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N Panté

Publications and source records attributed to N Panté.

At least 19 recordsLinked to original sources
PubMed1999

Nup192p is a conserved nucleoporin with a preferential location at the inner site of the nuclear membrane.

Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown. We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin. Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p. Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p. NUP192 is essential for cell growth. A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature. By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 +/- 15 nm from the central plane of the pore. This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane.

Amino Acid Sequence↗
PubMed1999

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

Adenosine Triphosphatases↗
PubMed1998

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

Animals↗
PubMed1998

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.

Escherichia coli↗
PubMed1997

Visualizing nuclear export of different classes of RNA by electron microscopy.

Export of RNA from the cell nucleus to the cytoplasm occurs through nuclear pore complexes (NPCs). To examine nuclear export of RNA, we have gold-labeled different types of RNA (i.e., mRNA, tRNA, U snRNAs), and followed their export by electron microscopy (EM) after their microinjection into Xenopus oocyte nuclei. By changing the polarity of the negatively charged colloidal gold, complexes with mRNA, tRNA, and U1 snRNA can be formed efficiently, and gold-tagged RNAs are exported to the cytoplasm with kinetics and specific saturation behavior similar to that of unlabeled RNAs. U6 snRNA conjugates, in contrast, remain in the nucleus, as does naked U6 snRNA. During export, RNA-gold was found distributed along the central axis of the NPC, within the nuclear basket, or accumulated at the nuclear and cytoplasmic periphery of the central gated channel, but not associated with the cytoplasmic fibrils. In an attempt to identify the initial NPC docking site(s) for RNA, we have explored various conditions that either yield docking of import ligands to the NPC or inhibit the export of nuclear RNAs. Surprisingly, we failed to observe docking of RNA destined for export at the nuclear periphery of the NPC under any of these conditions. Instead, each condition in which export of any of the RNA-gold conjugates was inhibited caused accumulation of gold particles scattered uniformly throughout the nucleoplasm. These results point to the existence of steps in export involving mobilization of the export substrate from the nucleoplasm to the NPC.

Animals↗
PubMed1996

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.

Animals↗
PubMed1996

Sequential binding of import ligands to distinct nucleopore regions during their nuclear import.

Protein import into nuclei is mediated by the nuclear pore complex (NPC) and by cellular factors. To structurally characterize this process, nuclear import of gold-labeled nucleoplasmin was followed by electron microscopy to identify NPC components interacting with the import ligand complex in vivo. Before translocation into the nucleus, nucleoplasmin sequentially bound to two distinct regions: first to the distal part of the cytoplasmic filaments and then at the cytoplasmic entry to the central gated channel. Evidence that the delivery of the import ligand from the first to the second binding region occurred by bending of the cytoplasmic filaments is presented here.

Animals↗
PubMed1996

Toward the molecular dissection of protein import into nuclei.

Transport of proteins, RNAs and ribonucleoprotein particles into and out of the nucleus is essential for many cellular functions to proceed. Recent progress in this area of research has led to the identification of a number of signals and cytosolic factors that mediate the nuclear import of proteins through the nuclear pore complexes. However, as the sites on the nuclear pore complex at which these signals and factors exert their function are still largely unidentified, the molecular mechanisms underlying this nuclear import pathway remain to be elucidated.

Animals↗
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↗
PubMed1995

A giant nucleopore protein that binds Ran/TC4.

Ran/TC4 is a small nuclear G protein that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref. 2). Loss of RCC1 causes defects in cell cycle progression, RNA export and nuclear protein import. Some of these can be suppressed by overexpression of Ran/TC4 (ref. 1), suggesting that Ran/TC4 functions downstream of RCC1. We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues. This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBP1-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin. The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins, and immunolocalization suggests that RanBP2 is a constituent of the NPC. The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC.

Amino Acid Sequence↗
PubMed1995

Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope.

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane, which binds directly to both lamin B1 and chromosomes in a mitotic phosphorylation-regulated manner. The biochemical and physiological properties of LAP2 suggest an important role in nuclear envelope re-assembly at the end of mitosis and/or anchoring of the nuclear lamina and interphase chromosomes to the nuclear envelope. We describe the cDNA cloning of LAP2 and characterization of its membrane topology and targeting to the nuclear envelope. The LAP2 cDNA sequence predicts a protein of 452 amino acids, containing a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane-spanning sequence at residues 410-433. Immunogold localization of an LAP2 epitope in isolated nuclear envelopes indicates that the large amino-terminal hydrophilic domain (residues 1-409) is exposed to the nucleoplasm. By expressing deletion mutants of LAP2 in cultured cells, we have identified multiple regions in its nucleoplasmic domain that promote localization at the nuclear envelope. These data suggest that targeting of LAP2 to the nuclear envelope is mediated by cooperative interactions with multiple binding sites at the inner nuclear membrane.

Amino Acid Sequence↗
PubMed1995

Nuclear pore complex proteins.

The nuclear envelope forms the boundary between the nucleus and the cytoplasm and as such regulates the exchange of macromolecules between the two compartments. The channels through the nuclear envelop that actually mediate this macromolecular traffic are the nuclear pore complexes. These are extremely elaborate structures which in vertebrate cells exhibit a mass of approximately 120 MDa. They are thought to be composed of as many as 100 distinct polypeptide subunits. A major challenge in the field of nucleocytoplasmic transport is to identify these subunits and to determine their functions and interactions in the context of the three-dimensional structure of the nuclear pore complex. It is the aim of this review to summarize what is currently known of the 20 or so nuclear pore complex proteins that have been described in either vertebrate or yeast cells.

Amino Acid Sequence↗
PubMed1995

Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.

Animals↗
PubMed1995

Exploring nuclear pore complex structure and function in molecular detail.

Bidirectional molecular trafficking between the nucleus and the cytoplasm of eukaryotic cells occurs through the nuclear pore complexes (NPCs), approximately 120 megadalton supramolecular assemblies embedded in the double-membraned nuclear envelope. Significant progress has been made in elucidating the three-dimensional (3-D) architecture of the NPC, and in identifying, characterizing, and cloning and sequencing NPC proteins. Several of these have now been localized within the 3-D structure of the NPC. Nevertheless, there still remain major questions relating to the conformation, molecular composition and functional roles of distinct NPC components. Here we review recent structural studies from our group and others which have contributed toward dissecting the molecular architecture of the NPC. We also present our results on the molecular characterization of some NPC components, and on the elucidation of their functional roles in mediated nucleocytoplasmic transport.

Animals↗
PubMed1994

Interactions and three-dimensional localization of a group of nuclear pore complex proteins.

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N-acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold-conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.

Amino Acid Sequence↗
PubMed1994

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.

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.

Amino Acid Sequence↗