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PubMed1994

The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm.

We have carried out partial amino acid sequence analysis of a putative nuclear pore complex protein (nucleoporin) of rat that reacts with wheat germ agglutinin and with the polyspecific monoclonal antibody 414. Surprisingly, these partial amino acid sequence data revealed a high degree of similarity with the human CAN protein, the complete cDNA-derived primary structure of which was reported by Von Lindern et al. [Von Lindern, M., Fornerod, M., van Baal, S., Jaegle, M., de Wit, T., Buijs, A. & Grosveld, G. (1992) Mol. Cell. Biol. 12, 1687-1697]. The CAN protein has been proposed to be a putative oncogene product associated with myeloid leukemogenesis. Its subcellular localization was not established. To confirm that the putative rat nucleoporin is indeed a homolog of the human CAN protein and to determine its subcellular localization, we expressed a 39-kDa internal segment of the 213,790-Da human CAN protein in Escherichia coli and raised monospecific antibodies, which reacted with the putative rat nucleoporin. Immunofluorescence microscopy of HeLa cells gave a punctate nuclear surface staining pattern characteristic of nucleoporins, and immunoelectron microscopy yielded specific decoration of the cytoplasmic side of the nuclear pore complex. This suggests that the protein is part of the short fibers that emanate from the cytoplasmic aspect of the nuclear pore complex. In agreement with previously proposed nomenclature for nucleoporins, we propose the alternative term nup214 (nucleoporin of 214 kDa) for the CAN protein.

Amino Acid Sequence↗
PubMed2004

Components of coated vesicles and nuclear pore complexes share a common molecular architecture.

Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107-160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a beta-propeller fold, an alpha-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107-160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes.

Biochemistry↗
PubMed1982

A large particle associated with the perimeter of the nuclear pore complex.

The three-dimensional structure of the nuclear pore complex has been determined to a resolution of approximately 90 A by electron microscopy using nuclear envelopes from Xenopus oocytes. It is shown to be an assembly of several discrete constituents arranged with octagonal symmetry about a central axis. There are apparent twofold axes perpendicular to the octad axis which suggest that the framework of the pore complex is constructed from two equal but oppositely facing halves. The half facing the cytoplasm is in some instances decorated by large particles, similar in appearance and size to ribosomes.

Animals↗
PubMed1998

The nuclear basket of the nuclear pore complex is part of a higher-order filamentous network that is related to chromatin.

We have achieved well-preserved nuclear basket structures in amphibian nuclear envelope spreads dried by the critical point method after tannic acid fixation, and we have compared these images with those obtained from conventional sections of amphibian oocyte nuclei. In cross sections, bundles of filaments from adjacent nuclear pore complexes were interconnected at regular distances, building up a higher-order network. Sometimes these bundles were observed to extend inward to amplified nucleoli located near the nuclear envelope. Furthermore, immunoelectron analysis indicated that DNA and histones were localized at these intranuclear filaments, suggesting a close relationship between chromatin and nuclear pore complexes. A model is proposed by which the intranuclear filaments associated with adjacent nuclear pore complexes create a regular higher-order network, which extends into the nucleus.

Animals↗
PubMed1992

Architecture and design of the nuclear pore complex.

A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how it is built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of ions and small molecules.

Animals↗
PubMed2005

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Animals↗
PubMed1999

Conformational changes of the in situ nuclear pore complex.

By bridging the double membrane separating the cell nucleus and cytoplasm, nuclear pore complexes (NPCs) are crucial pathways for the exchange of ions, proteins, and RNA between these two cellular compartments. A structure in the central lumen of the NPC, called the nuclear transport protein, central granule, or nuclear plug, appeared to gate diffusion of intermediate-sized molecules (10-40 kDa) across the nuclear membranes. Visualization of the NPC required drying and fixation of the specimen for electron and atomic force microscopy (AFM), a requirement that has raised doubts about the physiological relevance of the observation. Here we present AFM images of the outer nuclear membranes and NPCs of Xenopus laevis oocytes under more physiological conditions. Measured under a variety of Ca2+ depletion conditions, the central granule appeared to occupy and occlude the lumen of the pore in >80% of NPCs compared to <10% in controls. In a few instances images were obtained of the same NPCs as the solution was changed from control saline to store depletion conditions, and finally to store repletion conditions. We conclude that the central lumen of the nuclear pore complex undergoes a conformational change in response to depletion of nuclear cisternal Ca2+ levels.

Adenosine Triphosphate↗
PubMed1992

O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import.

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.

Animals↗
PubMed1992

Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein.

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.

Animals↗
PubMed2002

Proteomic analysis of the mammalian nuclear pore complex.

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.

Amino Acid Sequence↗
PubMed2003

Structural adaptation of the nuclear pore complex in stem cell-derived cardiomyocytes.

Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell-derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle-isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell-derived cardiomyocytes. Stem cell-derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell-derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell-derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.

Animals↗
PubMed2003

Polyproline type II conformation in the C-terminal domain of the nuclear pore complex protein gp210.

gp210 is a major constituent of the nuclear pore complex (NPC) with possible structural and regulatory roles. It interacts with components of the NPC via its C-terminal domain (CTD), which follows a transmembrane domain and a massive ( approximately 200 kDa) N-terminal region that resides in the lumen of the perinuclear space. Here, we report the solution structure of the human gp210 CTD as determined by various spectroscopic techniques. In water, the CTD adopts an extended, largely unordered conformation, which contains a significant amount of left-handed polyproline type II (PII) helical structure. The conformation of the CTD is altered by high pH, charged detergents, and the hydrogen bond-promoting reagent trifluoroethanol (TFE), which decrease the PII fraction of the fragment. TFE also induces a conformational change in a region containing an SPXX motif whose serine becomes specifically phosphorylated during mitosis. We propose that PII elements in the CTD may play a role in its interaction with the NPC and may serve as recognition sites for regulatory proteins bearing WW or other, unknown PII-binding motifs.

Amino Acid Sequence↗
PubMed2004

Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export.

Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus.

Active Transport, Cell Nucleus↗
PubMed1994

nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates. We have used mutational analysis to investigate the function of Nup1p. Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents. Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex. Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype. Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature. In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration. Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip. EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus. Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

Base Sequence↗
PubMed1993

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.

Amino Acid Sequence↗
PubMed1997

From nucleoporins to nuclear pore complexes.

One of the largest supramolecular assemblies in the eukaryotic cell, the nuclear pore complex, is now being dissected into its numerous molecular constituents. The combined use of biochemistry and genetics in yeast has made this rapid development possible. Although less is known about vertebrate nucleoporins, the first clues are now emerging about their in vivo function also. Much remains to be learned about nuclear pore complex assembly and function, however.

Amino Acid Sequence↗
PubMed2004

Nuclear pore complex structure and dynamics revealed by cryoelectron tomography.

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/transporter (CP/T) was variable in volume and could occupy different positions along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold.

Active Transport, Cell Nucleus↗
PubMed1998

Saccharomyces cerevisiae Ndc1p is a shared component of nuclear pore complexes and spindle pole bodies.

We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies (SPBs) revealed by our studies of the Saccharomyces cerevisiae NDC1 gene. Although both NPCs and SPBs are embedded in the nuclear envelope (NE) in yeast, their known functions are quite distinct. Previous work demonstrated that NDC1 function is required for proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743-751). Here, we show that Ndc1p is a membrane protein of the NE that localizes to both NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC component, Nup49p. Additionally, distinct spots of Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows that Ndc1p localizes to the regions of NPCs and SPBs that interact with the NE. The NPCs in ndc1-1 mutant cells appear to function normally at the nonpermissive temperature. Finally, we have found that a deletion of POM152, which encodes an abundant but nonessential nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show that Ndc1p is a shared component of NPCs and SPBs and propose a shared function in the assembly of these organelles into the NE.

Centrosome↗