAG chains, all corrected by Mn2+ (and, for galactosylation, galactose) supplementation [#2, #5, #13]. TMEM165 abundance is itself governed by Mn2+: high Golgi Mn2+ pumped by the P-type ATPase SPCA1 triggers lysosomal degradation of TMEM165 via the cytosolic ELGDK motif (E108), coupling the two transporters into a system that detoxifies cytosolic Mn2+ and maintains organellar Mn2+ homeostasis [#4, #9, #15]. The conserved UPF0016 E-φ-G-D-[KR]-[TS] motifs form a cytosolic acidic cation-binding site and are required for both transport/glycosylation and Mn2+-induced degradation [#11, #16]. Beyond the Golgi, a lysosomal pool of TMEM165 imports Ca2+ into the lysosomal lumen and mediates Ca2+-induced proton leakage, accelerating recovery from cytosolic Ca2+ overload and promoting cell survival [#17]. TMEM165 deficiency causes a congenital disorder of glycosylation, and in lactating mammary gland it is required for lactose biosynthesis and normal milk Ca2+/Mn2+ content [#0, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that TMEM165 is functionally required for Golgi glycosylation, defining it as a candidate for congenital disorders of glycosylation rather than an orphan membrane protein.\",\n      \"evidence\": \"siRNA knockdown in HEK cells with glycosylation readout\",\n      \"pmids\": [\"22683087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the molecular activity underlying the glycosylation requirement\", \"No localization or transport mechanism defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped TMEM165 to Golgi, plasma membrane, and late endosome/lysosome compartments and showed disease mutations mislocalize the protein, identifying the YNRL motif as a localization determinant.\",\n      \"evidence\": \"Fluorescence microscopy of WT/mutant constructs plus yeast gdt1Δ complementation\",\n      \"pmids\": [\"23575229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish the biochemical activity\", \"Functional significance of the non-Golgi pools left unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified Golgi Mn2+ homeostasis as the mechanistic basis of the glycosylation defect, since Mn2+ supplementation rescues both yeast and mammalian loss-of-function.\",\n      \"evidence\": \"Yeast/mammalian depletion with Mn2+ rescue and GPP130 Golgi Mn2+ sensor\",\n      \"pmids\": [\"27008884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate direct Mn2+ transport by TMEM165\", \"Stoichiometry and coupling ion unknown at this stage\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that the ortholog Gdt1p directly transports Ca2+ as a Ca2+/H+ antiporter, providing the first direct biochemical evidence for transport activity and linking it to cellular calcium stores.\",\n      \"evidence\": \"Heterologous expression in L. lactis with Ca2+ uptake assay plus yeast genetics\",\n      \"pmids\": [\"27075443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Mn2+ transport by the human protein not yet shown\", \"Physiological relevance of Ca2+ vs Mn2+ selectivity unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed TMEM165 abundance is controlled by Mn2+-induced lysosomal degradation through the cytosolic ELGDK motif (E108), revealing a regulatory feedback layer distinct from its transport function.\",\n      \"evidence\": \"MnCl2 exposure with WT/mutant western blots and lysosomal inhibitors\",\n      \"pmids\": [\"28270545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degradation machinery and Mn2+-sensing step not identified\", \"E108G separates degradation from glycosylation function but its transport effect was not tested here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the specific glycan lesions (hypogalactosylation, GalNAc-transfer defects) and showed both Mn2+ and galactose correct them in cells and patients, translating the Mn2+ model toward therapy.\",\n      \"evidence\": \"MS glycomics in KO HEK293, patient galactose treatment, transferrin IEF\",\n      \"pmids\": [\"28323990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which galactose bypasses the Mn2+ deficiency not fully resolved\", \"Long-term clinical efficacy not assessed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Characterized ER-localized splice isoforms with distinct tissue expression and altered glycosylation effects, indicating isoform-specific regulation of the protein.\",\n      \"evidence\": \"RT-PCR/RT-qPCR from brain tissue, isoform imaging, glycosylation western blots\",\n      \"pmids\": [\"28088503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological role of ER-retained isoforms unknown\", \"Whether isoforms transport ions untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Dissected the route of extracellular Mn2+ rescue, showing SERCA pumps rather than endocytosis or SPCA1 deliver rescuing Mn2+ in TMEM165 KO cells.\",\n      \"evidence\": \"Thapsigargin/CPA inhibition and SERCA2b overexpression in KO HEK293 cells\",\n      \"pmids\": [\"30307768\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of SERCA vs other pumps unresolved\", \"Relationship between SERCA-delivered ER Mn2+ and Golgi Mn2+ not detailed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided direct biochemical proof that human TMEM165 transports both Ca2+ and Mn2+ and that the disease variant E108G reduces transport, unifying the transport and disease mechanisms.\",\n      \"evidence\": \"Reconstitution in S. cerevisiae and L. lactis, Fura-2 Ca2+ assay, growth/glycosylation assays, mutagenesis\",\n      \"pmids\": [\"32047108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transport stoichiometry not quantified\", \"High-resolution structure of the transport site absent\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established a reciprocal regulatory circuit in which SPCA1-mediated Golgi Mn2+ pumping sustains TMEM165 by preventing constitutive lysosomal degradation.\",\n      \"evidence\": \"SPCA1-deficient Hap1 cells, SPCA1 transport-selective mutants, western blot, microscopy\",\n      \"pmids\": [\"31652305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular sensor coupling Golgi Mn2+ to TMEM165 degradation unidentified\", \"Whether SPCA1 and TMEM165 physically interact untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated a physiological requirement in vivo: mammary TMEM165 supports lactose biosynthesis and normal milk Ca2+/Mn2+, consistent with Ca2+/Mn2+-for-H+ exchange into the Golgi.\",\n      \"evidence\": \"Tissue-specific conditional knockout mice with milk composition and immunostaining\",\n      \"pmids\": [\"30622138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type contributions within mammary tissue not separated\", \"Systemic vs local Mn2+ effects not dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed the conserved UPF0016 motifs underlie both glycosylation and Mn2+-sensitivity, with individual residues differentially partitioning between the two activities.\",\n      \"evidence\": \"Site-directed mutagenesis of conserved motifs with glycosylation and degradation readouts\",\n      \"pmids\": [\"31351090\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of motif function not resolved here\", \"Coupling between ion binding and degradation signal unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended the SPCA1–TMEM165 link to disease cells, showing SPCA1 loss (Hailey-Hailey) sensitizes TMEM165 to Mn2+-induced degradation via cytosolic Mn2+ accumulation.\",\n      \"evidence\": \"HHD patient fibroblasts/keratinocytes, MnCl2 western blot, ICP-MS, GPP130 sensor\",\n      \"pmids\": [\"32335229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal contribution of TMEM165 loss to HHD phenotype unproven\", \"Patient-derived single-lab data\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed Mn2+ limitation from TMEM165 loss impairs proteoglycan GAG chain elongation and disrupts TGFβ/BMP signaling and chondrocyte maturation, broadening the functional consequences beyond N/O-glycans.\",\n      \"evidence\": \"KO cells, GAG chain analysis, Mn2+ rescue, TGFβ/BMP and chondrocyte differentiation assays\",\n      \"pmids\": [\"34930890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between GAG defects and signaling changes not fully traced\", \"In vivo skeletal phenotype not established here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the antiporter mechanism precisely, showing Gdt1p exchanges H+ for Ca2+/Mn2+ with reversible H+ directionality set by gradients and that it shapes Golgi pH.\",\n      \"evidence\": \"L. lactis pH recording with ion gradients plus genetically encoded cytosolic/Golgi pH probes in yeast\",\n      \"pmids\": [\"36963491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human TMEM165 reverses H+ direction in vivo not directly shown\", \"Quantitative coupling ratio unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Quantified the cooperative role of TMEM165 and SPCA1 in organellar Mn2+ filling and cytosolic Mn2+ detoxification, tying the degradation mechanism to Mn2+ handling.\",\n      \"evidence\": \"siRNA depletion of TMEM165/SPCA1, ICP-MS of organellar and cytosolic Mn2+, glycosylation assays\",\n      \"pmids\": [\"37062452\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab dataset\", \"Dynamic kinetics of Mn2+ partitioning not modeled\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Proposed a structural model in which the duplicated three-helix repeats place the conserved motifs face-to-face to form a cytosolic acidic cation-binding site, rationalizing distant patient mutations.\",\n      \"evidence\": \"AlphaFold2 model refined by molecular dynamics with cellular mutation validation\",\n      \"pmids\": [\"37416081\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational model lacks experimental structure\", \"Predicted binding-site residues not biochemically confirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a distinct lysosomal function: a lysosomal pool of TMEM165 imports Ca2+ and drives Ca2+-induced proton leakage, accelerating recovery from cytosolic Ca2+ overload and enhancing survival.\",\n      \"evidence\": \"Lysosomal patch-clamp electrophysiology, ion-flux imaging, depletion/overexpression, survival assays\",\n      \"pmids\": [\"40473625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between lysosomal and Golgi pools' regulation unresolved\", \"Physiological contexts triggering lysosomal Ca2+ handling not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM165 is partitioned and independently regulated between Golgi and lysosomal membranes, and whether its lysosomal Ca2+/H+ activity intersects with Golgi Mn2+ homeostasis, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental structure of the human transporter\", \"Mechanism sensing Golgi Mn2+ to trigger lysosomal degradation unidentified\", \"Determinants of dual Golgi/lysosomal targeting unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [3, 8, 14, 17]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [3, 8, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 2, 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\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 8, 14, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 5, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ATP2C1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}