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PubMed1997

Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments.

Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M(r) approximately 270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515-1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC-attached filaments and discuss its possible functions.

3T3 Cells↗
PubMed1996

A biochemical and immunological comparison of nuclear and cytoplasmic pore complexes.

Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.

Animals↗
PubMed1985

Gene gating: a hypothesis.

It is assumed that the genome of a higher eukaryotic organism is organized into a number of distinct three-dimensional (3-D) structures, each characteristic for a given differentiated state. These discrete 3-D structures are envisioned to develop in a hierarchical and largely irreversible manner from an omnipotent 3-D structure of the zygotic genome. The information for these processes is assumed to reside in the genome. The nuclear pore complexes, the peripheral nuclear lamina, and components of the nuclear core are proposed to be among the topologically most proximal organelles that interpret this information and thereby serve in the maintenance and the alteration of the 3-D structure of the genome during development, differentiation, and the cell cycle. The nuclear pore complexes are envisioned to serve as gene-gating organelles capable on interacting specifically with expanded (transcribable) portions of the genome. Their nonrandom distribution on the nuclear surface would reflect the underlying periodic organization of the genome into expanded and compacted domains, alternating with each other. All transcripts of a given gated gene would leave the nucleus by way of that pore complex to which the gene is gated. Implications for cell asymmetry and polarity are discussed and evolutionary considerations are presented.

Animals↗
PubMed2006

Introduction to nucleocytoplasmic transport: molecules and mechanisms.

Nucleocytoplasmic transport, the exchange of matter between nucleus and cytoplasm, plays a fundamental role in human and other eukaryotic cells, affecting almost every aspect of health and disease. The only gate for the transport of small and large molecules as well as supramolecular complexes between nucleus and cytoplasm is the nuclear pore complex (NPC). The NPC is not a normal membrane transport protein (transporter). Composed of 500 to 1000 peptide chains, the NPC features a mysterious functional duality. For most molecules, it constitutes a molecular sieve with a blurred cutoff at approx 10 nm, but for molecules binding to phenylalanine-glycine (FG) motifs, the NPC appears to be a channel of approx 50 nm diameter, permitting bidirectional translocation at high speed. To achieve this, the NPC cooperates with soluble factors, the nuclear transport receptors, which shuttle between nuclear contents and cytoplasm. Here, we provide a short introduction to nucleocytoplasmic transport by describing first the structure and composition of the nuclear pore complex. Then, mechanisms of nucleocytoplasmic transport are discussed. Finally, the still essentially unresolved mechanisms by which nuclear transport receptors and transport complexes are translocated through the nuclear pore complex are considered, and a novel translocation model is suggested.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

Calcium regulation of nuclear pore permeability.

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Animals↗
PubMed2002

Accelerating the rate of disassembly of karyopherin.cargo complexes.

Transport of macromolecules across the nuclear pore complex (NPC) occurs in seconds and involves assembly of a karyopherin.cargo complex and docking to the NPC, translocation of the complex across the NPC via interaction with nucleoporins (Nups), and dissociation of the complex in the nucleoplasm. To identify rate-limiting steps in the Kap95p.Kap60p-mediated nuclear import pathway of Saccharomyces cerevisiae, we reconstituted key intermediate complexes and measured their rates of dissociation and affinities of interaction. We found that a nuclear localization signal-containing protein (NLS-cargo) dissociates slowly from Kap60p monomers and Kap60p.Kap95p heterodimers with half-lives (t(12)) of 7 and 73 min, respectively; that Kap60p and Kap60p.NLS-cargo complexes dissociate slowly from Kap95p (t(12) = 36 and 73 min, respectively); and that Kap95p.Kap60p.NLS-cargo complexes and Kap95p.Kap60p heterodimers dissociate rapidly from the nucleoporin Nup1p (t(12) < or = 21 s) and other Nups. A search for factors that accelerate disassembly of the long-lived intermediates revealed that Nup1p and Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p.Kap95p heterodimers; that Gsp1p-GTP accelerates > or = 447-fold the rate of dissociation of Kap60p.NLS-cargo from Kap95p; and that Nup2p and the Cse1p.Gsp1p-GTP complex independently accelerate > or = 22- and > or = 39-fold the rate of dissociation of NLS-cargo from Kap60p. We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p.Kap60p.NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding partners. In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as karyopherin release factors (or KaRFs) in the nuclear basket structure of the S. cerevisiae NPC.

Active Transport, Cell Nucleus↗
PubMed1994

[Nuclear reconstitution around purified HeLa chromosomes in a cell-free system from Xenopus eggs].

Activated Xenopus egg extracts are capable of reconstituting nucleus around the added DNA or chromatin. To understand better the nuclear reconstitution process and the structure of the assembled nucleus, we have developed a cell-free system using activated Xenopus egg extracts and metaphase chromosomes of HeLa cells. We demonstrate that the extracts are efficient promoting the decondensation of the metaphase chromosomes and the nuclear reconstitution around the chromosomes, and that the nuclear reconstitution is a precise process. The reassembled nucleus possesses a typical nuclear envelope, pore complexes, nuclear lamina, nuclear matrix, chromatin, etc., as shown by electron microscopy, embedment and embedment-free electron microscopy, and immunofluorescence method. We also found that the annulate lamellae directly take part in nuclear envelope and pore complex reconstitution by fusing with the double membrane, having reassembled on the surface of chromatin/chromosomes.

Animals↗
PubMed2002

The path that RNA takes from the nucleus to the cytoplasm: a trip with some surprises.

It is now clear that nuclear context is playing an essential role in gene expression. For this reason we have developed methods to study gene expression in situ. Transcription takes place in discrete foci in the nucleoplasm of mammalian cells. These sites concentrate several RNA polymerases II and factors involved in the production of mRNA. Moreover, these sites also contain the active machinery to carry out protein synthesis. Once the mRNA leaves the transcription site, it interacts with the nuclear pore complex and the mRNA is exported using the filaments of the nuclear pore complex in to the middle part of the nuclear pore complex and finally is released in the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2001

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.

Active Transport, Cell Nucleus↗
PubMed1992

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.

Biological Evolution↗
PubMed1996

An RNA-export mediator with an essential nuclear export signal.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) mediates the translocation of viral messenger RNAs from the nucleus to the cytoplasm. In yeast, Rev can mediate the nuclear export of Rev response-element-containing RNAs. The export of Rev itself proceeds through the nuclear pore complex and requires a nuclear export signal (NES) and interaction with a cellular cofactor, the protein Rip1. Endogenous RNA export mediators that interact with Rip1 and harbour NESs are thought to exist but have yet to be identified. Here we report the characterization of a new and essential yeast protein, Gle1, which contains an NES and has a relative molecular mass of 62,000. Mutation of the NES in Gle1 prevents export of polyadenylated RNA from the nucleus. Gle1 interacts with Rip1 and the nucleoporin Nup100 and is localized predominantly at nuclear pore complexes. These properties indicate that Gle1 is an RNA-export factor and that Rev may mediate viral RNA export by mimicking the function of Gle1.

Amino Acid Sequence↗
PubMed2002

ERK2 enters the nucleus by a carrier-independent mechanism.

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.

Active Transport, Cell Nucleus↗
PubMed2000

Structure and assembly of the Nup84p complex.

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.

Alleles↗
PubMed1998

Active nuclear import of single-stranded oligonucleotides and their complexes with non-karyophilic macromolecules.

The objective of this investigation was to characterize intranuclear accumulation of oligonucleotides and their adducts with non-karyophilic compounds in cultured animal cells and thus to present a model system for nucleic acid-mediated nuclear import. In digitonin-permeabilized cells, nuclear uptake of 3'-FITC-labeled, single-stranded 25-mer oligodeoxyribonucleotides was independent of added cytosolic protein, largely energy-dependent, inhibitable by wheat germ agglutinin but not by N-ethylmaleimide, and a function of their base composition. When coupled to FITC-labeled streptavidin or streptavidin-bovine serum albumin conjugates, the oligonucleotides delivered the proteins to the nuclear interior with rates roughly proportional to their karyophilicity as free molecules. Transport activity was also demonstrated for single-stranded oligoribonucleotides. The transport was energy-dependent, inhibited by GMP-PNP and wheat germ agglutinin, but unaffected by N-ethylmaleimide. Nuclear import of oligo(dG)25/protein adducts needed 3 to 4 oligonucleotide signals per complex and the signal had to be at least 15 nucleotides long. Micro-injection experiments showed that the results obtained with digitonin-permeabilized cells are not artifacts of a quasi-intact cellular system. These data were confirmed by electron microscopy employing complexes of oligodeoxyribonucleotides with streptavidin-peroxidase-bovine serum albumin-1 nm gold. In permeabilized cells, the complexes docked to the cytoplasmic face of the nuclear pore complexes, were translocated through the central pore channel and accumulated in large quantities in the nuclear baskets before they were released into the nucleoplasm. These results demonstrate that nuclear uptake of oligonucleotides and their complexes is an active process mediated by nuclear pore complexes, which, at least regarding its cytoplasmic component, is different from the pathway requiring classical nuclear localization signals.

Animals↗
PubMed2005

Mechanisms of receptor-mediated nuclear import and nuclear export.

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.

Active Transport, Cell Nucleus↗
PubMed1996

Calcium transport pathways in the nucleus.

Due to the availability of new biophysical and biochemical techniques, there has recently been considerable progress in our understanding of Ca2+ transport inside, as well as into and out of, the nucleus. A number of Ca2+ transport pathways have been localized specifically in the outer or inner nuclear membrane and the Ca2+ permeability through the nuclear pore complex has been assessed. The nuclear envelope has characteristics similar to those of a leaky epithelium. The leak is through the nuclear pore complex. The outer nuclear membrane contains the Ca2+ ATPase whereas the functionally important inositol trisphosphate (IP3)-activated Ca2+ release channels are specifically localized in the inner nuclear membrane.

Animals↗
PubMed2002

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.

Active Transport, Cell Nucleus↗
PubMed2006

Nuclear pores form de novo from both sides of the nuclear envelope.

Nuclear pore complexes are multiprotein channels that span the double lipid bilayer of the nuclear envelope. How new pores are inserted into the intact nuclear envelope of proliferating and differentiating eukaryotic cells is unknown. We found that the Nup107-160 complex was incorporated into assembly sites in the nuclear envelope from both the nucleoplasmic and the cytoplasmic sides. Nuclear pore insertion required the generation of Ran guanosine triphosphate in the nuclear and cytoplasmic compartments. Newly formed nuclear pore complexes did not contain structural components of preexisting pores, suggesting that they can form de novo.

Active Transport, Cell Nucleus↗