Karyopherin enrichment at the nuclear pore complex attenuates Ran permeability.

Ran is a small GTPase whose nucleotide-bound forms cycle through nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport (NCT). Generally, Ran guanosine triphosphate (RanGTP) binds cargo-carrying karyopherin receptors (Kaps) in the nucleus and releases them into the cytoplasm following hydrolysis to Ran guanosine diphosphate (RanGDP). This generates a remarkably steep Ran gradient across the nuclear envelope that sustains compartment-specific cargo delivery and accumulation. However, because NPCs are permeable to small molecules of comparable size, it is unclear how an uncontrolled mixing of RanGTP and RanGDP is prevented. Here, we find that an NPC-enriched pool of karyopherin subunit beta 1 (KPNB1, hereafter referred to as Kapß1) selectively mediates Ran diffusion across the pore but not passive molecules of similar size (e.g. GFP). This is due to RanGTP having a stronger binding interaction with Kapß1 than RanGDP. For this reason, the RanGDP importer, nuclear transport factor 2, facilitates the return of RanGDP into the nucleus following GTP hydrolysis. Accordingly, the enrichment of Kapß1 at NPCs may function as a retention mechanism that preserves the sharp transition of RanGTP and RanGDP in the nucleus and cytoplasm, respectively.

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores.

Large multiprotein nanopores remain difficult to reconstitute in vitro, such as, for instance, the nuclear pore complex (NPC) that regulates macromolecular transport between the nucleus and cytoplasm in cells. Here, we report that two NPC pore membrane proteins self-assemble into ∼20 nm diameter nanopores following in vitro reconstitution into lipid bilayers. Pore formation follows from the assembly of Pom121 and Ndc1 oligomers, which arrange into ringlike membrane structures that encircle aqueous, electrically conductive pores. This represents a key step toward reconstituting membrane-embedded NPC mimics for biological studies and biotechnological applications.