A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (m^DcTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for m^DcTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and m^DcTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations.