Yeast and vertebrate nuclear-pore complexes: evolutionary conserved, yet divergent macromolecular assemblies

Summary The nuclear-pore complex (NPC), which consists of ca. 50 proteins called nucleoporins, is a huge macromolecular structure that spans the nuclear envelope and is an obligatory passage for molecules in transit between the cytoplasm and the nucleus. In the last years, major progress has allowed the characterization of the so-called soluble phase of nucleocytoplasmic transport, that involves transport substrates, import and export receptors of which some belong to the karyopherin- family, and the small GTPase Ran and its modulators. In addition, the knowledge of the NPC architecture, the identification of its constituents, and the determination of the hierarchy of interactions within the pore should help to understand how nucleoporins are assembled, and how they give rise to a functional NPC through interactions with specific transport factors. In this review, we will focus on recent insights into the stationary phase of nucleocytoplasmic transport (i.e., the NPCs) that have been gained from exploiting the benefits of several organisms, such asXenopus laevis oocytes, mammalian cell lines, and the yeastSaccharomyces cerevisiae.Abbreviations FXFG phenylalanine X phenylalanine glycine - GLFG glycine leucine phenylalanine glycine - NPC nuclear-pore complex - RLNE rat liver nuclear envelope - WGA wheat germ agglutinin     

Transport between the cytoplasm and the nucleus

Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC.     

Caspases target only two architectural components within the core structure of the nuclear pore complex

Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.     

Molecular trafficking across the nuclear pore complex.

The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge.     

GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.     

Nucleocytoplasmic transport: cargo trafficking across the border

Transport of macromolecules between the cytoplasm and the nucleus is mediated by at least three different classes of soluble transport receptors, members of the importin-beta protein family, the Mex67/Tap family and the small nuclear transport factor 2 (NFT2). All nuclear transport factors can bidirectionally traverse the nuclear pore complex through specific interactions with phenylalanine/glycine-rich nuclear pore complex components. Recent kinetic and structural analyses revealed novel insight into the details of these interactions. In addition, new biochemical and genetic studies have dramatically improved our understanding of ribosomal and messenger RNA export, unveiling a tight coupling between RNA processing and transport.     

Multiscale dynamics in nucleocytoplasmic transport

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.     

Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusion. We report here that depletion of calcium from the lumen of the endoplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal mediated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-mediated protein import and passive diffusion are rapidly restored when the drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate that loss of calcium from the lumen of the endoplasmic reticulum and nuclear envelope reversibly affects properties of pore complex components located on the nuclear/cytoplasmic membrane surfaces, and they provide direct functional evidence for conformational flexibility of the pore complex. These methods will be useful for achieving reversible inhibition of nucleocytoplasmic trafficking for in vivo functional studies, and for studying the structure of the passive diffusion channel(s) of the pore complex.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Transport pathways of macromolecules between the nucleus and the cytoplasm.

Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.     

Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle

Macromolecular transport between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC) and is mediated by multiple families of soluble transport factors. All these transport factors share the ability to translocate across the NPC through specific interactions with components of the nuclear pore. This review highlights advances in our understanding of the structure and function of the NPC and the shuttling transport receptors involved in nuclear transport. It discusses recently proposed models for the translocation of receptor-cargo complexes through the NPC channel and reviews how the small GTPase Ran functions as a positional marker of the genome to regulate multiple important aspects of the eukaryotic cell cycle.     

Regulation of Nuclear Transport: Central Role in Development and Transformation?

Transport of macromolecules into and out of the nucleus is generally effected by targeting signals that are recognized by specific members of the importin/exportin transport receptor family. The latter mediate passage through the nuclear envelope-embedded nuclear pore complexes (NPCs) by conferring interaction with NPC constituents, as well as with other components of the nuclear transport machinery, including the guanine nucleotide-binding protein Ran. Importantly, nuclear transport is regulated at multiple levels via a diverse range of mechanisms, such as the modulation of the accessibility and affinity of target signal recognition by importins/exportins, with phosphorylation/dephosphorylation as a major mechanism. Alteration of the level of the expression of components of the nuclear transport machinery also appears to be a key determinant of transport efficiency, having central importance in development, differentiation and transformation.     

Importin alpha: a multipurpose nuclear-transport receptor

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.     

Nucleocytoplasmic transport enters the atomic age

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors that shuttle between the nucleus and cytoplasm. Our understanding of the molecular interactions underlying this process has improved dramatically as a result of the elucidation of the crystal structures of several nuclear transport factors either alone or in a complex with other components of the nuclear transport machinery. Furthermore, a conserved family of proteins, which is distinct from the well characterized family of importin beta-like nuclear export receptors, is implicated in the export of messenger RNA to the cytoplasm.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Proteins which mediate the nuclear entry of goat uterine non activated estrogen receptor (naER) following naER internalization from the plasma membrane

The nuclear transport of the internalised naER is influenced by a 58 kDa protein, p58, that appears to recognize the nuclear localization signals on the naER. At the nuclear pore complex the naER-p58 complex binds to a 62 kDa protein, p62; p58 recognizes p62 in this interaction. It is further observed that p62 gets 'docked' at a 66 kDa nuclear pore complex protein, npcp66. The nuclear entry of naER is an ATP-dependent process. An ATP-dependent biphasic nuclear entry of naER, has been observed. It is possible that the docking of p58-naER complex at the nuclear pore complex and the eventual nuclear entry of naER following its dissociation from the p58 are influenced by two different ranges in the concentration of ATP. In this process, it appears that, the nuclear entry requires an additional quantum of energy, provided by the hydrolysed ATP, in contrast to the energy requirement associated with, the nuclear 'docking' event.     

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo–receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.     

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments

The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.     

Visualization of transport-related configurations of the nuclear pore transporter.

The transport of macromolecules between the cytoplasm and nucleus of the cell is mediated by the nuclear pore complex (NPC). In this study, details of the central transporter assembly within NPCs have been examined by cryoelectron microscopy, image processing, and classification analysis. The NPC transporter in isolated amphibian nuclei appears to adopt a minimum of four transport-related configurations including: (a) a putative closed form with a 90-100 A diameter central pore, (b) a docked form with material aligned over the pore, (c) an open form with substrates apparently caught "in transit," and (d) an open form with an enlarged pore. This data confirms previous observations on NPC transporters labeled with nucleoplasmin-gold (Akey, C.W., and D.S. Goldfarb. 1989. J. Cell Biol. 109:971-982) and allows a working model of the central NPC transporter to be proposed. The model is comprised of two supramolecular irislike assemblies which open asynchronously to provide an expanded pore for translocation while maintaining transport fidelity.