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.

能够以单一蛋白质-单一配体方式将特定蛋白质快速转运至质膜 (PM) 的化学遗传学方法是细胞生物学中的有用工具。我们最近开发了一种技术，其中使用合成的肉豆蔻酰-d -Cys -tethered trimethoprim (m ^D cTMP)将融合到携带 N 末端六赖氨酸残基的大肠杆菌二氢叶酸还原酶 (eDHFR) 变体的蛋白质从细胞质中募集到 PM配体。然而，该系统只有在 eDHFR 标签融合到蛋白质的 N 端时才能实现 PM 特异性易位，从而限制了其应用。^{在本报告中，我们为 m D}设计了一个通用的 PM 定位标签通过将六赖氨酸基序移植到 eDHFR 的环内区域中，cTMP 诱导的蛋白质易位。我们展示了新的循环工程 eDHFR 标签和 m ^D cTMP 对的广泛适用性，用于条件性 PM 募集和激活具有不同融合配置的各种标签融合信号蛋白，以及可逆和重复控制蛋白质定位以产生合成信号振荡。