TM9SF2

TM9SF2 is a conserved Golgi-resident nonaspanin that maintains Golgi integrity and governs glycosphingolipid and glycosaminoglycan biosynthesis by controlling the localization—rather than the catalytic activity—of relevant enzymes (PMID:30481169, PMID:30660999). It is required for the proper localization and stability of NDST1, the enzyme that N-sulfates heparan sulfate, so its loss reduces HS N-sulfation (PMID:28404855), and it positions Gb3 synthase correctly within the Golgi; in its absence Gb3 synthase activity is preserved but mislocalized, causing accumulation of the precursor lactosylceramide and depletion of Gb3 and other glycosphingolipids (PMID:30660999). TM9SF2 sustains Golgi architecture in part by regulating Golgi cholesterol, since its depletion drives abnormal Golgi cholesterol accumulation and Golgi fragmentation (PMID:40423301). Through these glycosylation, lipid, and trafficking functions TM9SF2 is co-opted by diverse pathogens and toxins, supporting CHIKV and vaccinia virus infection via heparan sulfate (PMID:28404855, PMID:30996093), AAV transduction (PMID:32280726), and Shiga toxin/EHEC T3SS effector entry and ricin retrograde transport (PMID:29921669, PMID:40423301). TM9SF2 additionally controls PD-L1 surface levels by recruiting PGK1 to promote PD-L1 plasma-membrane recycling while dismantling HIP1R-mediated lysosomal degradation, a complex destabilized by the ceramide species Cer(d18:1/26:0) (PMID:41888515). The Drosophila ortholog contributes to bacterial phagocytosis and actin regulation and interacts with the receptor PGRP-LC to restrain unstimulated signaling (PMID:18796536, PMID:25139117).

1. 2008 Medium

   Established the first cellular role for TM9SF2, showing it acts in pathogen internalization and actin cytoskeleton control, partially redundant with its paralog TM9SF4.

   Evidence RNAi knockdown and null mutants in Drosophila S2 cells and haemocytes with phagocytosis and actin readouts

   PMID:18796536

   Open questions at the time

   • Molecular mechanism linking TM9SF2 to actin remodeling not defined
   • Findings from Drosophila ortholog, not human protein
   • Redundancy with TM9SF4 not fully resolved
2. 2014 Medium

   Connected TM9SF2 to immune receptor regulation by showing it physically associates with PGRP-LC and suppresses inappropriate signaling without being required for receptor trafficking, distinguishing its role from TM9SF4.

   Evidence Reciprocal Co-IP, co-localization imaging, and RNAi in Drosophila S2 cells and fat body

   PMID:25139117

   Open questions at the time

   • Mechanism of signal suppression undefined
   • Drosophila ortholog only
   • No structural basis for the interaction
3. 2017 Medium

   Identified a glycosylation-control function in human cells, showing TM9SF2 is needed for NDST1 localization/stability and thus heparan sulfate N-sulfation, which determines CHIKV binding.

   Evidence Genome-wide haploid insertional mutagenesis screen with KO validation and viral binding/infectivity assays in HAP1 cells

   PMID:28404855

   Open questions at the time

   • Direct interaction between TM9SF2 and NDST1 not shown
   • How TM9SF2 stabilizes NDST1 mechanistically unknown
4. 2018 High

   Broadened TM9SF2 to a general regulator of Golgi glycosylation and endosomal trafficking by showing its loss depletes Gb3 and other glycosphingolipids and disrupts endosomal transport.

   Evidence Genome-wide CRISPR-Cas9 screen, mass spectrometry glycolipid profiling, and trafficking assays in KO cells

   PMID:30481169

   Open questions at the time

   • Direct enzyme partners not all identified
   • Link between Golgi and endosomal defects not mechanistically resolved
5. 2018 Medium

   Extended TM9SF2's glycosphingolipid role to toxin/pathogen entry, placing it at early steps of Shiga toxin binding and EHEC T3SS effector entry via Gb3 biosynthesis.

   Evidence Genome-wide CRISPR screen with KO validation, Stx binding and T3SS effector entry assays in intestinal epithelial cells

   PMID:29921669

   Open questions at the time

   • Whether T3SS effect is solely Gb3-dependent unresolved
   • No direct toxin-TM9SF2 interaction tested
6. 2019 High

   Resolved that TM9SF2 acts by controlling enzyme localization rather than activity, demonstrating Gb3 synthase retains activity but is mislocalized with precursor accumulation, a conserved TM9SF-family function.

   Evidence Genome-wide CRISPR/Cas9 KO screen in HeLa, Gb3 synthase activity assays, localization studies, and glycolipid profiling

   PMID:30660999

   Open questions at the time

   • How TM9SF2 directs glycosyltransferase localization unknown
   • Whether mechanism is direct binding or compartmental
7. 2019 Medium

   Independently confirmed TM9SF2's role in heparan sulfate expression as the basis for its contribution to vaccinia virus infection.

   Evidence Genome-wide haploid insertional mutagenesis screen with KO validation and HS expression assays in HAP1 cells

   PMID:30996093

   Open questions at the time

   • Mechanism overlaps with prior NDST1 finding but not further dissected
8. 2020 Medium

   Implicated TM9SF2 in AAV intracellular trafficking, showing it is broadly required for efficient transduction across AAV serotypes.

   Evidence Genome-wide pooled CRISPR screens with flow cytometry and imaging validation across at least 8 AAV serotypes in U-2 OS cells

   PMID:32280726

   Open questions at the time

   • Specific trafficking step affected not pinpointed
   • Whether effect is glycan- or trafficking-mediated unclear
9. 2024 Low

   Linked TM9SF2 to macrophage phagocytic signaling, placing it upstream of PLC-γ1 activation during phagocytosis.

   Evidence siRNA knockdown in THP-1-derived M2 macrophages with phagocytosis and PLC-γ1 activation assays

   PMID:38218914

   Open questions at the time

   • Single lab, siRNA only without rescue
   • Mechanism connecting TM9SF2 to PLC-γ1 undefined
   • No direct interaction shown
10. 2025 Medium

    Defined a cholesterol-dependent basis for TM9SF2's Golgi maintenance, showing its loss causes Golgi cholesterol accumulation and fragmentation that impairs ricin retrograde transport, reversible by restoring cholesterol metabolism.

    Evidence TM9SF2 KD with cholesterol trafficking assays, Golgi morphology imaging, ricin cytotoxicity assays, and pharmacological rescue (A939572, avasimibe)

    PMID:40423301

    Open questions at the time

    • How TM9SF2 regulates Golgi cholesterol mechanistically unknown
    • Direct cholesterol-handling activity not demonstrated
11. 2025 Low

    Identified TM9SF2 as a negative regulator of type I interferon signaling, with knockdown enhancing IFN-β induction and IRF3 phosphorylation.

    Evidence siRNA knockdown in A549 cells, VSV-GFP infection, plaque assay, RT-qPCR, and IRF3 phosphorylation Western blot

    PMID:40525335

    Open questions at the time

    • Single lab, siRNA without rescue
    • Mechanism of IFN pathway suppression undefined
    • No direct pathway-component interaction tested
12. 2026 Medium

    Established a direct protein-complex function for TM9SF2 in immune checkpoint regulation, showing it recruits PGK1 to recycle PD-L1 and block HIP1R-driven lysosomal degradation, a complex lipid-sensitive to ceramide.

    Evidence CRISPR screen of glycosphingolipid metabolism, Co-IP of TM9SF2-PGK1, PD-L1 trafficking and lysosomal degradation assays, and ceramide treatment

    PMID:41888515

    Open questions at the time

    • Structural basis of TM9SF2-PGK1 binding unknown
    • How ceramide destabilizes the complex mechanistically unresolved
    • Single lab

• The biochemical mechanism by which a multipass Golgi membrane protein directs glycosyltransferase localization, regulates Golgi cholesterol, and scaffolds cytosolic partners like PGK1 remains undefined.
• No structure or direct biochemical activity established
• Whether localization control reflects direct enzyme binding vs. compartmental effects is unresolved
• Unifying molecular function across glycosylation, lipid, and trafficking roles not defined