Fig. 1: Design of the feeding–fishing strategy and selection of optimal proximity labeling enzyme.
From: Membrane editing with proximity labeling reveals regulators of lipid homeostasis
a, Feeding–fishing strategy couples membrane editing and proximity labeling for elucidation of regulators of PA homeostasis on different organelle membranes. An optogenetic superPLD is targeted to a membrane of interest to feed it with PA, and a proximity labeling enzyme is anchored to the same membrane to enable biotinylation to fish out proteins recruited to the lipid-edited membrane. PL, proximity labeling. b, Comparison of APEX2 and TurboID for proximal organelle membrane proteomics, evaluating their ability to selectively label a model protein (optoPLD) associated with the same versus different membranes. APEX2 labeling was performed using a 30-min incubation with 500 μM biotin-phenol followed by 1 min with H2O2. TurboID labeling was performed using a 10-min incubation with 500 μM biotin. PM, plasma membrane. c, Quantification of biotinylated optoPLD in b (n = 3 for APEX2 and n = 2 for TurboID, where n indicates independent biological replicates). d,e, Immunofluorescence images of cells expressing APEX2 (d) or TurboID (e) targeted to plasma membrane or lysosomes. Shown are representative images from two independent experiments. Scale bars, 10 μm. f, Western blots of cells expressing TurboID targeted to either the plasma membrane (KRAS CAAX domain19), lysosomes (p18/LAMTOR1 lysosomal-targeting sequence20 or TMEM192 (ref. 65)) or the ER (P450 2C1 (residues 1–29)66). Labeling was performed by 10-min incubation with 500 μM biotin. The rightmost two lanes indicate cells without expressing TurboID treated with or without biotin. Shown are representative blots from two independent experiments. g, One-shot transduction strategy enables efficient and transient coexpression of the feeding–fishing components. h,i, Immunofluorescence images of cells coexpressing TurboID and superPLD targeted to the plasma membrane (h) or lysosomes (i; Lyso). Shown are representative images from three independent experiments. hv, blue light (470 nm). Scale bars, 20 μm.
