PRINTS, a tool for subcellular protein and organelle translocation and its application in screening LLPS disrupting peptides

Protein subcellular localization is dynamically regulated, requiring new tools to control protein translocation for elucidating its biological significance and eventually enabling synthetic biology applications. Here, we developed a new method, Protein Rerouting via INtein-mediated Trans-Splicing (PRINTS), for precise control of protein translocation across diverse subcellular compartments. By reconstituting functional signaling peptides, PRINTS enables robust relocalization to varied destinations, including the 26S proteasome, nucleus, mitochondria, plasma membrane, endomembrane organelles, and liquid-liquid phase separation (LLPS) condensates. Moreover, integration of the CRY2clust light-responsive dimerization domain into PRINTS achieved optically controlled protein entry into the cell membrane and LLPS condensates. Additionally, PRINTS allow light-controlled organelle relocalization, such as LLPS condensates or mitochondria to the plasma membrane. We also applied this targeted delivery system to screen and validate de novo designed short peptides that disrupt LLPS. As a versatile platform, PRINTS facilitate precise manipulation of protein and organelle dynamics, offering a powerful tool to study the regulatory coordination between membranous and membraneless compartments in response to physiological needs.