Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells
- 1St. Jude Children's Research Hospital
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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BibTeX
@misc{beyers2026biotinoperated,
title = {{Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells}},
author = {W. C. Beyers and S. E. Plutkis and K. R. Rutcher and J. L. Sun and M. R. Haas and M. S. Hutton and H. K. Kim and A. Chavez and S. M. Pruett-Miller and J. D. Vevea and L. Lavis and C.-L. Chang},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.02.756291},
url = {https://www.biorxiv.org/content/10.64898/2026.10.02.756291v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells
AU - W. C. Beyers
AU - S. E. Plutkis
AU - K. R. Rutcher
AU - J. L. Sun
AU - M. R. Haas
AU - M. S. Hutton
AU - H. K. Kim
AU - A. Chavez
AU - S. M. Pruett-Miller
AU - J. D. Vevea
AU - L. Lavis
AU - C.-L. Chang
PY - 2026
DA - 2026/10/02
PB - bioRxiv
DO - 10.64898/2026.10.02.756291
UR - https://www.biorxiv.org/content/10.64898/2026.10.02.756291v1
AB - 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.
ER -