Fig. 3. De novo designed Ras-dependent LOCKR-based proximity labeler (Ras-LOCKR-PL) identifies Ras targets in signaling microdomains.

a, Schematic of optimized Ras-LOCKR-PL consisting of split TurboID tethered to Cage and Key; GTP-Ras binds to RasBD, promoting reconstitution of functional TurboID and biotinylation of neighboring proteins. As in Fig. 1b, numbers correspond to regions that were optimzied during the development of Ras-LOCKR-PL. b, Predicted structure of Ras-LOCKR-PL with mutations highlighted. c, Bar graph of biotinylation levels of CIAR-PM-293 cells, which were transfected with Ras-LOCKR-PL candidates, and 500 μM biotin was added for 16 h without (−stimulation) or with (+stimulation) 250 nM A115 (n = 4 experiments per condition). Ratio is +stimulation divided by −stimulation. The numbers shown over the bar graphs represent the number of key:cage weakening mutations. d, Representative western blots of CIAR-PM-293 cells transfected with Ras-LOCKR-PL WT or a mutant without RasBD (ΔRasBD), and treated with 500 μM biotin with or without 250 nM A115 (labeled ‘A’) for 16 h (n = 3 experimental repeats). e,f, Representative epifluorescence images from three biologically independent experiments. CIAR-PM-293 cells were transfected with subcellularly localized Ras-LOCKR-PL or full-length TurboID expressed, were treated with 500 μM and 250nM A115 for f and underwent immunostaining with antibodies for established localization markers (e) or fluorescent dye-conjugated streptavidin (f). Bar graphs represent mean ± s.e.m. **P < 0.01, unpaired two-tailed Student’s t-test. Scale bars, 10 μm. All LOCKR-PL candidates and associated experimental results are listed in Supplementary Table 2.