Higher nucleoporin-Importinβ affinity at the nuclear basket increases nucleocytoplasmic import.

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex--suggesting that the affinity gradient seen in vitro is highly optimized

Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex.

The nuclear pore complex (NPC) is the portal for bidirectional transportation of cargos between the nucleus and the cytoplasm. While most of the structural elements of the NPC, i.e. nucleoporins (Nups), are well characterized, the exact transport mechanism is still under much debate. Many of the functional Nups are rich in phenylalanine-glycine (FG) repeats and are believed to play the key role in nucleocytoplasmic transport. We present a bioinformatics study conducted on more than a thousand FG Nups across 252 species. Our results reveal the regulatory role of polar residues and specific sequences of charged residues, named 'like charge regions' (LCRs), in the formation of the FG network at the center of the NPC. Positively charged LCRs prepare the environment for negatively charged cargo complexes and regulate the size of the FG network. The low number density of charged residues in these regions prevents FG domains from forming a relaxed coil structure. Our results highlight the significant role of polar interactions in FG network formation at the center of the NPC and demonstrate that the specific localization of LCRs, FG motifs, charged, and polar residues regulate the formation of the FG network at the center of the NPC

Efficiency, selectivity and robustness of the nuclear pore complex transport

All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. A central feature of this transport is the binding of cargo-carrying soluble transport factors to flexible, unstructured proteinaceous filaments called FG-nups that line the NPC. We have modeled the dynamics of transport factors and their interaction with the flexible FG-nups as diffusion in an effective potential, using both analytical theory and computer simulations. We show that specific binding of transport factors to the FG-nups facilitates transport and provides the mechanism of selectivity. We show that the high selectivity of transport can be accounted for by competition for both binding sites and space inside the NPC, which selects for transport factors over other macromolecules that interact only non-specifically with the NPC. We also show that transport is relatively insensitive to changes in the number and distribution of FG-nups in the NPC, due mainly to their flexibility; this accounts for recent experiments where up to half of the total mass of the NPC has been deleted, without abolishing the transport. Notably, we demonstrate that previously established physical and structural properties of the NPC can account for observed features of nucleocytoplasmic transport. Finally, our results suggest strategies for creation of artificial nano-molecular sorting devices.Comment: 38 pages, six figure

Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function

Effects of multiple occupancy and inter-particle interactions on selective transport through narrow channels: theory versus experiment

Many biological and artificial transport channels function without direct input of metabolic energy during a transport event and without structural rearrangements involving transitions from a 'closed' to an 'open' state. Nevertheless, such channels are able to maintain efficient and selective transport. It has been proposed that attractive interactions between the transported molecules and the channel can increase the transport efficiency and that the selectivity of such channels can be based on the strength of the interaction of the specifically transported molecules with the channel. Herein, we study the transport through narrow channels in a framework of a general kinetic theory, which naturally incorporates multi-particle occupancy of the channel and non-single-file transport. We study how the transport efficiency and the probability of translocation through the channel are affected by inter-particle interactions in the confined space inside the channel, and establish conditions for selective transport. We compare the predictions of the model with the available experimental data - and find good semi-quantitative agreement. Finally, we discuss applications of the theory to the design of artificial nano-molecular sieves.Comment: 27 pages, 6 figures, 1 Appendix, in press in Biophysical Journa

Brownian Dynamics Simulation of Nucleocytoplasmic Transport: A Coarse-Grained Model for the Functional State of the Nuclear Pore Complex

The nuclear pore complex (NPC) regulates molecular traffic across the nuclear envelope (NE). Selective transport happens on the order of milliseconds and the length scale of tens of nanometers; however, the transport mechanism remains elusive. Central to the transport process is the hydrophobic interactions between karyopherins (kaps) and Phe-Gly (FG) repeat domains. Taking into account the polymeric nature of FG-repeats grafted on the elastic structure of the NPC, and the kap-FG hydrophobic affinity, we have established a coarse-grained model of the NPC structure that mimics nucleocytoplasmic transport. To establish a foundation for future works, the methodology and biophysical rationale behind the model is explained in details. The model predicts that the first-passage time of a 15 nm cargo-complex is about 2.6±0.13 ms with an inverse Gaussian distribution for statistically adequate number of independent Brownian dynamics simulations. Moreover, the cargo-complex is primarily attached to the channel wall where it interacts with the FG-layer as it passes through the central channel. The kap-FG hydrophobic interaction is highly dynamic and fast, which ensures an efficient translocation through the NPC. Further, almost all eight hydrophobic binding spots on kap-β are occupied simultaneously during transport. Finally, as opposed to intact NPCs, cytoplasmic filaments-deficient NPCs show a high degree of permeability to inert cargos, implying the defining role of cytoplasmic filaments in the selectivity barrier

Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores

Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles

Biochemical Analysis of the Protein-Protein Interactions Involved in Karyopherin-Mediated Transport Across the Nuclear Pore Complex

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a highly symmetric ~50 MDa complex consisting of approximately 30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa such as proteins, mRNAs, and ribosomal subunits must be aided across by shuttle proteins (karyopherins, or Kaps). Kap-mediated transport involves FG-nups, a family of NPC proteins. While much has been learned about the mechanism of nucleocytoplasmic transport, many details are still unknown; perhaps among the most important missing details is the binding kinetics of almost all the transport relevant interactions, due to significant technical challenges. The aim of this work is to analyze the protein-protein interactions involved in Kap-mediated transport across the NPC, using biochemical, biophysical, and cell biological approaches. Yeast karyopherins, model cargoes, and full-length FG-nups are enriched from bacteria, and their affinities are studied quantitatively. The presence of competitor proteins and changes in bait protein distribution are seen to effect apparent affinity of these interactions. The relevance of the in vitro Kap/NLS-cargo binding measurements is confirmed with a nucleocytoplasmic import assay that allows quantitative measurements of import to be made within single living cells. Trends observed in vitro for Kap/FG-nup interactions were consistent with ex vivo observations of interactions of transport factors with Xenopus oocyte NPCs and also with in vitro measurements of transport through a synthetic NPC-based filter. This work has suggested a role for factors such as non-specific competition in determining the kinetics and selectivity of transport

Evolution of the Karyopherin-β Family of Nucleocytoplasmic Transport Factors; Ancient Origins and Continued Specialization

Macromolecular transport across the nuclear envelope (NE) is achieved through nuclear pore complexes (NPCs) and requires karyopherin-βs (KAP-βs), a family of soluble receptors, for recognition of embedded transport signals within cargo. We recently demonstrated, through proteomic analysis of trypanosomes, that NPC architecture is likely highly conserved across the Eukaryota, which in turn suggests conservation of the transport mechanisms. To determine if KAP-β diversity was similarly established early in eukaryotic evolution or if it was subsequently layered onto a conserved NPC, we chose to identify KAP-β sequences in a diverse range of eukaryotes and to investigate their evolutionary history.Thirty six predicted proteomes were scanned for candidate KAP-β family members. These resulting sequences were resolved into fifteen KAP-β subfamilies which, due to broad supergroup representation, were most likely represented in the last eukaryotic common ancestor (LECA). Candidate members of each KAP-β subfamily were found in all eukaryotic supergroups, except XPO6, which is absent from Archaeplastida. Phylogenetic reconstruction revealed the likely evolutionary relationships between these different subfamilies. Many species contain more than one representative of each KAP-β subfamily; many duplications are apparently taxon-specific but others result from duplications occurring earlier in eukaryotic history.At least fifteen KAP-β subfamilies were established early in eukaryote evolution and likely before the LECA. In addition we identified expansions at multiple stages within eukaryote evolution, including a multicellular plant-specific KAP-β, together with frequent secondary losses. Taken with evidence for early establishment of NPC architecture, these data demonstrate that multiple pathways for nucleocytoplasmic transport were established prior to the radiation of modern eukaryotes but that selective pressure continues to sculpt the KAP-β family