Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-moleculetriggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-ofprinciple work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

Automated summary

Scientists have successfully constructed synthetic protein condensate systems that can recruit and release proteins in mammalian cells in response to small molecules or light, allowing control over cellular processes and protein activity. This represents a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells, a crucial step towards tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.