Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.Eukaryotic cell nuclei are separated from the cytoplasm by a double lipid bilayer system known as the nuclear envelope (NE).2 Exchange of material between the two compartments proceeds through nuclear pore complexes (NPCs), large protein assemblies that span the NE and provide the sole medium for exchange. The vertebrate NPC has a molecular mass of ϳ125 MDa (1) and is made up of ϳ30 different proteins, called nucleoporins, most of which are present in multiples of eight (2, 3). The core of the NPC consists of a symmetrical framework, measuring ϳ120 ϫ 90 nm, which is made of two coaxial rings sandwiching a wheel-like array of eight spoke-shaped domains. The spoke-ring assembly encircles the central pore channel, which resembles an hourglass 45-50 nm wide at its waist (4 -7). In addition to the central framework, NPCs contain peripheral structures, which are anchored to the ring moieties of the spoke-ring assembly and serve as docking sites for nuclear transport receptors and effectors. These structures include eight short (ϳ50 nm) filaments that protrude toward the cytoplasm and a massive, fish trap-like structure, termed the nuclear basket, which extends into the nucleus (4 -11). Yeast NPCs have an overall similar architecture but are smaller (3,(12)(13)(14).Transport across the NPC has been reviewed in detail (15-22) and can be divided into two modes. Small molecules, such as ions, metabolites, and intermediate-sized macromolecules, can pass unassisted by diffusion which becomes increasingly restricted as the particle approaches a size limit of ϳ10 nm in diameter (23,24). In contrast, proteins, RNAs, and their complexes are ushered selectively by dedicated soluble transport receptors, which recognize specific import (NLS) or nuclear export signal (NES) peptides displayed by the cargo. In most cases, assembly and disassembly of transport complexes in the appropriate cellular compartment are coordinated by the small GTPase Ran, which performs these activities by binding to transport receptors in its GTP-bound form. Facilitated translocation is often coupled to an input of metabolic energy that permits transport ...

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and pliant interaction network between cargo and FG repeats.

The extensive and multifaceted traffic between nucleus and cytoplasm is handled by a single type of macromolecular assembly called the nuclear pore complex (NPC). While being readily accessible to ions and metabolites, the NPC imposes stringent selectivity on the passage of proteins and RNA, tightly regulating their traffic between the two major cellular compartments. Here we discuss how shuttling carriers, which mediate the transport of macromolecules through NPCs, cross its permeability barrier. We also discuss the co-existence of receptor-mediated macromolecular transport with the passive diffusion of small molecules in the context of the various models suggested for the permeability barrier of the NPC. Finally, we speculate on how nuclear transport receptors negotiate the dependence of their NPC-permeating abilities on hydrophobic interactions with the necessity of avoiding these promiscuous interactions in the cytoplasm and nucleus.