A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin beta, CRM1 and RanBP1

Transport of macromolecules between the nucleus and cytoplasm involves the recognition of intrinsic localization signals by either import or export receptors. The interaction of the receptors with their cargo is regulated by the small GTPase Ran in its GTP bound state. We have investigated the interaction of RanGTP with the import factor, importin beta, the export factor, CRM1, and the Ran binding protein, RanBP1, in solution. Importin beta specifically protected residues in the switch regions and basic patch region of Ran against proteolytic cleavage, whereas RanBP1 protected the C terminus. Moreover, the binding of importin beta induced a conformational change in the structure of Ran leading to an exposure of the C terminus and stimulated the binding of RanBP1. Mutating the basic patch (HRKK(142)) of Ran resulted in an increased binding of RanBP1 and weakened importin beta binding. In contrast to wild-type Ran, the mutant Ran could be released from importin beta independently of importin alpha. These data provide experimental support for a model in which the accessibility of the C terminus of Ran is influenced by an intramolecular interaction between the basic patch and the C-terminal acidic DEDDDL(216) motif. Binding of importin beta probably disrupts this interaction causing an exposure of the C-terminal extension, which is favorable for RanBP1 binding. Interestingly, basic patch mutations abolish CRM1 interaction, indicating that the determinants for RanGTP binding to the export factor, CRM1, is different from the import factor, importin beta.     

Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an approximately 20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state.     

Molecular dissection of the importin beta1-recognized nuclear targeting signal of parathyroid hormone-related protein

Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin beta1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin beta using a series of alanine mutated PTHrP peptides and truncated human importin beta1 derivatives. Our results indicate that PTHrP amino acids 83-93 (KTPGKKKKGK) are absolutely essential for importin beta1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of importin beta1 are required for the interaction. Binding of importin beta1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin beta is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin beta1-recognized nuclear targeting signal.     

The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a closed conformation incompatible with cargo binding

Cse1 mediates nuclear export of importin alpha, the nuclear localization signal (NLS) import adaptor. We report the 3.1 A resolution structure of cargo-free Cse1, representing this HEAT repeat protein in its cytosolic state. Cse1 is compact, consisting of N- and C-terminal arches that interact to form a ring. Comparison with the structure of cargo-bound Cse1 shows a major conformational change leading to opening of the structure upon cargo binding. The largest structural changes occur within a hinge region centered at HEAT repeat 8. This repeat contains a conserved insertion that connects the RanGTP and importin alpha contact sites and that is essential for binding. In the cargo-free state, the RanGTP binding sites are occluded and the importin alpha sites are distorted. Mutations that destabilize the N- to C-terminal interaction uncouple importin alpha and Ran binding, suggesting that the closed conformation prevents association with importin alpha.     

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.     

Anillin-mediated targeting of peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.     

Dissociation of nuclear import cargo complexes by the protein Ran: a fluorescence correlation spectroscopy study

In nucleated cells, proteins designed for nuclear import form complexes with soluble nuclear transport receptors prior to translocation across the nuclear envelope. The directionality of transport is due to the asymmetric distribution of the protein Ran, which dissociates import cargo complexes only in its nuclear RanGTP form. Using fluorescence correlation spectroscopy, we have studied the stability of cargo complexes in solution in the presence and in the absence of RanGTP. We find that RanGTP has a higher affinity for the major import receptor, the importin alpha/beta heterodimer, when importin alpha does not carry a cargo, suggesting that some nuclear transport targets might be preferentially released.     

Nuclear import of the U1A splicesome protein is mediated by importin alpha /beta and Ran in living mammalian cells

U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.     

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.     

Biochemical characterization of a family of proteins that stabilizes a plant Ran protein in its GTP-bound conformation.

Ran-binding proteins (RanBP) are a group of proteins that bind to Ran (Ras-related nuclear small G-protein) and thus control the GTP/GDP-bound states of the Ran and couple the Ran GTPase cycle to cellular processes. In an effort to identify potential downstream effectors for PsRan1-dependent cellular processes, we detected a group of pea Ran (PsRan1)-binding proteins and characterized their biochemical activities. A Ran overlay assay using [(32)P-GTP]-labeled PsRan1 revealed three PsRan1-binding proteins (33, 45, and 85kDa in size) from total protein extracts of dark-grown pea plumules. These proteins bound preferentially to the Ran-GTP over Ran-GDP conformation and subsequently stabilized its GTP-bound status. We propose that they are a family of proteins that maintain the Ran protein in the active conformation and are potential downstream mediators for PsRan1-dependent cellular processes. Our report provides the basis for characterizing and dissecting Ran downstream targets and Ran-mediated events, and it thus facilitates our understanding about the roles played by Ran/RanBP signaling pathways during plant growth and development.     

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.     

Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5′-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5′-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-β. Here, we show that RanGDP and importin-β form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-β binding partners such as importin-α. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.     

An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain

In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor A-type domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile(316), wedged into a hydrophobic socket in the closed form, but displaced from it in the open structure. Both mutants crystallized in the open conformation, and the Ile(316) --> Gly-modified integrin displayed high affinity. Structural differences between the low and high affinity forms were detected in solution. These data establish the structure-function correlates for the CD11b A-domain, and define a ligand-independent isoleucine-based allosteric switch intrinsic to this domain that controls its conformation and affinity.     

Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.     

Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

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.     

Downregulation of the small GTPase Ras-related nuclear protein accelerates cellular ageing

Background

The small GTPase Ran, Ras-related nuclear protein, plays important roles in multiple fundamental cellular functions such as nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, by binding to either GTP or GDP as a molecular switch. Although it has been clinically demonstrated that Ran is highly expressed in multiple types of cancer cells and specimens, the physiological significance of Ran expression levels is unknown.

Methods

During the long-term culture of normal mammalian cells, we found that the endogenous Ran level gradually reduced in a passage-dependent manner. To examine the physiological significance of Ran reduction, we first performed small interfering RNA (siRNA)-mediated abrogation of Ran in human diploid fibroblasts.

Results

Ran-depleted cells showed several senescent phenotypes. Furthermore, we found that nuclear accumulation of importin α, which was also observed in cells treated with siRNA against CAS, a specific export factor for importin α, occurred in the Ran-depleted cells before the cells showed senescent phenotypes. Further, the CAS-depleted cells also exhibited cellular senescence. Indeed, importin α showed predominant nuclear localisation in a passage-dependent manner.

Conclusions

Reduction in Ran levels causes cytoplasmic decrease and nuclear accumulation of importin α leading to cellular senescence in normal cells.

General significance

The amount of intracellular Ran may be critically related to cell fate determination, such as malignant transformation and senescence. The cellular ageing process may proceed through gradual regression of Ran-dependent nucleocytoplasmic transport competency.     

Mechanism of the stress-induced collapse of the Ran distribution

The small GTPase Ran plays a central role in several key nuclear functions, including nucleocytoplasmic transport, cell cycle progression, and assembly of the nuclear envelope. In a previous study, we showed that cellular stress induces the nuclear accumulation of importin alpha, and that this appears to be triggered by a collapse in the Ran gradient, leading to the down-regulation of classical nuclear transport. We report here that a decrease in stress-induced ATP is associated with an increase in cytoplasmic Ran levels. A luciferin-luciferase assay showed that cellular stress decreased the intracellular levels of ATP. Treatment of the cells with ATP-depleting agents altered the distribution of Ran. Furthermore, when exogenous ATP was introduced in oxidative stress-treated cells, a normal distribution of Ran was restored. In addition, a pull-down experiment with an importin beta1 variant that binds to RanGTP showed that oxidative stress was accompanied by a decrease in intracellular RanGTP levels. These findings indicate that the decrease in ATP levels induced by cellular stress causes a decrease in RanGTP levels and a collapse of Ran distribution.     

Nucleoporin Nup50 stabilizes closed conformation of armadillo repeat 10 in importin α5

The human genome encodes six isoforms of importin α that show greater than 60% sequence similarity and remarkable substrate specificity. The isoform importin α5 can bind phosphorylated cargos such as STAT1 and Epstein-Barr Virus Nuclear Antigen 1, as well as the influenza virus polymerase subunit PB2. In this work, we have studied the interaction of the nucleoporin Nup50 with importin α5. We show that the first 47 residues of Nup50 bind to the C terminus of importin α5 like a "clip," stabilizing the closed conformation of ARM 10. In vitro, Nup50 binds with high affinity either to empty importin α5 or to a preassembled complex of importin α5 bound to the C-terminal domain of the import cargo PB2, resulting in a trimeric complex. By contrast, PB2 can only bind with high affinity to importin α5 in the absence of Nup50. This suggests that Nup50 primary function may not be to actively displace the import cargo from importin α5 but rather to prevent cargo rebinding in preparation for recycling. This is the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor.