Figure 1. Development of a universal protein tag for PM-specific SLIPT. (a) Chemical structure 2 of mDcTMP. (b) Design of loop-engineered eDHFR constructs based on the topology of PM-3 anchored eDHFR. In the schematic illustration, the crystal structure of eDHFR complexed with 4 methotrexate (PDB: 1RG7[26]) was used and the cartoon model was created with UCSF 5 ChimeraX[47] (methotrexate is not shown). The structure is depicted in a manner such that eDHFR 6 is anchored on the putative PM by mDcTMP. The N- and C-termini are shown in magenta and 7 cyan, respectively. The K6 motif was inserted between D69 and D70 or between A145 and Q146 8 of wild-type eDHFR to generate eDHFR69K6 and eDHFR145K6, respectively. (c–f) mDcTMP-9 induced translocation of eDHFR69K6-EGFP (c), EGFP-eDHFR69K6 (d), eDHFR145K6-EGFP (e), 10 and EGFP-eDHFR145K6 (f). Confocal fluorescence images of HeLa cells expressing the indicated 11 construct were taken before (left) and 30 min after the addition of mDcTMP (10 µM) (right). Scale 12 bars, 10 µm. For the time-lapse movie of EGFP-eDHFR69K6 translocation, see Movie S1. (g) Time 13 course of PM translocation (quantification of data shown in c–f and Figure S2). The normalized 14 fluorescence intensity in the cytoplasm was plotted as a function of time. Data are presented as 15 the mean ± SD (n = 5 cells). 16 17 18
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