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TCF25 emerges as a cytoplasmic regulator of GPRASP2 stability

Liana HardyiD, Haziq K. MoinudeeniD, Laura Cathleen GrzegorzekiD, J. Fernando BazániD, Svetlana V. KhoronenkovaiD

Abstract

TCF25 is a conserved eukaryotic protein whose cellular localisation and functional context remain poorly understood. Here, we show that under basal conditions, TCF25 is predominantly cytoplasmic across several human cell types, with a digitonin-resistant perinuclear pool indicative of association to membrane-linked components. Exploration of TCF25's proximal network using proximity labelling further supports enrichment of cytoplasmic and intracellular membrane-associated proteins. Guided by the analysis of public datasets and the proximal proteome data, we show that TCF25 associates with G-protein coupled receptor-associated sorting protein, GPRASP2, and maintains its basal abundance by protecting it from proteasomal degradation. Using structural modelling, we identify a putative interaction interface between the two proteins, involving two closely-spaced helices in the C terminal region of TCF25 that dock to the helical repeat segment of GPRASP2. Consistent with this prediction, deletion of TCF25 amino acids 604-664 disrupted the relationship between TCF25 and GPRASP2. Together, these findings establish a cytoplasmic, membrane-proximal context for TCF25 and identify a C-terminal tract required for its association with GPRASP2. These findings implicate TCF25 in regulating GPRASP2 stability, providing a framework for investigating its role in cellular homeostasis and disease.

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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (22 Sept 2026) · CC BY 4.0

TCF25 is mostly cytoplasmic with a perinuclear membrane-proximal pool, stabilises GPRASP2 against proteasomal degradation, and its C-terminal residues 604-664 are required for their association.

TCF25 has been reported as nuclear in reporter and overexpression systems but cytoplasmic in endogenous studies, leaving its compartment and partners unclear. This work combines endogenous imaging, fractionation, proximity labelling and structure prediction to place TCF25 in the cytoplasm and link it to GPRASP2 stability.

Key results

  1. ShownEndogenous TCF25 is predominantly cytosolic across several human cell types, with a smaller perinuclear, membrane-proximal pool that resists digitonin extraction.

    RPE-1 differential ultracentrifugation: ~70% of total TCF25 in cytosol, ~30% in ER-, ribosome-enriched and microsomal fractions. U2-OS retains perinuclear TCF25 after digitonin. Line scans show TCF25 adjacent to, not coincident with, TOM20, calnexin and EEA1. · TCF25 localises to the cytoplasm with the…; Figure legends

  2. SuggestedProximity labelling in RPE-1 cells gave an exploratory TCF25 neighbourhood, possibly under-powered, enriched for cytoplasmic and membrane compartments; pulldown and co-IP supported a GPRASP2 association.

    73 reproducible candidates (Log2 fold change > 2 in both of two replicates); NEMF enriched, LTN1 and VCP/p97 not; GPRASP2 ~4-fold in both replicates but narrowly below threshold. FLAG-TCF25 co-immunoprecipitated endogenous GPRASP2 in U2-OS. · Data mining and proximity labelling define the…; TCF25 associates with GPRASP2 and influences its…

  3. ShownLoss of TCF25 lowers GPRASP2 protein without changing its mRNA. The two proteins correlate positively across cell lines; decoupling occurs preferentially in translationally active lines, consistent with MCF7 deviating from the trend.

    TCF25 siRNA did not alter GPRASP2 mRNA (RT-qPCR, n = 4). Per-cell IF: 1555 control vs 1478 knockdown cells, linear discriminant accuracy 90% (single biological replicate). Bayesian regression across 215 CCLE cell lines gave a positive relationship. · TCF25 associates with GPRASP2 and influences its…; Figure legends

  4. SuggestedTCF25 protects GPRASP2 from proteasomal turnover: knockout speeds GPRASP2 decay, and proteasome inhibition rescues it more strongly in knockout cells.

    In HEK293-T cycloheximide chases, GPRASP2 half-life exceeded 24 h in WT and was shorter in TCF25 KO (n = 3). MG132 raised GPRASP2 and bafilomycin A1 alone did not; the MG132 effect was larger in KO than in WT. · TCF25 regulates GPRASP2 protein stability in a…; Figure legends

  5. ShownStructure prediction mapped a two-helix C-terminal segment of TCF25 onto the GPRASP2 helical repeat region, and deleting residues 604-664 abolished GPRASP2 co-immunoprecipitation.

    AlphaFold 3 pTM 0.80, ipTM 0.84; Protenix-v2 pTM 0.83, ipTM 0.85, ipSAE 0.85. FLAG-WT TCF25 pulled down endogenous GPRASP2 in KO HEK-293T cells; the deletion mutant, expressed at a comparable level, did not. · The C-terminal region of TCF25 is required for…; Figure legends

Methods, data and reproducibility

Endogenous immunofluorescence, digitonin extraction and differential ultracentrifugation define localisation. TurboID proteomics, public interaction data, cycloheximide chases, co-IP and AlphaFold 3/Protenix-v2 modelling with deletion mutagenesis probe the GPRASP2 link.

  • Human cell lines (TIG1, HEK293-T incl. TCF25 KO clone #E9, U2-OS, RPE-1, MCF7) and iPSC-derived cardiomyocytes for imaging, fractionation and immunoblots · human
  • TurboID-TCF25 vs no-bait controls in RPE-1, LC-MS/MS (DIA-PASEF) · human · n = two independent biological replicates per condition · MassIVE MSV000103297
  • DepMap Harmonized Public Proteomics 24Q4 (CCLE) TCF25 and GPRASP2 abundance · human · n = 215 cell lines
  • Per-cell immunofluorescence of TCF25 and GPRASP2 after siRNA (single biological replicate) · human · n = 1555 siCTRL cells; 1478 siTCF25 cells
Methods & tools
TurboID proximity labelling with timsTOF Pro2 DIA-PASEF, DIA-NN v1.8, Perseus, g:Profiler, STRING v11.5, Human Predictome, BioGRID, AlphaFold 3, Protenix-v2, AlphaMissense, PyMC v5.23.0, GSEApy v1.1.4
Reagents & environment
DIA-NN v1.8; STRING v11.5; PyMC v5.23.0; GSEApy v1.1.4; GraphPad Prism v9.4.1; DepMap Harmonized Public Proteomics 24Q4 and DepMap Public 26Q1 metadata.
Reproducibility gaps
No code repository stated. AlphaFold 3 and Protenix-v2 output files and additional data are available only on request from the corresponding author.
Limitations (5)
  • The TurboID experiment had only two replicates and may be under-powered; known RQC partners LTN1 and VCP/p97 were not detected, and GPRASP2 narrowly missed the reproducibility threshold. · stated by the authors
  • Direct interaction between TCF25 and GPRASP2 is not demonstrated; the authors state it needs further evidence, and the mechanism of stabilisation remains open. · stated by the authors
  • The 604-664 deletion could instead damage a structural element used by TCF25 for other partners, not a GPRASP2-specific site; MCF7 decoupling needs testing in a broader panel of proliferative lines. · stated by the authors
  • Knockout work relies on one monoclonal HEK293-T clone (#E9) and a single deletion mutant, with no point mutants or folding controls to separate loss of an interface from a broader structural defect. · noted in this summary
  • Per-cell IF quantification comes from a single biological replicate, and GPRASP2 ubiquitination was not measured directly, so the proteasome-protection model rests on inhibitor and turnover data. · noted in this summary

Who should read:Read this if you study ribosome-associated quality control proteins, GPCR sorting, or want a worked example combining proximity labelling, CCLE proteomics and AlphaFold 3 to guide a deletion experiment.

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