Nuclear pore complexes permit rapid passage of cargoes bound to nuclear transport receptors, but otherwise suppress nucleocytoplasmic fluxes of inert macromolecules >/=30 kilodaltons. To explain this selectivity, a sieve structure of the permeability barrier has been proposed that is created through reversible cross-linking between Phe and Gly (FG)-rich nucleoporin repeats. According to this model, nuclear transport receptors overcome the size limit of the sieve and catalyze their own nuclear pore-passage by a competitive disruption of adjacent inter-repeat contacts, which transiently opens adjoining meshes. Here, we found that phenylalanine-mediated inter-repeat interactions indeed cross-link FG-repeat domains into elastic and reversible hydrogels. Furthermore, we obtained evidence that such hydrogel formation is required for viability in yeast.

The permeability barrier of nuclear pore complexes (NPCs) controls the exchange between nucleus and cytoplasm. It suppresses the flux of inert macromolecules > or = 30 kDa but allows rapid passage of even very large cargoes, provided these are bound to appropriate nuclear transport receptors. We show here that a saturated hydrogel formed by a single nucleoporin FG-repeat domain is sufficient to reproduce the permeability properties of NPCs. Importin beta and related nuclear transport receptors entered such hydrogel >1000x faster than a similarly sized inert macromolecule. The FG-hydrogel even reproduced import signal-dependent and importin-mediated cargo influx, allowing importin beta to accelerate the gel entry of a large cognate cargo more than 20,000-fold. Intragel diffusion of the importin beta-cargo complex occurred rapidly enough to traverse an NPC within approximately 12 ms. We extend the "selective phase model" to explain these effects.

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve-like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross-linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant-negative importin b 45-462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC-passing molecule. They have little effect on small species, inhibit the passage of green fluorescent proteinsized objects 410-fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.

The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the Nterminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular β-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain β-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.Beta-sheet | nuclear pore complex | Nup | Prion | solid-state NMR N uclear pore complexes (NPCs) control all nucleocytoplasmic exchange (1-4). Their permeability barrier allows free passage of small molecules but suppresses the flux of macromolecules larger than 30 kDa and thereby prevents an uncontrolled intermixing of nuclear and cytoplasmic contents. However, the permeability barrier also permits a rapid passage of even large cargoes, provided these are bound to appropriate nuclear transport receptors (NTRs) (2-5). NTRs thereby supply nuclei with proteins and the cytoplasm with ribosomes and other nuclear products.FG repeat domains are essential building blocks of NPCs (6). They are considered to be natively unfolded and contain up to 50 repeat units, in which a characteristic hydrophobic patch, typically with the sequence FG, FxFG, or GLFG, is surrounded by more hydrophilic spacer sequences (7-9). These hydrophobic patches transiently bind NTRs during facilitated NPC passage (10-15).Recent evidence suggests that the permeability barrier is a hydrogel derived from FG repeat domains (FG hydrogel) (13,(15)(16)(17)(18). FG hydrogels have been predicted by the selective phase model (15,16,19) and indeed have been reconstituted from the FG/FxFG domain of the yeast nucleoporin Nsp1p (13,16) or the GLFG domains from Nup49p and Nup57p (17). All these gels showed permeability properties very similar to those of NPCs themselves: they allowed an up to 20,000...

Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptorsTo gain insight into the mechanisms behind transport of molecules across the nuclear pore complex, Richter and co-workers have developed ultrathin films of nucleoporin FG repeat domains and quantify how these films bind dedicated shuttle molecules—the so-called nuclear transport receptors (NTRs). They find that NTRs can efficiently permeate the films, but do not affect their global morphology, which suggests that the FG repeat domains form a dense meshwork of entangled or transiently crosslinked polymers.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport through an FG domain-controlled barrier. We now explore how surface-features of a mobile species determine its NPC passage rate. Negative charges and lysines impede passage. Hydrophobic residues, certain polar residues (Cys, His), and, surprisingly, charged arginines have striking translocation-promoting effects. Favorable cation-π interactions between arginines and FG-phenylalanines may explain this apparent paradox. Application of these principles to redesign the surface of GFP resulted in variants that show a wide span of transit rates, ranging from 35-fold slower than wild-type to ∼500 times faster, with the latter outpacing even naturally occurring nuclear transport receptors (NTRs). The structure of a fast and particularly FG-specific GFP variant illustrates how NTRs can expose multiple regions for binding hydrophobic FG motifs while evading non-specific aggregation. Finally, we document that even for NTR-mediated transport, the surface-properties of the "passively carried" cargo can strikingly affect the translocation rate.