Retromer is a protein complex involved in cargo sorting and recycling within the endolysosomal system. Its recruitment to membranes is mediated in part by the small GTPase Rab7. Subsequent Retromer dissociation is facilitated by the Rab7 GTPase-activating protein (GAP) TBC1D5, which binds at the Vps29–Vps35 interface via a well characterised Pro-Leu (PL) motif (Jia et al., 2016). More recently, another GAP, TBC1D13, has been shown to interact with Retromer (Antón-Plágaro et al., 2024). TBC1D13 is the only other human GAP known to contain this PL motif insert and it also possesses a unique β-hairpin insert that may enhance binding affinity for Retromer. Notably, AlphaFold predicts this β-hairpin to occupy the same site as the Retromer-stabilising bis-1,3-phenyl guanylhydrazone 2a, raising the possibility of competitive interactions. This project seeks to characterise the interaction between TBC1D13 and Retromer using structural and biochemical approaches. The complex has been purified and its assembly validated using size-exclusion chromatography and mass photometry. Conditions for structural analysis by negative stain and cryo-electron microscopy are currently being optimised. The functional relevance of both the PL motif and β-hairpin insert is explored by mutagenesis and peptide-binding assays. Further, TBC1D13 remains poorly characterised and its Rab partner(s) are unknown. GAP activity assays, isothermal titration calorimetry (ITC), and a GFP pulldown approach are being used to screen the human Rabs for interaction. This project intends to define the molecular basis of Retromer regulation by TBC1D13 and expand on understanding of Rab–GAP specificity within the endosomal system.
Antón-Plágaro, Carlos, et al. "Mapping of endosomal proximity proteomes reveals Retromer as a hub for RAB GTPase regulation." Nature Communications 16.1 (2025): 6990.
Jia, D., Zhang, J. S., Li, F., Wang, J., Deng, Z., White, M. A., Osborne, D. G., Phillips-Krawczak, C., Gomez, T. S., Li, H., Singla, A., Burstein, E., Billadeau, D. D., & Rosen, M. K. (2016). Structural and mechanistic insights into regulation of the retromer coat by TBC1d5. Nat Commun, 7, 13305. https://doi.org/10.1038/ncomms13305