The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly

The small GTPase Ran is a key regulator of nucleocytoplasmic transport during interphase. The asymmetric distribution of the GTP-bound form of Ran across the nuclear envelope--that is, large quantities in the nucleus compared with small quantities in the cytoplasm--determines the directionality of many nuclear transport processes. Recent findings that Ran also functions in spindle formation and nuclear envelope assembly during mitosis suggest that Ran has a general role in chromatin-centred processes. Ran functions in these events as a signal for chromosome position.     

The mechanism of ran import into the nucleus by nuclear transport factor 2

The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.     

MYCBP2 Is a Guanosine Exchange Factor for Ran Protein and Determines Its Localization in Neurons of Dorsal Root Ganglia

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway.     

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.     

RAN1基因过表达抑制嗜热四膜虫大核无丝分裂

Ran GTPase通过RanGTP/RanGDP循环的形式,参与调控多种细胞增殖方式：包括有丝分裂和减数分裂.敲减RAN1基因可导致嗜热四膜虫大核内微管组装紊乱,从而抑制大核无丝分裂.为进一步分析Ran1在无丝分裂中的功能,本研究将野生型Ran1以及模拟GTP（Ran1Q70L）和GDP（Ran1T25N）锁定形式的Ran1突变体在嗜热四膜虫中过量表达,均导致四膜虫细胞增殖速率下降,并引起大核无丝分裂异常,且这种核异常细胞比率与Ran1过表达量呈正相关.免疫荧光定位结果显示,过表达的HA-Ran1在整个细胞中弥散分布,破坏了正常的Ran1分布形式;而过表达的HA-Ran1Q70L明显集中在大核核膜和胞质中,HA-Ran1T25N则主要定位在大核和小核内,分别与Ran1GTP/Ran1GDP循环的辅助调节因子定位模式一致.以上结果表明,过表达Ran1及其突变体可能影响嗜热四膜虫细胞中正常的Ran1GTP/Ran1GDP循环,进而导致大核无 丝分裂异常.     

Nucleocytoplasmic transport of Alp7/TACC organizes spatiotemporal microtubule formation in fission yeast

Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled‐coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during mitosis, although its underlying mechanism remains elusive. Here, we investigate the behaviour of Alp7 and its binding partner, Alp14/TOG, throughout the cell cycle. Interestingly, Alp7 enters the nucleus during interphase but is subsequently exported to the cytoplasm by the Exportin‐dependent nuclear export machinery. The continuous nuclear export of Alp7 during interphase is essential for maintaining the array‐like cytoplasmic microtubule structure. The mitosis‐specific nuclear accumulation of Alp7 seems to be under the control of cyclin‐dependent kinase (CDK). These results indicate that the spatiotemporal regulation of microtubule formation is established by the Alp7/TACC–Alp14/TOG complex through the coordinated interplay of Ran and CDK.     

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.     

Ran GTPase regulates Mad2 localization to the nuclear pore complex

In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.     

A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells

A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.     

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.     

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.     

嗜热四膜虫Ran结合蛋白1的表达对大小核分裂的影响

Ran是细胞内的一种具有GTP酶活性的功能蛋白,可以调节染色体稳定性、细胞核组建以及核质运输等多种细胞进程．Ran结合蛋白1（Ran-binding protein 1, Rbp1p ）是Ran的必要调控因子,促进Ran-GTP水解为Ran-GDP．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的Ran结合蛋白基因RBP1(TTHERM_00158040, http://www.ciliate.org)．实时荧光定量PCR表明,RBP1在四膜虫营养生长和有性生殖过程中都有表达,且在有性生殖过程中表达水平提高．免疫荧光定位表明,在营养 生长期Rbp1p定位于细胞质中．过表达RBP1或敲减RBP1后,细胞生长速率下降,大核的无丝分裂异常,细胞分裂末期产生了无大核的异常细胞,同时过表达RBP1导致了多小核的产生．结果表明,Rbp1p影响四膜虫细胞核的分裂进程,它的正常表达对细胞增殖过程起到重要的调节作用．     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.     

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.     

Role of importin-beta in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.     

Both ran and importins have the ability to function as nuclear mRNA export factors

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.     

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.     

Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import

The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min). Cotransfection with epitope-tagged, wild- type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.