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bioRxiv · 10.64898/2026.10.02.756291

Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells

Beyers, W. C.·Plutkis, S. E.·Rutcher, K. R.·Sun, J. L.·Haas, M. R.·Hutton, M. S.·Kim, H. K.·Chavez, A.·Pruett-Miller, S. M.·Vevea, J. D.·Lavis, L.·Chang, C.-L.
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Abstract

Controlling protein localization through chemical biology tools is essential for mechanistic interrogations, functional perturbations, and therapeutic development. While existing tools cover a wide range of applications, no single approach simultaneously enables inducible trapping, controlled release, and visualization of a protein of interest (POI). To address this gap, we developed Biotin-Operated Ligand Trapping (BOLT), which combines a HaloTag-fused POI with a streptavidin trap. Desthiobiotin-conjugated Janelia Fluor HaloTag ligands enable POI visualization, trapping, and biotin-operated release. We demonstrate BOLT's capacity in synchronized secretory cargo release, microtubule-directed organelle movement and hitchhiking, and ectopically localizing nuclear transcription factor. Importantly, pulse-chase BOLT further reveals bidirectional endoplasmic reticulum-Golgi trafficking of an endogenous Golgi enzyme and distinguishes recycled from newly synthesized enzyme populations using distinct HTLs. Altogether, BOLT provides an all-in-one platform to interrogate how subcellular localization regulates protein function across HaloTag-compatible systems.

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Beyers, W. C., Plutkis, S. E., Rutcher, K. R., Sun, J. L., Haas, M. R., Hutton, M. S., Kim, H. K., Chavez, A., Pruett-Miller, S. M., Vevea, J. D., Lavis, L., Chang, C.-L.. 2026-10-06. Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells. https://doi.org/10.64898/2026.10.02.756291

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