Fig. 6: Selectivity of FG phases assembled from Pro-containing perfect repeats.
From: Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide
a An ultimately simplified FG domain (prf.GLFG52x12) was generated by deleting the GLEBS domain from prf.GLFG52x12[+GLEBS]. b FG phases were challenged with different protein probes for selectivity. Scanning settings/image brightness were adjusted individually due to the large range of signals. Note that GLEBS-free FG phases (Mac98A ΔGLEBS and prf.GLFG52x12) allowed for a stronger accumulation of NTRs, NTR·cargo complexes, and the NTR-like sffrGFP4. However, the conversion of the Mac98A FG domain to perfect repeats caused only small changes in NTR-accumulation. Scale bar: 10 μm. sc: Saccharomyces cerevisiae; hs: Homo sapiens. c In total, 10 µM of indicated FG domain variants were allowed to phase separate and the FG phases were pelleted by ultracentrifugation. Equivalent ratios of pellets and supernatants were analyzed by SDS-PAGE/Coomassie-staining. Critical concentrations for phase separation were taken as the concentrations that remained in the supernatants (“Methods”). The experiment was repeated independently three times with similar results, and representative images are shown. Full scans of gels with molecular weight markers were provided as a Source Data file. d FG particles spiked with 2% Alexa488-(covalently) labeled FG domains (of the same species) were photobleached. Fluorescence recovery after photobleaching (FRAP) was recorded over time. Scale bar: 3 μm. e Recovery curves corresponding to (d). Y axis: recovery was normalized to 1 for a complete recovery. The numbers are translational diffusion coefficients derived from fitting the datasets to theoretical recovery curves (“Methods”). The GLEBS-containing Mac98A and prf.GLFG52x12[+GLEBS] FG domains were essentially immobile. Source data are provided as a Source Data file. f Alexa488-labeled Ran was converted either to the RanGTP form (by pre-incubation with RanGEF and an ATP/GTP-regenerating system), or to the RanGDP form (by pre-incubation with RanGAP). Panels show the partitioning of these Ran forms into the ultimately simplified prf.GLFG52x12 phase. Where indicated, unlabeled NTF2 (2 µM dimer concentration) was also added. g prf.GLFG52x12 phase was challenged with import (IBB-EGFP) and export cargoes (EGFP fused to a strong Xpo1-dependent nuclear export signal (NES) + RanQ69L, a mutant of Ran locked in the GTP form) in the absence or presence of the corresponding NTRs: Impβ/Xpo1. h prf.GLFG52x12 phase was challenged with an 80 kDa import cargo carrying two orthogonal import signals, an IBB (recognized by Impβ) and an M9 domain (recognized by transportin, Trn). Note that strong intra-phase accumulation was only observed in the presence of both NTRs, recapitulating the requirement for large cargo transport through NPCs. Numbers refer to GFP fluorescence ratios in the central regions of the particles to that in the surrounding buffer.
