Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran

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.     

A new role for nuclear transport factor 2 and Ran: nuclear import of CapG

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin-permeabilized cells, neither GTPγS nor the GTP hydrolysis-deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2–Ran complex. NTF2–Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.     

The interaction between Ran and NTF2 is required for cell cycle progression

The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus. Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly. In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state. Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus. Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle. A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature. Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G(2) in a MAD2 checkpoint-dependent manner. These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo. Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest.     

Nuclear import of Ran is mediated by the transport factor NTF2

A concentration gradient of the GTP-bound form of the GTPase Ran across nuclear pores is essential for the transport of many proteins and nucleic acids between the nuclear and cytoplasmic compartments of eukaryotic cells [1], [2], [3] and [4]. The mechanisms responsible for the dynamics and maintenance of this Ran gradient have been unclear. We now show that Ran shuttles between the nucleosol and cytosol, and that cytosolic Ran accumulates rapidly in the nucleus in a saturable manner that is dependent on temperature and on the guanine-nucleotide exchange factor RCC1. Nuclear import in digitonin-permeabilized cells in the absence of added factors was minimal. The addition of energy and nuclear transport factor 2 (NTF2) [5] was sufficient for the accumulation of Ran in the nucleus. An NTF2 mutant that cannot bind Ran [6] was unable to facilitate Ran import. A GTP-bound form of a Ran mutant that cannot bind NTF2 was not a substrate for import. A dominant-negative importin-β mutant inhibited nuclear import of Ran, whereas addition of transportin, which accumulates in the nucleus, enhanced NTF2-dependent Ran import. We conclude that NTF2 functions as a transport receptor for Ran, permitting rapid entry into the nucleus where GTP-GDP exchange mediated by RCC1 [7] converts Ran into its GTP-bound state. The Ran–GTP can associate with nuclear Ran-binding proteins, thereby creating a Ran gradient across nuclear pores.     

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.     

Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

Nuclear-Cytoplasmic Trafficking of NTF2, the Nuclear Import Receptor for the RanGTPase, Is Subjected to Regulation

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).     

Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo

Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus.     

Hyperosmotic stress signaling to the nucleus disrupts the Ran gradient and the production of RanGTP

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10-20 min) and late (30-60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response.     

The 1.6 Å Resolution Crystal Structure of Nuclear Transport Factor 2 (NTF2)

Nuclear transport factor 2 (NTF2) facilitates protein transport into the nucleus and interacts with both the small Ras-like GTPase Ran and nucleoporin p62. We have determined the structure of bacterially expressed rat NTF2 at 1.6 Å resolution using X-ray crystallography. The NTF2 polypeptide chain forms an α + β barrel that opens at one end to form a distinctive hydrophobic cavity and its fold is homologous to that of scytalone dehydratase. The NTF2 hydrophobic cavity is a candidate for a potential binding site for other proteins involved in nuclear import such as Ran and nucleoporin p62. In addition, the hydrophobic cavity contains a putative catalytic Asp-His pair, which raises the possibility of an unanticipated enzymatic activity of the molecule that may have implications for the molecular mechanism of nuclear protein import.     

Nucleocytoplasmic protein transport and recycling of Ran

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.     

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.     

The mammalian Mog1 protein is a guanine nucleotide release factor for Ran

Ran is a Ras-related GTPase that is essential for the transport of protein and RNA between the nucleus and the cytoplasm. Proteins that regulate the GTPase cycle and subcellular distribution of Ran include the cytoplasmic GTPase-activating protein (RanGAP) and its co-factors (RanBP1, RanBP2), the nuclear guanine nucleotide exchange factor (RanGEF), and the Ran import receptor (NTF2). The recent identification of the Saccharomyces cerevisiae protein Mog1p as a suppressor of temperature-sensitive Ran mutations suggests that additional regulatory proteins remain to be characterized. Here, we describe the identification and biochemical characterization of murine Mog1, which, like its yeast orthologue, is a nuclear protein that binds specifically to RanGTP. We show that Mog1 stimulates the release of GTP from Ran, indicating that Mog1 functions as a guanine nucleotide release factor in vitro. Following GTP release, Mog1 remains bound to nucleotide-free Ran in a conformation that prevents rebinding of the guanine nucleotide. These properties distinguish Mog1 from the well characterized RanGEF and suggest an unanticipated mechanism for modulating nuclear levels of RanGTP.     

Stress-mediated inhibition of the classical nuclear protein import pathway and nuclear accumulation of the small GTPase Gsp1p.

Stress modifies all aspects of cellular physiology, including the targeting of macromolecules to the nucleus. To determine how distinct types of stress affect classical nuclear protein import, we followed the distribution of NLS-GFP, a reporter protein containing a classical nuclear localization sequence (NLS) fused to green fluorescent protein GFP. Nuclear accumulation of NLS-GFP requires import to be constitutively active; inhibition of import redistributes NLS-GFP throughout the nucleus and cytoplasm. In the yeast Saccharomyces cerevisiae, starvation, heat shock, ethanol and hydrogen peroxide rapidly inhibited classical nuclear import, whereas osmotic stress had no effect. To define the mechanisms underlying the inhibition of classical nuclear import, we located soluble components of the nuclear transport apparatus. Failure to accumulate NLS-GFP in the nucleus always correlated with a redistribution of the small GTPase Gsp1p. Whereas predominantly nuclear under normal conditions, Gsp1p equilibrated between nucleus and cytoplasm in cells exposed to starvation, heat, ethanol or hydrogen peroxide. Furthermore, analysis of yeast strains carrying mutations in different nuclear transport factors demonstrated a role for NTF2, PRP20 and MOG1 in establishing a Gsp1p gradient, as conditional lethal alleles of NTF2 and PRP20 or a deletion of MOG1 prevented Gsp1p nuclear accumulation. On the basis of these results, we now propose that certain types of stress release Gsp1p from its nuclear anchors, thereby promoting a collapse of the nucleocytoplasmic Gsp1p gradient and inhibiting classical nuclear protein import.     

Functional analysis of the yeast Ran exchange factor Prp20p: in vivo evidence for the RanGTP gradient model

Numerous cellular processes rely on the movement of macromolecules into and out of the nucleus. The primary regulator of this movement is the small GTPase Ran. Like other small GTPases, the nucleotide-bound state of Ran is regulated by effectors that enhance the rate of nucleotide exchange or hydrolysis. Current models for vectorial nuclear transport suggest that it is the strict compartmentalization of these Ran effector molecules that generates a gradient of RanGTP between the nucleus and the cytoplasm to impart directionality to the transport process. Here we investigate the mechanism by which the Ran exchange factor is targeted to the nucleus, and test the impact of disrupting this nuclear compartmentalization on nucleocytoplasmic transport in vivo. Our results indicate that in Saccharomycces cerevisiae the nucleotide exchange factor Prp20p can be targeted to the nucleus via a classical nuclear localization sequence. This transport mechanism is dependent both on Ran and the receptor that recognizes the nuclear localization sequence, importin alpha. Mutations in the evolutionarily conserved nuclear localization sequence only partially inhibit nuclear import of Prp20p, suggesting the existence of a secondary mechanism for this critical nuclear targeting. In an in vivo test of the RanGTP gradient model, we demonstrate that overexpression of a functional cytoplasmic exchange factor inhibits cell growth and blocks both protein import and RNA export in wild-type cells that contain the endogenous nuclear Prp20 protein. Taken together, our results provide in vivo evidence for the idea that the compartmentalization of the exchange factor serves as a mechanism for establishing directional nuclear transport.     

Interaction between the small GTPase Ran/Gsp1p and Ntf2p is required for nuclear transport.

Bidirectional movement of proteins and RNAs across the nuclear envelope requires Ran, a Ras-like GTPase. A genetic screen of the yeast Saccharomyces cerevisiae was performed to isolate conditional alleles of GSP1, a gene that encodes a homolog of Ran. Two temperature-sensitive alleles, gsp1-1 and gsp1-2, were isolated. The mutations in these two alleles map to regions that are structurally conserved between different members of the Ras family. Each mutant strain exhibits various nuclear transport defects. Both biochemical and genetic experiments indicate a decreased interaction between Ntf2p, a factor which is required for protein import, and the mutant GSP1 gene products. Overexpression of NTF2 can suppress the temperature sensitive phenotype of gsp1-1 and gsp1-2 and partially rescue nuclear transport defects. However, overexpression of a mutant allele of NTF2 with decreased binding to Gsp1p cannot rescue the temperature sensitivity of gsp1-1 and gsp1-2. Taken together, these data demonstrate that the interaction between Gsp1p and Ntf2p is critical for nuclear transport.     

Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis

Background: The transport of macromolecules between the nucleus and cytoplasm is an energy-dependent process. Substrates are translocated across the nuclear envelope through nuclear pore complexes (NPCs). Translocation requires nucleocytoplasmic transport receptors of the importin β family, which interact both with the NPC and, either directly or via an adaptor, with the transport substrate. Although certain receptors have recently been shown to cross the NPC in an energy-independent manner, translocation of substrate–receptor complexes through the NPC has generally been regarded as an energy-requiring step.Results: We describe an in vitro system that is based on permeabilised cells and supports nuclear export mediated by leucine-rich nuclear export signals. In this system, export is dependent on exogenous CRM1/Exportin1 – a nuclear export receptor – the GTPase Ran and nucleotide triphosphates (NTPs), and is further stimulated by Ran-binding protein 1 (RanBP1) and nuclear transport factor 2 (NTF2). Unexpectedly, non-hydrolysable NTP analogues completely satisfy the NTP requirements for a single-round of CRM1-mediated translocation of protein substrates across the NPC. Similarly, single transportin-mediated nuclear protein import events are shown not to require hydrolysable NTPs and to occur in the absence of the Ran GTPase.Conclusions: Our data show that, contrary to expectation and prior conclusions, the translocation of substrate–receptor complexes across the NPC in either direction occurs in the absence of NTP hydrolysis and is thus energy independent. The energy needed to drive substrate transport against a concentration gradient is supplied at the step of receptor recycling in the cytoplasm.     

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.