Fig. 1: Site-specific labelling of NUP98 in the functional state inside the NPC and comparison with phase-separated condensates in vitro.

a, Schematic of site-specific labelling of target NUP98 inside the NPC. The genetic code was exclusively expanded for target NUP98. ncAAs were introduced into NUP98 at specific sites by synthetic orthogonal translating organelle-enabled genetic code expansion (OTO-GCE). Tetrazine-modified dye molecules were added to the cells and reacted with ncAAs (click chemistry). b, Passive exclusion (70-kDa dextran was excluded) and facilitated assay, or active transport assay (IBB–MBP–GFP supplied with transport mixture was imported) showing that the NPCs were functional with site-specific labelled NUP98 in permeabilized COS-7 cells (with the plasma membrane selectively permeabilized with low-dosage digitonin). Scale bars, 20 μm. c, Purified NUP98 FG domain was phase-separated in vitro by rapidly diluting a denatured highly concentrated stock solution into physiological buffer. The permeability of the droplet-like condensates formed was measured rapidly where they still obeyed liquid-like characteristics²⁶. The droplets recapitulated the function of the permeability barrier, as shown in the passive exclusion and facilitated transport assays. Scale bars, 5 μm. For b,c, n = 3 experiments were repeated independently with the same conclusion.