posted on 2020-03-20, 16:03authored byAkinobu Nakamura, Choji Oki, Shunsuke Sawada, Tatsuyuki Yoshii, Keiko Kuwata, Andrew K. Rudd, Neal K. Devaraj, Kentaro Noma, Shinya Tsukiji
Inducing protein
translocation to the plasma membrane (PM) is an
important approach for manipulating diverse signaling molecules/pathways
in living cells. We previously devised a new chemogenetic system,
in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from
the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing
ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation
turned out to be transient and spontaneously reversed within 1 h,
limiting its application. Here, we first demonstrated that the spontaneous
reverse translocation was caused by cellular degradation of mgcTMP,
presumably by proteases. To address this problem, we newly developed
a proteolysis-resistant SL, mDcTMP. This mDcTMP
now allows sustained PM localization of eDHFR-fusion proteins (over
several hours to a day), and it was applicable to inducing prolonged
signal activation and cell differentiation. mDcTMP also
worked in live nematodes, making it an attractive new tool for probing
and controlling living systems.