scholarly journals Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

2004 ◽  
Vol 15 (9) ◽  
pp. 4261-4277 ◽  
Author(s):  
Sandra Krull ◽  
Johan Thyberg ◽  
Birgitta Björkroth ◽  
Hans-Richard Rackwitz ◽  
Volker C. Cordes
Keyword(s):  
Nuclear Pore Complex ◽  
Coiled Coil ◽  
Radial Symmetry ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Architectural Element ◽  
Genes Encoding ◽  
Complex Core ◽  
Core Elements ◽  
Amino Terminal Domain

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

scholarly journals Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p.

1996 ◽  
Vol 133 (6) ◽  
pp. 1141-1152 ◽  
Author(s):  
U Zabel ◽  
V Doye ◽  
H Tekotte ◽  
R Wepf ◽  
P Grandi ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Pore Formation ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Physical Interaction ◽  
Nonpermissive Temperature ◽  
Null Mutant ◽  
Amino Terminal ◽  
Allele Specific ◽  
Amino Terminal Domain

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.

scholarly journals Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

1996 ◽  
Vol 16 (5) ◽  
pp. 2025-2036 ◽  
Author(s):  
M A Kenna ◽  
J G Petranka ◽  
J L Reilly ◽  
L I Davis
Keyword(s):  
Nuclear Pore Complex ◽  
Complementation Group ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Localization Sequence ◽  
Complementation Groups ◽  
Amino Terminal Domain ◽  
Yeast Homolog

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.

scholarly journals Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex.

1994 ◽  
Vol 127 (6) ◽  
pp. 1515-1526 ◽  
Author(s):  
D A Byrd ◽  
D J Sweet ◽  
N Panté ◽  
K N Konstantinov ◽  
T Guan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Liver ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Coiled Coil ◽  
In Vitro Translation ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Cytoplasmic Surface

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.

scholarly journals Autoantibodies from patients with primary biliary cirrhosis preferentially react with the amino-terminal domain of nuclear pore complex glycoprotein gp210.

1995 ◽  
Vol 182 (4) ◽  
pp. 1159-1162 ◽  
Author(s):  
J Wesierska-Gadek ◽  
H Hohenauer ◽  
E Hitchman ◽  
E Penner
Keyword(s):  
Primary Biliary Cirrhosis ◽  
Nuclear Pore Complex ◽  
Cytoplasmic Tail ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Transmembrane Segment ◽  
Amino Terminal ◽  
Screening Assays ◽  
Amino Terminal Domain ◽  
Lumenal Domain

Patients with primary biliary cirrhosis frequently develop autoantibodies directed to gp210, a major glycoprotein of the nuclear pore complex. This protein contains a large glycosylated cisternal domain, a single transmembrane segment, and a short cytoplasmic tail. It has been previously shown that autoantibodies from primary biliary cirrhosis patients exclusively react with the cytoplasmic tail. We demonstrate that autoantibodies against gp210 recognize at least two different epitopes. 4 out of 12 anti-gp210 positive sera reacted with the fragment consisting of the cytoplasmic tail, and 8 sera targeted a novel epitope located within the large glycosylated lumenal domain. Moreover, our data prove that carbohydrate moieties are an essential part of this novel epitope. We propose, therefore, that future screening assays should be performed with antigens possessing both epitopes to detect all sera with anti-gp210 specificity.

scholarly journals Enzymes of the SUMO Modification Pathway Localize to Filaments of the Nuclear Pore Complex

2002 ◽  
Vol 22 (18) ◽  
pp. 6498-6508 ◽  
Author(s):  
Hong Zhang ◽  
Hisato Saitoh ◽  
Michael J. Matunis
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Studies ◽  
Sumo Modification ◽  
Amino Terminal ◽  
Trimeric Complex ◽  
Cytoplasmic Filaments ◽  
Amino Terminal Domain

ABSTRACT SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.

scholarly journals Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

2003 ◽  
Vol 14 (5) ◽  
pp. 1923-1940 ◽  
Author(s):  
Manuela E. Hase ◽  
Volker C. Cordes
Keyword(s):  
Nuclear Pore Complex ◽  
Coiled Coil ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Binding Partner ◽  
Architectural Element ◽  
Direct Binding ◽  
Late Telophase ◽  
Substitution Mutations ◽  
Two Hybrid

Tpr is a 267-kDa protein forming coiled coil-dominated homodimers that locate at the nucleoplasmic side of the nuclear pore complex (NPC). The proteins that tether Tpr to this location are unknown. Moreover, the question whether Tpr itself might act as a scaffold onto which other NPC components need to be assembled has not been answered to date. To assess Tpr's role as an architectural element of the NPC, we have studied the sequential disassembly and reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of Tpr as a result of posttranscriptional tpr gene silencing by RNA interference (RNAi). NPC assembly and recruitment of several nucleoporins, including Nup50, Nup93, Nup96, Nup98, Nup107, and Nup153, in anaphase/early telophase is shown to precede NPC association of Tpr in late telophase. In accordance, cellular depletion of Tpr by RNAi does not forestall binding of these nucleoporins to the NPC. In a search for proteins that moor Tpr to the NPC, we have combined the RNAi approach with affinity-chromatography and yeast two-hybrid interaction studies, leading to the identification of nucleoporin Nup153 as the binding partner for Tpr. The specificity of this interaction is demonstrated by its sensitivity to Tpr amino acid substitution mutations that abolish Tpr's ability to adhere to the NPC and affect the direct binding of Tpr to Nup153. Accordingly, cellular depletion of Nup153 by RNAi is shown to result in mislocalization of Tpr to the nuclear interior. Nup153 deficiency also causes mislocalization of Nup50 but has no direct effect on NPC localization of the other nucleoporins studied in this investigation. In summary, these results render Tpr a protein only peripherally attached to the NPC that does not act as an essential scaffold for other nucleoporins.

Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex

1993 ◽  
Vol 106 (1) ◽  
pp. 23-29 ◽  
Author(s):  
D.C. Chugani ◽  
L.H. Rome ◽  
N.L. Kedersha
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Radial Symmetry ◽  
Nuclear Pore ◽  
Isolated Nuclei ◽  
Tissue Sections ◽  
Ribonucleoprotein Particles ◽  
Eukaryotic Species ◽  
Central Plug

Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.

scholarly journals Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

1998 ◽  
Vol 143 (7) ◽  
pp. 1801-1812 ◽  
Author(s):  
Peter Bangs ◽  
Brian Burke ◽  
Christine Powers ◽  
Roger Craig ◽  
Aruna Purohit ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Nuclear Interior ◽  
Mammalian Cell Lines ◽  
Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

scholarly journals nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

1994 ◽  
Vol 127 (2) ◽  
pp. 319-332 ◽  
Author(s):  
A M Bogerd ◽  
J A Hoffman ◽  
D C Amberg ◽  
G R Fink ◽  
L I Davis
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Mutational Analysis ◽  
Complex Function ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Amino Terminal ◽  
Carboxy Terminal

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.

scholarly journals Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

1993 ◽  
Vol 178 (6) ◽  
pp. 2237-2242 ◽  
Author(s):  
R E Nickowitz ◽  
H J Worman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Primary Biliary Cirrhosis ◽  
Antigenic Determinants ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

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