Atypical RanGAP drives nucleocytoplasmic transport in a parasitic Alveolate
Abstract
Transport of macromolecules between the nucleus and cytoplasm requires a gradient of the small GTPase, Ran. Ran:GTP marks the nucleus because Ran activity requires a cytoplasmic GTPase activating protein (RanGAP) for GTP hydrolysis. As expected for such central and essential cellular machinery, both Ran and RanGAP are conserved across the vast majority of eukaryotes. Many alveolates, including apicomplexan parasites, however, lack a canonical RanGAP. Here, we biochemically purify RanGAP activity from the model alveolate Toxoplasma gondii. We demonstrate that this activity is provided by a neofunctionalized RabGAP-fold protein, called TBC9. We find that TBC9 is sufficient to provide RanGAP activity in yeast, and is absolutely required to maintain active nucleocytoplasmic transport in Toxoplasma. By purifying Toxoplasma Ran and TBC9, we demonstrate that TBC9 is a robust and specific GAP for Ran. Finally, we delineate an essential, conserved low complexity motif in the C-terminus of TBC9 that drives interaction with Ran and is required for its full RanGAP activity. We use this C-terminal motif to identify TBC9 orthologs in all non-ciliate clades of Alveolata, which suggests that the canonical RanGAP has been replaced by the neofunctionalized TBC9 RanGAP in these lineages.
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (6 Aug 2026) · CC BY 4.0
A RabGAP-fold protein, TBC9, supplies the missing RanGAP activity in Toxoplasma; its acidic C-terminal motif promotes Ran binding and marks TBC9 orthologs across non-ciliate alveolates.
Ran and its GAP are conserved in most eukaryotes, yet many alveolates, including apicomplexans, lack an identifiable LRR-type RanGAP, so the source of Ran GTP hydrolysis was unknown. This work identifies a repurposed TBC-fold protein as that activity and shows how a low complexity motif may seed a new function.
Key results
ShownActivity-guided fractionation of Toxoplasma lysate with quantitative proteomics singled out TBC9 as a RanGAP candidate, and TBC9 rescued a yeast RanGAP mutant as well as human RanGAP did.
More than 1000 proteins were identified and 14 covaried with activity (PCC ≥ 0.95); TBC9 PCC=0.98, second to DRG GTPase. TBC9 and human LRR-RanGAP rescued rna1-1 at 37°C; DRG and vector did not. · Purification of RanGAP activity from Toxoplasma…
ShownAuxin-induced degradation of TBC9 quickly disrupts the Ran gradient and nuclear retention of a reporter protein, so TBC9 is needed for nucleocytoplasmic transport in the parasite.
TBC9-AID was lost within 2-3 h of IAA. After 2 h of auxin, mRuby3-Ran and the TGGT1_258850-3xFLAG reporter shifted to the cytosol (n=100 parasites per n=3 biological replicates; p<0.001, unpaired t-test with Benjamini-Hochberg). · Loss of Toxoplasma TBC9 quickly disrupts…; Figure legends
ShownPurified TBC9 strongly activates Toxoplasma Ran in vitro but shows no GAP activity on four Rabs previously reported to bind it, and the Rabs do not alter its Ran activity.
TBC9 activated 20 μM TgRan:GTP (linear, pseudo-zeroth order kinetics). Rab1b, Rab2, Rab11a and Rab11b were not activated, even at 10-fold higher TBC9 concentration. TgRan Q70L had ~3-fold higher basal activity than wild-type. · Toxoplasma TBC9 is a robust and specific…; Figure legends
ShownThe conserved acidic C-terminal motif is required for full RanGAP activity and for rescue of TBC9 loss, and it appears to engage a basic patch on Ran.
TBC9 ΔCT (residues 1-300) failed yeast rescue and plaque rescue. It had ~4-fold lower kcat/Km than full-length TBC9 on TgRan Q70L, but only ~2-fold lower on the R96A/K100A/R107A mutant, which was itself ~2-fold poorer for full-length TBC9 than Q70L. · A conserved C-terminus in Toxoplasma TBC9 is…; Toxoplasma TBC9 is a robust and specific…; Discussion
SuggestedThe C-terminal motif identifies TBC9 orthologs in all non-ciliate alveolates and in haptophytes, which lack an LRR-RanGAP, whereas trypanosome TBC9-like protein did not rescue yeast.
Motif conserved in apicomplexan and other myzozoan TBC9 proteins but missing from most TBC9-like proteins outside alveolates. Trypanosome TBC9-like, and a chimera carrying residues 301-315 of the Toxoplasma C-terminus, failed to rescue rna1-1. · A conserved C-terminus in Toxoplasma TBC9 is…; Discussion; Bioinformatic analysis
Methods, data and reproducibility
Activity-guided fractionation of Toxoplasma lysate with TMT proteomics and activity-abundance correlation, then yeast rna1-1 complementation, auxin-inducible degradation with imaging reporters, in vitro GTPase kinetics, AlphaFold3 modeling and phylogenetics.
- Ten anion-exchange fractions of soluble Toxoplasma lysate with RanGAP activity, analyzed by TMT 10plex proteomics · Toxoplasma gondii · n = 10 fractions · PXD077025
- Temperature-sensitive rna1-1 yeast (W303) complementation with candidate GAPs, growth at 24°C vs 37°C · Saccharomyces cerevisiae
- TBC9-AID parasite strains with mRuby3-Ran or nuclear 3xFLAG reporter, imaged after 2 h auxin · Toxoplasma gondii (grown in human foreskin fibroblasts) · n = n=100 parasites per n=3 biological replicates
- Plaque assays of TBC9-AID with wild-type or ΔCT rescue copies · Toxoplasma gondii · n = n=3 technical replicates for each of n=3 biological replicates
- Recombinant Toxoplasma Ran, Ran mutants, four Rabs and TBC9 variants for GTPase kinetics · Toxoplasma gondii (expressed in E. coli)
- TBC domain (PF00566) and LRR protein sequences, plus EukProt v3 SAR and haptophyte proteomes, for phylogenetics · Multiple eukaryotes
- Methods & tools
- IQ-TREE2, MAFFT, Clustal Omega, PSI-BLAST, HMMER v3.4, AlphaFold3, Proteome Discoverer v.3.0 SP1, TMT 10plex, GTPase-Glo assay, MESG/PNP continuous phosphate assay, Fiji, SciPy and Statsmodels
- Reagents & environment
- Proteome Discoverer v.3.0 SP1; HMMER v3.4; ToxoDB v68; EukProt v3; Inkscape v1.4; Python with matplotlib, seaborn, SciPy and Statsmodels (other versions not stated).
- Reproducibility gaps
- No code repository is stated. Accessions, alignments and trees are in SI Data S1-S9 rather than public URLs; proteomics deposited under PXD077025.
Limitations (5)
- Kinetic parameters could not be obtained for wild-type Ran because assay sensitivity prevented lowering the substrate concentration. Parameters were estimated only for the Q70L mutant, which has altered affinity for TBC9. · stated by the authors
- TBC9 ΔCT gave only a partial nucleocytoplasmic transport phenotype but failed to rescue essentiality, so additional C-terminal functions cannot be excluded. The Ran:TBC9 complex is highly dynamic and its structural basis awaits future work. · stated by the authors
- The basic patch on Ran does not account for all of the binding energy of the TBC9 C-terminus; further basic residues on Ran likely contribute through multiple binding conformations of the acidic motif. · stated by the authors
- Orthology across non-ciliate alveolates and haptophytes is inferred from sequence motifs and absence of LRR-RanGAP; RanGAP activity was tested only for Toxoplasma TBC9, with a negative result for trypanosome TBC9-like. · noted in this summary
- Yeast complementation is reported only as growth comparisons at 24°C versus 37°C, with no quantification or statistics described for these assays in the text. · noted in this summary
Who should read:Read this if you study small GTPase regulation, nucleocytoplasmic transport or apicomplexan cell biology, or want an example of neofunctionalization via a low complexity motif.
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