Ran-Binding Protein 3 Is a Cofactor for Crm1-Mediated Nuclear Protein Export

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex posseses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1–RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase–activating protein.

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A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.8.12.2591 ◽

1997 ◽

Vol 8 (12) ◽

pp. 2591-2604 ◽

Cited By ~ 13

Author(s):

Gretchen A. Murphy ◽

Mary Shannon Moore ◽

George Drivas ◽

Pablo Pérez de la Ossa ◽

Alicia Villamarin ◽

...

Keyword(s):

Cell Cycle Progression ◽

Protein Import ◽

Nuclear Protein ◽

Binding Protein ◽

Nuclear Pore ◽

Nucleotide Exchange ◽

Wild Type ◽

Direct Role ◽

Permeabilized Cell ◽

Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

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Targeting of Ran: variation on a common theme?

Journal of Cell Science ◽

10.1242/jcs.114.18.3233 ◽

2001 ◽

Vol 114 (18) ◽

pp. 3233-3241 ◽

Cited By ~ 1

Author(s):

Markus Künzler ◽

Ed Hurt

Keyword(s):

Nuclear Envelope ◽

Mitotic Spindle ◽

Nucleocytoplasmic Transport ◽

Common Theme ◽

Spindle Formation ◽

Ran Gtpase ◽

Cellular Processes ◽

Important Target ◽

Transport Receptors ◽

Nuclear Envelope Formation

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin β family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin β as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

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The Yeast Nucleoporin Nup53p Specifically Interacts with Nic96p and Is Directly Involved in Nuclear Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3885 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3885-3896 ◽

Cited By ~ 22

Author(s):

Birthe Fahrenkrog ◽

Wolfgang Hübner ◽

Anna Mandinova ◽

Nelly Panté ◽

Walter Keller ◽

...

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Nuclear Protein ◽

Genomic Library ◽

Nuclear Periphery ◽

Nucleocytoplasmic Transport ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Protein Import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ∼30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen withNIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

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Getting across the nuclear pore complex: new insights into nucleocytoplasmic transportThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y06-001 ◽

2006 ◽

Vol 84 (3-4) ◽

pp. 499-507 ◽

Cited By ~ 16

Author(s):

Daniel Stoffler ◽

Kyrill Schwarz-Herion ◽

Ueli Aebi ◽

Birthe Fahrenkrog

Keyword(s):

Nuclear Pore Complex ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Special Issue ◽

The Past ◽

Transport Receptors ◽

A Cell ◽

New Evidence ◽

Retention Signal ◽

Selection Of

Small ions and molecules can traverse the nuclear pore complex (NPC) simply by diffusion, whereas larger proteins and RNAs require specific signals and factors that facilitate their passage through the NPC. Our understanding of the factors that participate and regulate nucleocytoplasmic transport has increased tremendously over the past years, whereas the actual translocation step through the NPC has remained largely unclear. Here, we present and discuss recent findings on the interaction between the NPC and transport receptors and provide new evidence that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargos.

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Molecular determinants of large cargo transport into the nucleus

eLife ◽

10.7554/elife.55963 ◽

2020 ◽

Vol 9 ◽

Author(s):

Giulia Paci ◽

Tiantian Zheng ◽

Joana Caria ◽

Anton Zilman ◽

Edward A Lemke

Keyword(s):

Nuclear Import ◽

Nucleocytoplasmic Transport ◽

Biophysical Model ◽

Nuclear Pore ◽

Cargo Transport ◽

Transport Receptors ◽

Molecular Determinants ◽

Quantitative Understanding ◽

Kinetics Of

Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex (NPC). Among the thousands of molecules that cross the NPC, even very large (>15 nm) cargoes such as pathogens, mRNAs and pre-ribosomes can pass the NPC intact. For these cargoes, there is little quantitative understanding of the requirements for their nuclear import, especially the role of multivalent binding to transport receptors via nuclear localisation sequences (NLSs) and the effect of size on import efficiency. Here, we assayed nuclear import kinetics of 30 large cargo models based on four capsid-like particles in the size range of 17–36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for nuclear transport can be recapitulated by a simple two-parameter biophysical model that correlates the import flux with the energetics of large cargo transport through the NPC. Together, our results reveal key molecular determinants of large cargo import in cells.

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Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors

eLife ◽

10.7554/elife.00745 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 46

Author(s):

Kasper R Andersen ◽

Evgeny Onischenko ◽

Jeffrey H Tang ◽

Pravin Kumar ◽

James Z Chen ◽

...

Keyword(s):

Nuclear Transport ◽

Evolutionary Relationship ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Rotational Axis ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Structural And Functional Properties ◽

Transport Receptors ◽

Pore Complexes

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery.

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The dynamics of karyopherin-mediated nuclear transport

Biochemistry and Cell Biology ◽

10.1139/o01-149 ◽

2001 ◽

Vol 79 (5) ◽

pp. 603-612 ◽

Cited By ~ 18

Author(s):

Marcello Marelli ◽

David J Dilworth ◽

Richard W Wozniak ◽

John D Aitchison

Keyword(s):

Biochemical Characterization ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Complex Interplay ◽

Conventional View ◽

Cytoplasmic Transport ◽

Transport Receptors ◽

Pore Complexes

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.Key words: Nucleocytoplasmic transport, nuclear pore complex, nucleoporin, karyopherin, Nup2p.

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Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

Molecular and Cellular Biology ◽

10.1128/mcb.17.9.5087 ◽

1997 ◽

Vol 17 (9) ◽

pp. 5087-5096 ◽

Cited By ~ 63

Author(s):

R Deane ◽

W Schäfer ◽

H P Zimmermann ◽

L Mueller ◽

D Görlich ◽

...

Keyword(s):

Nuclear Transport ◽

Binding Protein ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleotide Exchange ◽

Transport Pathway ◽

Importin Beta ◽

Pore Complexes

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

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RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import.

The Journal of Cell Biology ◽

10.1083/jcb.135.3.559 ◽

1996 ◽

Vol 135 (3) ◽

pp. 559-569 ◽

Cited By ~ 126

Author(s):

N C Chi ◽

E J Adam ◽

G D Visser ◽

S A Adam

Keyword(s):

Protein Import ◽

Nuclear Protein ◽

Gel Filtration ◽

Nuclear Pore ◽

Binding Assays ◽

Permeabilized Cell ◽

Trimeric Complex ◽

Cell Extracts ◽

Nuclear Protein Import ◽

Ran Gtp

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.

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The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants

Journal of Cell Science ◽

10.1242/jcs.111.20.3017 ◽

1998 ◽

Vol 111 (20) ◽

pp. 3017-3026 ◽

Cited By ~ 2

Author(s):

M. Hughes ◽

C. Zhang ◽

J.M. Avis ◽

C.J. Hutchison ◽

P.R. Clarke

Keyword(s):

Dna Replication ◽

Nuclear Import ◽

Cell Cycle Control ◽

Critical Role ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Wild Type ◽

Ran Gtpase ◽

Free System ◽

Nuclear Assembly

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

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