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.

中文翻译：

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设计师基于棕榈酰化基序的自定位配体，用于持续控制活细胞和秀丽隐杆线虫中的蛋白质定位。

诱导蛋白质易位至质膜 (PM) 是操纵活细胞中多种信号分子/通路的重要方法。我们之前设计了一种新的化学遗传学系统，其中融合到大肠杆菌二氢叶酸还原酶 (eDHFR) 的蛋白质可以使用基于甲氧苄啶 (TMP) 的自定位配体 (SL) mgcTMP 快速从细胞质转移到 PM。然而，mgcTMP 诱导的蛋白质易位结果是短暂的，并在 1 小时内自发逆转，限制了其应用。在这里，我们首先证明了自发的反向易位是由 mgcTMP 的细胞降解引起的，可能是由蛋白酶引起的。为了解决这个问题，我们新开发了一种抗蛋白水解的 SL，mDcTMP。这种 mDcTMP 现在允许 eDHFR 融合蛋白的持续 PM 定位（超过几个小时到一天），它适用于诱导延长的信号激活和细胞分化。mDcTMP 也适用于活线虫，使其成为探测和控制生命系统的有吸引力的新工具。