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PubMed1995

The nuclear pore complex.

The nuclear pore complex (NPC) creates an aqueous channel across the nuclear envelope through which macromolecular transport between nucleus and cytoplasm occurs. Nucleocytoplasmic traffic is bidirectional and involves diverse substrates, including protein and RNA. It is unclear whether import and export are mechanistically similar, but evidence suggests that numerous pathways may be involved. The discovery of filaments that extend out from each side of the NPC suggests that the NPC may also have a structural role, perhaps providing a connection between cytoskeletal elements of the nucleus and cytoplasm. If this suggestion is valid, it remains to be determined whether this aspect of NPC function is related to its role in nuclear transport. This review discusses recent developments regarding the structure of the NPC, characterization of its constituent proteins (nucleoporins), the mechanism by which transport occurs, the function of individual nucleoporins, and the pathway of NPC assembly and disassembly.

Amino Acid Sequence↗
PubMed1999

Getting across the nuclear pore complex.

The nuclear pore complex (NPC) connects the cytoplasm and nucleus through the nuclear envelope and serves as the pipeline for moving material between the two compartments. Macromolecules that move through the NPC range in size from the very small (for example, ions and ATP) to the very large (for example, ribonucleoprotein particle complexes). Unlike translocation across other organelle membranes, proteins do not have to be unfolded to be transported through the NPC, and the NPC also routinely transports large, multicomponent substrates in both directions. This review focuses on current understanding of the different mechanisms by which macromolecules move across the NPC.

Animals↗
PubMed1997

High-resolution field-emission scanning electron microscopy of nuclear pore complex.

The nuclear pore complex (NPC) is a large macromolecular assembly inserted into the nuclear envelope (NE). It controls the traffic of proteins, RNA, and RNA proteins between nucleus and cytoplasm. It chemical composition and function are now intensively investigated in many organisms. To understand this unique membrane transport system, we must know the supramolecular organization of the NPC. In recent years, high-resolution field-emission scanning electron microscopy has made important contributions to our knowledge of NPC structure. It provided the first images of the complex and beautiful fish trap-like structure of its intranuclear surface, documented in this review. It also has provided the first images of a new intranuclear structure, a system of branching hollow cables connecting the nuclear interior with the NPCs at the nuclear surface. Most likely this is an intranuclear transport system, assuring efficient exchange between the nuclear interior and the NE, especially in large nuclei.

Animals↗
PubMed1995

Structural plasticity of the nuclear pore complex.

The nuclear pore complex (NPC) is strategically located at continuous junctions of the inner and outer nuclear membranes to catalyze macromolecular transport, without impending the diffusion of small molecules. In this paper, the structural plasticity of 4412 NPCs in isolated nuclear envelopes has been evaluated, utilizing correspondence analysis, classification and difference mapping. The data are grouped into seven clusters comprising two major groups, based on the degree of radial compaction within spokes and the symmetry of the inner spoke ring. The results have been correlated with differences in spoke domain packing observed in two published three-dimensional maps suggesting that symmetrical detergent-extracted NPCs are similar, but not identical to the most probable in vivo structure. A model is proposed in which spoke architecture is responsive to changes in the turgor pressure of the nuclear envelope. For example, detergent extraction may allow the outward facing domains of each spoke to adopt a radially-extended configuration while osmotic swelling may induce an inwards displacement, resulting in a radially compact spoke. Difference maps between approximately 822 symmetric projections of NPCs in membranes and after detergent-extraction have localized the nuclear envelope border. The data place limits on the radial and circumferential dimensions of diffusion channels (approximately 0 to 20 A x 190 A), proposed to reside at the pore periphery. The results confirm the observation that each spoke penetrates the nuclear envelope, linking up with the radial arms to form a "lumenal ring". Finally, putative closed, open and in-transit forms of the transporter are found with the same relative frequency in membrane-associated NPCs with radially compact or extended spokes; hence spoke deformations in isolated envelopes may be induced by experimental factors. However, concerted movements of the spoke domains (if reversible) may be utilized in the biological function of the NPC and some examples are given.

Analysis of Variance↗
PubMed1991

Toward a more complete 3-D structure of the nuclear pore complex.

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.

Actins↗
PubMed1996

Molecular dissection of the nuclear pore complex.

The nuclear pore complex (NPC) is an approximately 120 megadalton (MDa) supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that mediates bidirectional molecular trafficking between the cytoplasm and the nucleus of interphase eukaryotic cells. The structure of the NPC has been studied extensively by electron microscopy (EM), and a consensus model of its basic framework has emerged. Over the past few years, there has been significant progress in dissecting the molecular constituents of the NPC and in identifying distinct NPC subcomplexes. The combination of well-characterized antibodies with different EM specimen preparation methods has allowed localization of several of these proteins within the three-dimensional (3-D) architecture of the NPC. Thus, the molecular dissection of the NPC is definitely on its way to being elucidated. Here, we review these findings and discuss the emerging structural concepts.

Amino Acid Sequence↗
PubMed2001

Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import.

The trafficking of macromolecules between cytoplasm and nucleus through nuclear pore complexes is mediated by specific carrier molecules such as members of the importin-beta family. Nuclear pore proteins (nucleoporins) frequently contain sequence repeats based on FG cores and carriers appear to move their cargo through the pores by hopping between successive FG cores. A major question is why some macromolecules are transported while others are not. This selectivity may be generated by the ability to bind FG repeats, a local concentration of carrier-cargo complexes near the entrance to the pore channel, and steric hindrance produced by high concentrations of nucleoporins in the channel.

Active Transport, Cell Nucleus↗
PubMed1993

Intranuclear filaments containing a nuclear pore complex protein.

Nuclear pore complexes (NPCs) are anchoring sites of intranuclear filaments of 3-6 nm diameter that are coaxially arranged on the perimeter of a cylinder and project into the nuclear interior for lengths varying in different kinds of cells. Using a specific monoclonal antibody we have found that a polypeptide of approximately 190 kD on SDS-PAGE, which appears to be identical to the recently described NPC protein "nup 153," is a general constituent of these intranuclear NPC-attached filaments in different types of cells from diverse species, including amphibian oocytes where these filaments are abundant and can be relatively long. We have further observed that during mitosis this filament protein transiently disassembles, resulting in a distinct soluble molecular entity of approximately 12.5 S, and then disperses over most of the cytoplasm. Similarly, the amphibian oocyte protein appears in a soluble form of approximately 16 S during meiotic metaphase and can be immunoprecipitated from egg cytoplasmic supernatants. We conclude that this NPC protein can assemble into a filamentous form at considerable distance from the nuclear envelope and discuss possible functions of these NPC-attached filaments, from a role as guidance structure involved in nucleocytoplasmic transport to a form of excess storage of NPC proteins in oocytes.

Animals↗
PubMed1999

Function and assembly of nuclear pore complex proteins.

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. The current view of NPC organization features a massive symmetrical framework that is embedded in the double membranes of the nuclear envelope. It embraces a central channel of as yet ill-defined structure but which may accommodate particles with diameters up to 26 nm provided that they bear specific import/export signals. Attached to both faces of the central framework are peripheral structures, short cytoplasmic filaments, and a nuclear basket assembly, which interact with molecules transiting the NPC. The mechanisms of assembly and the nature of NPC structural intermediates are still poorly understood. However, mutagenesis and expression studies have revealed discrete sequences within certain NPC proteins that are necessary and sufficient for their appropriate targeting. In addition, some details are emerging from observations on cells undergoing mitosis where the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized to form nuclear envelopes in the two daughter cells. To date, it has been possible to define a time course of postmitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral inner nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a dynamic component of the nuclear basket, associates with chromatin towards the end of anaphase coincident with, although independent of, the inner nuclear membrane protein, LAP2. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, p54, p45) during mitosis, and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates and which may therefore represent an essential component of the central framework of the NPC.

Animals↗
PubMed1998

Transport routes through the nuclear pore complex.

The nuclear pore complex can be considered to be the stationary phase of bidirectional traffic between the nucleus and the cytoplasm. The mobile phase consists of karyopherins, transport substrates, and the small GTPase Ran and its modulators. Recently, the family of karyopherins was expanded with the recognition of numerous open reading frames with limited homology to karyopherin beta 1. In several cases, the specific substrates transported by the new karyopherins have been identified, allowing the characterization of new pathways into and out of the nucleus. However, the mechanisms of transport, particularly the role of Ran, remain poorly understood.

Animals↗
PubMed1994

Pores for thought: nuclear pore complex proteins.

Nuclear pore complexes (NPCs) are enormous macromolecular structures that mediate the active exchange of proteins and RNPs between the nucleus and cytoplasm. Recent work has resulted in a windfall of identified NPC polypeptides, many with unique sequences. Several of the proteins have been shown to be part of extended cytoplasmic and nucleoplasmic NPC filaments. Biochemical, structural and genetic studies on NPC proteins are just beginning to allow an understanding of how they associate into a functional organelle.

Journal Article↗
PubMed1997

The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.

BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts. Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae. The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function. Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import. Crm1p has recently been shown to contribute to nuclear export in vertebrate systems. Here, we have studied this mechanism of nuclear to cytoplasmic transport. RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S. cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism. Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p. CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

Biological Transport↗
PubMed2006

Isolation and fractionation of rat liver nuclear envelopes and nuclear pore complexes.

The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins.

Animals↗
PubMed1996

Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins.

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.

Amino Acid Sequence↗
PubMed1981

Temperature-induced changes in nuclear pore complex frequencies, nuclear envelope surface areas, and nuclear volumes in light-synchronized Euglena.

An autotrophic culture of Euglena, synchronized using a day:night (D:N), 14:10-h cycle, was subjected to a 21.5 leads to 31.5 degrees C temperature shift and then to a reversed shift in temperature after three D:N cycles at 31.5 degrees C. Nuclear pore complex (NPC) number per square micrometre and nuclear surface area and volume determinations were made on G1 cells at various intervals. Cells sampled immediately prior to the 21.5 leads to 31.5 degrees C shift had a mean value of 37.68 NPC . micron-2 nuclear envelope surface area, 30.40 NPCs/micron2 after three D:N cycles at 31.5 degrees C and 39.98 NPCs/micron2 after three D:N cycles at the resumed culture temperature of 21.5 degrees. Thus temperature changes affect NPC numbers per square micrometre and these changes are reversible. Mean nuclear surface area was 125.76 micron2 immediately prior to the 21.5 leads to 31.5 degrees C shift, and decreased over two D:N cycles at 31.5 degrees C to 101.30 micron2 by the end of the third D:N cycle. Nuclear envelope surface area, one and two D:N cycles after the 31.5 leads to 21.5 degrees C shift, was approximately equal that prior to the 21.5 leads to 31.5 degrees C shift. After the third D:N cycle, however, nuclear surface area had increased to 173.05 micron2. The changes in nuclear surface area resulted in large differences in the estimates of the total number of NPCs per nucleus. Euglena immediately prior to the 21.5 leads to 31.5 degrees C temperature shift had 4739 NPCs/nucleus; immediately prior to the 31.5 leads to 21.5 degrees C shift had 3079 NPCs/nucleus; and had 6919 NPCs/nucleus at 21.5 degrees C and three D:N cycles after the 31.5 leads to 21.5 degrees C shift. Estimates of the number of NPCs per cubic micrometre of nuclear volume were almost identical between these samples.

Animals↗
PubMed1993

Nuclear pore complex antigens delineate nuclear envelope dynamics in vegetative and conjugating Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the nucleus undergoes dramatic shape changes during mitosis and mating. We have studied nuclear envelope dynamics during the processes of mitosis and conjugation using nuclear pore complexes as a marker for the nuclear envelope in wild-type cells and several cell-division-cycle (cdc) mutants. Three monoclonal antibodies are described that recognize nuclear pore complex-related antigens in S. cerevisiae. One of these antibodies, RL1, has been extensively characterized by Gerace and colleagues and recognizes nuclear pore complexes in mammalian and amphibian cells. By indirect immunofluorescence of yeast cells, all three antibodies yield a discontinuous nuclear rim stain. All three react with multiple nuclear-enriched proteins in immunoblots, including the nucleoporin protein encoded by the NSP1 gene. When the antibodies were used in immunofluorescence experiments on mating cells, the nuclear pore complex staining pattern proved to be a sensitive indicator of nuclear fusion. Nuclei with closely apposed spindle pole bodies and unfused nuclear envelopes could be readily distinguished. Marked shape changes were observed in nuclei during fusion and segregation of the diploid nucleus into the zygotic bud. In cdc14 and cdc15 mutants that arrest late in mitosis, the elongated nuclear envelope extension that stretches between daughter nuclei during telophase was preserved. In cytokinesis-defective mutants (cdc3, cdc10, cdc11 and cdc12), the elongated nuclear envelope was usually resolved into two daughter nuclei in the absence of cytokinesis. These results indicate that nuclear envelope division is mechanically distinguishable from chromosome segregation, nucleolar segregation and cytokinesis.

Amino Acid Sequence↗
PubMed1998

Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.

Cell Line↗
PubMed2002

Isolation and characterization of new Saccharomyces cerevisiae mutants perturbed in nuclear pore complex assembly.

BACKGROUND: Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature. RESULTS: A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects. CONCLUSION: The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly.

Genetic Complementation Test↗