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#35835118 2022/07/06 To Up
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 (mcTMP) 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 mcTMP-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 mcTMP 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.
Sachio Suzuki, Akinobu Nakamura, Yuka Hatano, Masaru Yoshikawa, Tatsuyuki Yoshii, Shunsuke Sawada, Kyoko Atsuta-Tsunoda, Kazuhiro Aoki, Shinya Tsukiji