The yeast nuclear pore complex: composition, architecture, and transport mechanism

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.     

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.     

Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors

Nuclear pore complexes (NPCs) are highly selective gates that mediate the exchange of all proteins and nucleic acids between the cytoplasm and the nucleus. Their selectivity relies on a supramolecular assembly of natively unfolded nucleoporin domains containing phenylalanine–glycine (FG)‐rich repeats (FG repeat domains), in a way that is at present poorly understood. We have developed ultrathin FG domain films that reproduce the mode of attachment and the density of FG repeats in NPCs, and that exhibit a thickness that corresponds to the nanoscopic dimensions of the native permeability barrier. By using a combination of biophysical characterization techniques, we quantified the binding of nuclear transport receptors (NTRs) to such FG domain films and analysed how this binding affects the swelling behaviour and mechanical properties of the films. The results extend our understanding of the interaction of FG domain assemblies with NTRs and contribute important information to refine the model of transport across the permeability barrier.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.     

Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.     

蛋白质相互作用调控蛋白质核转运及其功能

蛋白质的核转运是真核生物细胞内发生的重要过程之一,是一大群蛋白质发挥其功能的前提,与细胞正常功能的维持密切相关。蛋白质的核运输通常采用核受体介导的方式进行。此过程非常复杂,需要多种蛋白质的参与,涉及到大量的蛋白质相互作用。本文将综合近年来本领域取得的进展,就蛋白质相互作用参与蛋白质核转运来调节蛋白质的亚细胞定位,进一步在多方面影响细胞以及生物体生理功能的变化进行阐述。     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

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.     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Cytosolic import factor- and Ran-independent nuclear transport of ribosomal protein L5

Ribosomal protein L5 is a shuttling protein that, in Xenopus oocytes, is involved in the nucleocytoplasmic transport of 5S rRNA. As demonstrated earlier, L5 contains three independent nuclear import signals (NLSs), which function in oocytes as well as in somatic cells. Upon physical separation, these NLSs differ in respect to their capacity to bind to nuclear import factors in vitro and to mediate the nuclear import of a heterologous RNP in vivo. As reported in this communication, analysis of the in vitro nuclear import activity of these three NLSs reveals that they also differ in respect to their requirements for cytosolic import factors and Ran. Nuclear import mediated by the N-terminal and the central NLS depends on cytosolic import factor(s) and Ran, whereas import via the C-terminal NLS occurs independently from these factors. Thus, the presence of multiple NLSs in ribosomal protein L5 appears to allow for efficient nuclear transport via utilisation of multiple, mechanistically different import pathways.     

Characterisation of nuclear pore complex oxalate binding protein from human kidney

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4-dithiocyanostilbene-2,2-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton–high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl–Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 M oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.     

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.     

Protein Tpr is required for establishing nuclear pore‐associated zones of heterochromatin exclusion

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel‐ and cone‐like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC‐associated protein Tpr and its large coiled coil‐forming domain. RNAi‐mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub‐compartment and delimiting heterochromatin distribution.     

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.