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The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules but allows facilitated passage of nuclear transport receptors that shuttle cargoes into or out of nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains, including foremost the charge-depleted FG domain of Nup98. We found that Nup98 FG domains show an LCST-type phase separation, and we provide comprehensive and orthogonal experimental datasets for a quantitative description of this behaviour. A derived thermodynamic model correlates saturation concentration with repeat number, temperature, and ionic strength. It allows estimating the enthalpy, entropy, and ΔG (0.2 kJ/mol, 0.1 k B · T ) contributions per repeat to phase separation and inter-repeat cohesion. While changing the cohesion strength strongly impacts the strictness of barrier, these numbers provide boundary conditions for in-depth modelling not only of barrier assembly but also of NPC passage. The nuclear pore complex (NPC) barrier is a selective phase assembled from disordered but cohesive FG domains. The authors provide a thermodynamic description of an FG phase that is ultimately simplified and yet closely recapitulates NPC transport selectivity.

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic ‘FG particles’. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore. Cells of eukaryotic species—which include plants, animals, and fungi—have a nucleus that harbours the organism's genome. Two membrane layers surround the nucleus and separate its contents from the cytoplasm, where proteins are made. This separation is essential for a correct interpretation of the genetic information. Yet, various molecules, such as proteins, need to move into or out of the nucleus for the cell to work properly. This transit has to occur without an uncontrolled mixing of the contents of the nucleus and the cytoplasm happening. Structures called nuclear pore complexes span the double membrane and allow material to be exchanged between the nucleus and the cytoplasm. Small molecules can freely pass through these complexes, while larger molecules can only be transported when bound as “cargo” to so-called nuclear transport receptors. Nuclear pore complexes are large assemblies of proteins called nucleoporins. FG nucleoporins are special in that they contain regions with a repeating pattern of two amino acids, phenylalanine (‘F’) and glycine (‘G’). These regions are called FG domains. They bind to nuclear transport receptors and have been suspected to form a barrier that decides which molecules may pass through the nuclear pore complex. Exactly how this control is exerted has been a matter of debate. Versions of a particular FG nucleoporin called Nup98 are found in all branches of eukaryotic life, i.e. in animals, fungi, plants, amoebas, and even in the evolutionarily most distant protozoans. When Schmidt and Görlich dispersed small amounts of Nup98 FG domains in an aqueous solution, the domains rapidly attracted each other to form ‘FG particles’, regardless of which species the proteins came from. These FG particles were so dense that they repelled ‘normal’ macromolecules, yet they allowed nuclear transport receptors, along with their bound cargoes, to rapidly enter. Taken together, the work of Schmidt and Görlich suggests that such FG particles form the transport barrier in nuclear pore complexes. Based on these findings, Schmidt and Görlich refine a model where the FG domains form a mesh in the nuclear pore complexes that acts like a ‘smart sieve’. Smaller molecules can move through gaps in the meshwork, but larger molecules are hindered. Schmidt and Görlich suggest that nuclear transport receptors help large molecules to move through nuclear pore complexes by ‘melting’ the FG meshwork locally, creating a path for the molecule to move through. The reconstitution of these smart barriers in the laboratory will now allow researchers to analyse the process of receptor-mediated nuclear pore passage in unprecedented (mechanistic) detail.

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 nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with ‘DNA-specific’ DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport. The permeability barrier of nuclear pore complexes blocks passage of inert macromolecules but allows rapid, receptor-mediated, and RanGTPase-driven transport of cargoes up to ribosome size. The authors now show that such a barrier can be faithfully recapitulated by an ultimately simplified FG phase assembled solely from a tandemly repeated 12mer GLFG peptide.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel 1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase 2 that is assembled from cohesively interacting phenylalanine–glycine (FG) repeats 3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans -acting NTRs would increase the diameter by 10 nm or more, we suggest that such a ‘self-translocating’ capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection. The HIV-1 capsid behaves like a nuclear transport receptor entering and traversing an FG phase, with its interior serving as a cargo container, bypassing an otherwise effective barrier to viral infection.

Xpo4 is a bidirectional nuclear transport receptor that mediates nuclear export of eIF5A and Smad3 as well as import of Sox2 and SRY. How Xpo4 recognizes such a variety of cargoes is as yet unknown. Here we present the crystal structure of the RanGTP·Xpo4·eIF5A export complex at 3.2 Å resolution. Xpo4 has a similar structure as CRM1, but the NES-binding site is occluded, and a new interaction site evolved that recognizes both globular domains of eIF5A. eIF5A contains hypusine, a unique amino acid with two positive charges, which is essential for cell viability and eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4 and is thus a critical element of eIF5A’s complex export signature. This further suggests that Xpo4 recognizes other cargoes differently, and illustrates how Xpo4 suppresses – in a chaperone-like manner – undesired interactions of eIF5A inside nuclei. Xpo4 imports Sox2 and other proteins into the cell nucleus, while exporting eIF5A or Smad3; how it recognizes these proteins has been unclear. Here, the authors solved the crystal structure of the RanGTP, Xpo4 and eIF5A complex and investigate how Xpo4 identifies its major export cargo.

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy. The permeability barrier of nuclear pores is formed by disordered and yet self-interacting FG repeat domains, whose sequence heterogeneity is a challenge for mechanistic insights. Here the authors overcome this challenge and characterize the protein’s dynamics by applying NMR techniques to an FG phase system that has been simplified to its essentials.

CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction. Animals, plants and other eukaryotic organisms subdivide their cells into compartments that carry out specific tasks. For example, the cell nucleus hosts the genome and handles the genetic information, whereas the surrounding cytoplasm is specialized in making proteins. These proteins are then either used in the cytoplasm or transported to the nucleus and other cell compartments. Since proteins are not made in the nucleus, all proteins in this compartment must be imported from the cytoplasm. Two layers of membrane separate the nucleus and cytoplasm from each other. Any exchange of material must therefore proceed through channels called nuclear pore complexes, or NPCs for short. The NPCs have filters that allow only small molecules a free transit, while larger ones are typically rejected. However, larger proteins may also rapidly pass through the nuclear pore complexes if loaded onto dedicated shuttle molecules; for example, “exportins” transport proteins out of the nucleus. Kırlı, Karaca et al. used an approach called proteomics to measure the levels of 5,000 different proteins within the nucleus and the cytoplasm. Such a census helps to predict where a given protein works and where it might cause problems. Further experiments also used proteomics to identify which proteins are carried by an exportin called CRM1. This revealed that a remarkably large number of different proteins (around 1,000) are exported by CRM1 from either yeast, human or frog cell nuclei. Most of these “cargo” proteins were previously thought to never leave the cytoplasm. It now seems, however, that these proteins can leak into the nucleus, but CRM1 recognizes them as cytoplasmic proteins and expels them from the nucleus. These findings suggest that the border control at NPCs is less perfect than was previously believed. If not remedied, this would pose a serious problem for the cell, because the accumulation of \"wrong\" proteins inside the nucleus would disturb the processes that occur there and could destabilize the genome. Kırlı, Karaca et al. propose that the export of such accidentally displaced proteins by CRM1 is a crucial measure to protect the nucleus.

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phase-entry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFP NTR -variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR·FG interactions is key to transport selectivity. The permeability barrier of the nuclear pore assembles from cohesive FG repeats. By systematic engineering and testing repeat variants, the authors pinpointed the sequence features that rule barrier assembly and transport selectivity.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Here, we report a structural analysis of the FG Nup62•58•54 complex, which is a crucial component of the transport system. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. We further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. We suggest that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.