scholarly journals Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p

1998 ◽  
Vol 143 (7) ◽  
pp. 1813-1830 ◽  
Author(s):  
Marcello Marelli ◽  
John D. Aitchison ◽  
Richard W. Wozniak
Keyword(s):  
Nuclear Pore Complex ◽  
Core Protein ◽  
Specific Binding ◽  
Protein A ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Binding Assays ◽  
Reporter Protein ◽  
Physiological Relevance

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.

scholarly journals The small GTPase Gsp1p binds to the repeat domain of the nucleoporin Nsp1p

1998 ◽  
Vol 330 (1) ◽  
pp. 421-427 ◽  
Author(s):  
Ursula STOCHAJ ◽  
Mehrdad HÉJAZI ◽  
Pierre BELHUMEUR
Keyword(s):  
Nuclear Pore Complex ◽  
Cell Cycle Progression ◽  
Protein Complexes ◽  
Distinct Type ◽  
Small Gtpase ◽  
Binding Domain ◽  
Nuclear Pore ◽  
Amino Acid Residues

The small GTPase Gsp1p of Saccharomyces cerevisiae and its homologue Ran play essential roles in several nuclear processes, such as cell-cycle progression, nuclear organization and nucleocytoplasmic traffic of RNA and proteins. Gsp1p/Ran is an abundant nuclear protein that interacts with different cytoplasmic and nuclear factors. Several of the previously identified Ran-binding proteins located at the nuclear-pore complex carry a specific Ran-binding domain. So far, direct interactions between the GTPase and other proteins have been mostly characterized in higher eukaryotes. Here we report that the yeast protein Gsp1p can directly bind to the nucleoporin Nsp1p in vitro. Nsp1p does not contain a Ran-binding domain and therefore represents a distinct type of nucleoporin that associates with Gsp1p. We demonstrate that the middle domain of Nsp1p is sufficient to mediate this interaction. Importantly, we show that a conserved cluster of positively charged amino acid residues of Gsp1p located at positions 142-144 is essential for the binding reaction. Thus we have identified Nsp1p as a new candidate protein located at the nuclear pore complex of the yeast S. cerevisiae that interacts directly with Gsp1p. We further demonstrate that both Gsp1p and Nsp1p are components of larger protein complexes in vivo, supporting the idea that the association between both proteins takes place in growing cells.

The Formation, Structure, Distribution and Frequency of Nuclear Pores

1971 ◽  
Vol 29 ◽  
pp. 518-519
Author(s):  
G. G. Maul
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Dynamic Structure ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Filter Function ◽  
Etching Technique ◽  
Selective Filter ◽  
Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

scholarly journals Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Balamurugan Shanmugaraj ◽  
Kaewta Rattanapisit ◽  
Suwimon Manopwisedjaroen ◽  
Arunee Thitithanyanont ◽  
Waranyoo Phoolcharoen
Keyword(s):  
Monoclonal Antibodies ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Protein A ◽  
Single Step ◽  
Affinity Column ◽  
Plant Expression ◽  
Plant Expression System

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in Nicotiana benthamiana leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity in vitro. To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.

The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro

1996 ◽  
Vol 74 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Werner Barth ◽  
Ursula Stochaj
Keyword(s):  
Nuclear Localization ◽  
Specific Binding ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Sv40 T Antigen ◽  
Nuclear Targeting ◽  
Nuclear Localization Sequences

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Gal4. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 × 107 M−1 and 5 × 107 M−1. An association constant of 5 × 107 M−1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.Key words: Nsp1, nuclear pore complex, nucleoporin, nuclear localization sequence, protein targeting, yeast.

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 A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export

1999 ◽  
Vol 145 (4) ◽  
pp. 645-657 ◽  
Author(s):  
Ralph H. Kehlenbach ◽  
Achim Dickmanns ◽  
Angelika Kehlenbach ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Activated T Cells ◽  
Permeabilized Cells ◽  
Binding Domains ◽  
Biochemical Fractionation ◽  
Nuclear Export Receptor ◽  
Gtpase Ran

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

scholarly journals Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

2003 ◽  
Vol 185 (6) ◽  
pp. 1808-1816 ◽  
Author(s):  
Victor McAlister ◽  
Chao Zou ◽  
Robert H. Winslow ◽  
Gail E. Christie
Keyword(s):  
Rna Polymerase ◽  
Serratia Marcescens ◽  
Specific Binding ◽  
Protein A ◽  
Upstream Region ◽  
Late Gene Expression ◽  
In Vitro Characterization ◽  
Template Dna

ABSTRACT NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P nucA and to the sequence upstream of the P2 late promoter P F is accompanied by DNA bending. NucC protects about 25 nucleotides of the P F upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P F in vitro.

scholarly journals Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

2011 ◽  
Vol 286 (18) ◽  
pp. 16308-16320 ◽  
Author(s):  
Hong-Juan Peng ◽  
Karen M. Henkels ◽  
Madhu Mahankali ◽  
Mary C. Dinauer ◽  
Julian Gomez-Cambronero
Keyword(s):  
Specific Binding ◽  
Strong Association ◽  
Living Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Deletion Mutants ◽  
Ph Domain ◽  
Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

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.

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