TMEM165

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).

1. 2012 High

   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

   PMID:22683087

   Open questions at the time

   • Transport mechanism unknown
   • Substrate ion not identified
   • No structure available
2. 2013 Medium

   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

   PMID:23575229

   Open questions at the time

   • Mechanism of YNRL-mediated sorting not defined
   • Relative functional contribution of each compartment not established
3. 2016 High

   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

   PMID:27008884 PMID:27075443

   Open questions at the time

   • Direct Mn²⁺ transport by human TMEM165 not yet demonstrated
   • Stoichiometry of Ca²⁺/Mn²⁺:H⁺ exchange unknown
4. 2017 High

   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

   PMID:28270545 PMID:31351090

   Open questions at the time

   • Identity of the Mn²⁺ sensor that recognizes E108 is unknown
   • Whether degradation is ubiquitin-dependent not tested
5. 2017 High

   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

   PMID:28323990

   Open questions at the time

   • Long-term clinical efficacy of galactose therapy not established
   • Mechanism by which galactose bypasses Mn²⁺ deficiency not fully resolved
6. 2019 High

   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

   PMID:32047108

   Open questions at the time

   • No reconstitution in proteoliposomes for kinetic characterization
   • H⁺ counterflux not directly measured for the human protein at this stage
7. 2019 High

   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

   PMID:31652305

   Open questions at the time

   • Whether TMEM165 and SPCA1 physically interact is unknown
   • Mechanism by which SPCA1 Mn²⁺ transport prevents TMEM165 degradation not defined
8. 2019 High

   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

   PMID:30622138

   Open questions at the time

   • Systemic phenotype of whole-body knockout not reported
   • Contribution to bone/cartilage phenotype in mice not tested
9. 2021 Medium

   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

   PMID:34930890

   Open questions at the time

   • In vivo cartilage-specific knockout not performed
   • Whether all glycosyltransferases are equally Mn²⁺-dependent through TMEM165 is unclear
10. 2023 High

    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

    PMID:36963491

    Open questions at the time

    • Bidirectionality not yet confirmed for human TMEM165 directly
    • Impact on Golgi pH in mammalian cells not measured
11. 2023 Medium

    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

    PMID:37416081

    Open questions at the time

    • No experimental structure (cryo-EM, X-ray) available
    • Ion-binding site not validated by mutagenesis of predicted coordinating residues beyond known motifs
12. 2025 High

    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

    PMID:40473625

    Open questions at the time

    • 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

• 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.
• No experimental structure
• Mn²⁺ sensor/E3 ligase for TMEM165 degradation unidentified
• Relative physiological importance of Ca²⁺ versus Mn²⁺ transport in different tissues not resolved