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Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1’s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis.HighlightsNups form native cytosolic condensatesNsp1 reduction mimics NPC aging phenotypesNsp1 acts as phase state modulator of FG-NupsNsp1 shares surveillance function with classical chaperones

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular-dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

The intrinsically disordered FG-Nups in the NPC central channel can undergo liquid-liquid phase sepration (LLPS) into liquid condensates that display NPC-like permeability barrier properties. Here we present LLPS characteristics of each of the disordered FG-Nups of the yeast NPC. Using molecular dynamics simulations at amino acid resolution, FG-Nup condensates are studied and the main physicochemical driving forces for FG-Nup LLPS are identified. We show that FG-motifs that are predominantly present in the disordered domain of FG-Nups act as highly-dynamic hydrophobic stickers that are essential for the formation of stable liquid-like condensates. Next to that, we study LLPS of an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple types of FG-Nups. We find that the LLPS of this NPC condensate is also driven by FG–FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed LLPS behavior, we categorize the different FG-Nups of the yeast NPC into two classes: The GLFG-Nups located in the central channel of the NPC phase separate into liquid-like condensates, forming a high-density cohesive barrier that can exclude inert particles. The FG-Nups at the entry and exit of the NPC channel, containing no GLFG-motifs, do not phase separate and possibly form a repulsive barrier by entropically excluding inert particles.