Barrier properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phaseentry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-p interactions. Although incompatible with NTR center dot cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFP(NTR)-variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR center dot FG interactions is key to transport selectivity.

Automated summary

The Nup98 FG repeat domains in the nuclear pore complex (NPC) capture nuclear transport receptors (NTRs) and condense into a selective phase with barrier properties. Shortening the FG spacers enhances phase cohesion and barrier tightness, while arginines promote phase entry through hydrophobic interactions/hydrogen-bonding. Mutations in the FG motifs affect NTR entry, and the antagonism between cohesion and FG interactions is crucial for transport selectivity.