Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of ''natively unfolded'' proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.T he nuclear pore complex (NPC) spans the nuclear envelope of eukaryotes and serves as the conduit for all nucleocytoplasmic transport (1). In Saccharomyces cerevisiae, the NPC is composed of multiple copies of 30 different nucleoporins (Nups) that form an octagonal pore structure of 60 MDa measuring 95 nm in diameter and 35 nm in height (2). The FG family of Nups contain extensive regions of FG repeats (typically 200-700 aa in length) and facilitate the passage of karyopherin-cargo complexes through the NPC by binding directly to karyopherins (Kaps͞importins͞exportins͞ transportins) (3, 4), the soluble receptors that recognize nucleocytoplasmic transport signals within cargo (5).Although the mechanism of Kap translocation through NPCs is poorly understood, Kap-FG Nup interactions are central to the process. It is therefore likely that the structures of FG Nups play important roles in directing the movement of Kap-cargo complexes as they traverse the NPC; however, little is known about the structure of the individual FG Nups within the NPC. The FG repeat region of the mammalian Nup p62 was initially hypothesized to adopt a ␤-conformation (6). More recent models of NPC organization have favored disordered conformations for the FG Nups (7, 8), despite a lack of conclusive evidence. In support of these models, Bayliss et al. (9) observed that a fragment of the Nsp1p FG repeat region (a yeast homolog of p62) lacks ␣-helical structure and may be disordered. Also, our recent biophysical analyses of the fulllength S. cerevisiae FG Nup Nup2p characterized it as a natively unfolded protein (10). Natively unfolded proteins, such as Nup2p, lack significant ␣-helical and ␤-sheet secondary structure and exhibit high flexibility under physiological conditions. They also exhibit low overall hydrophobicity, high net charge, and low compactness͞nonglobularity (11, 12). Here, we perform structural analyses of additional FG Nups to test whether unfolded structures are a conserved feat...

Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 Å) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA.More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K D ‫؍‬ 1.5 M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine–glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin–cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

A highly sensitive, equilibrium-based binding assay termed "Bead Halo" was used here to identify and characterize interactions involving components of the nucleocytoplasmic transport machinery in eukaryotes. Bead Halo uncovered novel interactions between the importin Kap95 and the nucleoporins (nups) Nic96, Pom34, Gle1, Ndc1, Nup84, and Seh1, which likely occur during nuclear pore complex biogenesis. Bead Halo was also used to characterize the molecular determinants for binding between Kap95 and the family of nups that feature multiple phenylalanine-glycine motifs (FG nups). Binding was sensitive to the number of FG motifs present and to amino acid (AA) residues immediately flanking the FG motifs. Also, binding was reduced but not abolished when phenylalanine residues in all FG motifs were replaced by tyrosine or tryptophan. These results suggest flexibility in the binding pockets of Kap95 and synergism in binding FG motifs. The hypothesis that Nup53 and Nup59 bind directly to membranes through a C-terminal amphipathic alpha helix and to DNA via an RNA recognition motif domain was also tested and validated using Bead Halo. The results support a role for these nups in nuclear pore membrane biogenesis and in gene expression. Finally, Bead Halo detected binding of the nups Gle1, Nup60, and Nsp1 to phospholipid bilayers. This may reflect the known interaction between Gle1 and phosphoinositides and suggests similar interactions for Nup60 and Nsp1. As the Bead Halo assay detected molecular interactions in cell lysates, as well as between purified components, it can be adapted for large-scale proteomic studies using automated robotics and microscopy. Molecular & Cellular Proteomics 7:121-131, 2008.In the context of the cellular milieu, protein interactions of high and low affinity are key for the survival of organisms. Current proteomic studies aim to uncover all such interactions with the ultimate goal of reconstructing, understanding and predicting all cellular behavior at a system-wide level. Despite the great advances in cellular proteomics, our ability to detect protein interactions of low affinity, in particular, is limited. Current proteomic analyses using biochemical tools, such as Protein A-tag (also termed ZZ-tag; TAP tag) pull-downs or co-immunoprecipitations, are limited to only detecting high affinity interactions. This is because the necessary wash steps that remove nonspecific interactors (i.e. from the agarose or magnetic beads used as solid support during the isolations) also remove specific low affinity interactions. It is estimated that pull-down methods can identify protein interactions stronger than the K d ϳ5 M range (1, 2). The sensitivity can be improved through extended incubation times followed by rapid collection and washing of the beads, but nonspecific interactions also increase (3). To detect protein interactions of low affinity (beyond the K d ϳ5 M range), we recently reported the development of an assay termed Bead Halo, which can detect molecular interactions of high and low affinity in...