scholarly journals GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

1995 ◽  
Vol 131 (3) ◽  
pp. 571-581 ◽  
Author(s):  
F Melchior ◽  
T Guan ◽  
N Yokoyama ◽  
T Nishimoto ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Binding Studies ◽  
Early Step ◽  
Permeabilized Cells ◽  
Complex Protein ◽  
Ran Gtp

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

scholarly journals Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

2000 ◽  
Vol 11 (2) ◽  
pp. 703-719 ◽  
Author(s):  
Susanne M. Steggerda ◽  
Ben E. Black ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

scholarly journals Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.

1995 ◽  
Vol 129 (4) ◽  
pp. 925-937 ◽  
Author(s):  
B M Paschal ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Multicomponent System ◽  
Nuclear Pore ◽  
Transport Activity ◽  
Complex Protein ◽  
Multistep Process ◽  
Cytosolic Factor ◽  
Cytosolic Factors

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.

scholarly journals Targeting and function in mRNA export of nuclear pore complex protein Nup153.

1996 ◽  
Vol 134 (5) ◽  
pp. 1141-1156 ◽  
Author(s):  
R Bastos ◽  
A Lin ◽  
M Enarson ◽  
B Burke
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Terminal Domain ◽  
General Decline ◽  
Complex Protein ◽  
Pore Complexes ◽  
And Function

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

scholarly journals The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

2002 ◽  
Vol 158 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Tobias C. Walther ◽  
Helen S. Pickersgill ◽  
Volker C. Cordes ◽  
Martin W. Goldberg ◽  
Terry D. Allen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Slight Reduction ◽  
Cytoplasmic Filaments ◽  
Localization Sequence ◽  
Importin Α

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

Carrier-Independent Nuclear Import of the Transcription Factor PU.1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3561-3561
Author(s):  
Nabeel R. Yaseen ◽  
Akiko Takeda ◽  
Reza Nazari ◽  
Helen Shio ◽  
Gunter Blobel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Ultrastructural Studies ◽  
Ets Family ◽  
Ets Domain ◽  
Necessary And Sufficient

Abstract PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using GFP-PU.1 fusions, we show that the Ets DNA-binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted with the FG repeats of nucleoporins Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, dramatically increased in the presence of RanGMPPNP, indicating the formation of a PU.1/RanGTP/Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Since Nup62 is located close to the midplane of the nuclear pore complex (NPC) while Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 towards the nuclear side of the NPC by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

scholarly journals Nup2 performs diverse interphase functions inAspergillus nidulans

2018 ◽  
Vol 29 (26) ◽  
pp. 3144-3154 ◽  
Author(s):  
Subbulakshmi Suresh ◽  
Sarine Markossian ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Proteins ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Nuclear Movement ◽  
Multifunctional Protein ◽  
Long Period ◽  
Complex Protein ◽  
Mitotic Chromatin

The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket–associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.

scholarly journals Protein import through the nuclear pore complex is a multistep process.

1989 ◽  
Vol 109 (3) ◽  
pp. 971-982 ◽  
Author(s):  
C W Akey ◽  
D S Goldfarb
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Binding Sites ◽  
Protein Import ◽  
Nuclear Pore ◽  
Cryo Electron Microscopy ◽  
Transport Inhibitor ◽  
Multistep Process ◽  
Subsequent Step ◽  
Transport Of Macromolecules

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.

scholarly journals Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p.

1994 ◽  
Vol 126 (3) ◽  
pp. 619-630 ◽  
Author(s):  
K D Belanger ◽  
M A Kenna ◽  
S Wei ◽  
L I Davis
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Sequence Similarity ◽  
Nuclear Architecture ◽  
Nuclear Pore ◽  
Beta Catenin ◽  
Complex Protein ◽  
Oligomeric Complex ◽  
Physical Interactions

Nup1p is a yeast nuclear pore complex protein (nucleoporin) required for nuclear protein import, mRNA export and maintenance of normal nuclear architecture. We have used a genetic approach to identify other proteins that interact functionally with Nup1p. Here we describe the isolation of seventeen mutants that confer a requirement for Nup1p in a background in which this protein is normally not essential. Some of the mutants require wild-type Nup1p, while others are viable in combination with specific nup1 alleles. Several of the mutants show nonallelic noncomplementation, suggesting that the products may be part of a hetero-oligomeric complex. One is allelic to srp1 which, although it was identified in an unrelated screen, was shown to encode a protein that is localized to the nuclear envelope (Yano, R., M. Oakes, M. Yamaghishi, J. A. Dodd, and M. Nomura. 1992. Mol. Cell. Biol. 12:5640-5651). We have used immunoprecipitation and fusion protein precipitation to show that Srp1p forms distinct complexes with both Nup1p and the related nucleoporin Nup2p, indicating that Srp1p is a component of the nuclear pore complex. The distant sequence similarity between Srp1p and the beta-catenin/desmoplakin family, coupled with the altered structure of the nuclear envelope in nup1 mutants, suggests that Srp1p may function in attachment of the nuclear pore complex to an underlying nuclear skeleton.

Nuclear import of DNA in digitonin-permeabilized cells

1997 ◽  
Vol 110 (18) ◽  
pp. 2323-2331 ◽  
Author(s):  
J.E. Hagstrom ◽  
J.J. Ludtke ◽  
M.C. Bassik ◽  
M.G. Sebestyen ◽  
S.A. Adam ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Staining Pattern ◽  
Nucleocytoplasmic Transport ◽  
Cell System ◽  
Nuclear Pore ◽  
Permeabilized Cell ◽  
Permeabilized Cells ◽  
Energy Dependent ◽  
Punctate Staining

DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.

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