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1.
We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP. These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.  相似文献   

2.
The 1.9 A resolution X-ray crystal structure of Ran-binding protein Mog1p shows that it has a unique fold based on a six-stranded antiparallel beta-sheet backed on both sides by an extensive alpha-helix. The topology of some elements of Mog1p secondary structure resemble a portion of nuclear transport factor 2 (NTF2), but the hydrophobic cavity and surrounding negatively charged residues that are important in the NTF2-RanGDP interaction are not conserved in Mog1p. In addition to binding RanGTP, Mog1p forms a 1:1 complex with RanGDP and so binds Ran independent of its nucleotide state. Mog1p and NTF2 compete for binding to RanGDP indicating that their binding sites on RanGDP are sufficiently close to prevent both proteins binding simultaneously. Although there may be some overlap between the Mog1p and NTF2 binding sites on RanGDP, these sites are not identical. Sequence analysis of Mog1p homologues from Schizosaccharomyces pombe, human, and Caenorhabditis elegans in the context of the Mog1p crystal structure indicates the presence of a cluster of highly conserved surface residues consistent with an interaction site for Ran.  相似文献   

3.
Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1], [2], [3], [4]. Import receptors such as importin β or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin α/β heterodimer, coupled directly to importin β release from the NPCs. If the importin β –RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5], [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin–substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin–substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin–substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin β independently of Ran and energy.  相似文献   

4.
D Grlich  N Pant  U Kutay  U Aebi    F R Bischoff 《The EMBO journal》1996,15(20):5584-5594
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.  相似文献   

5.
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.  相似文献   

6.
Nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. The simplicity and specialization of this system, combined with the availability of crystal structures of NTF2, RanGDP and their complex, has facilitated the investigation of the molecular mechanism of its trafficking. NTF2 binds to both RanGDP and FxFG repeat-containing nucleoporins. Mutants engineered on the basis of structural information together with determination of binding constants have been used to dissect the roles of these interactions in transport. Thus, NTF2 binds to RanGDP sufficiently strongly for the complex to remain intact during transport through NPCs, but the interaction between NTF2 and FxFG nucleoporins is much more transient, which would enable NTF2 to move through the NPC by hopping from one repeat to another. An analogous nucleoporin hopping mechanism may also be used by carrier molecules of the importin-beta family to move through NPCs.  相似文献   

7.
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.  相似文献   

8.
Interactions with nucleoporins containing FxFG-repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here the 1.9 A resolution crystal structure of yeast NTF2-N77Y bound to a FxFG-nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking. The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2. Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild-type protein in yeast. Comparison with the crystal structure of FG-nucleoporin cores bound to importin-beta and TAP/p15 identified a number of common features of their binding sites. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.  相似文献   

9.
Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.  相似文献   

10.
Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231–239, 1998.© 1998 Wiley-Liss, Inc.  相似文献   

11.
12.
A new role for nuclear transport factor 2 and Ran: nuclear import of CapG   总被引:1,自引:0,他引:1  
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
Here, we analyse the RanGTPase system and its coupling to receptor-mediated nuclear transport. Our simulations predict nuclear RanGTP levels in HeLa cells to be very sensitive towards the cellular energy charge and to exceed the cytoplasmic concentration approximately 1000-fold. The steepness of the RanGTP gradient appears limited by both the cytoplasmic RanGAP concentration and the imperfect retention of nuclear RanGTP by nuclear pore complexes (NPCs), but not by the nucleotide exchange activity of RCC1. Neither RanBP1 nor the NPC localization of RanGAP has a significant direct impact on the RanGTP gradient. NTF2-mediated import of Ran appears to be the bottleneck for maximal capacity of Ran-driven nuclear transport. We show that unidirectional nuclear transport can be faithfully simulated without the assumption of a vectorial NPC passage; transport receptors only need to reversibly cross NPCs and switch their affinity for cargo in response to the RanGTP gradient. A significant RanGTP gradient after nuclear envelope (NE) breakdown can apparently exist only in large cytoplasm. This indicates that RanGTP gradients can provide positional information for mitotic spindle and NE assembly in early embryonic cells, but hardly any in small somatic cells.  相似文献   

16.
The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2''s E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP''s affinity to RanBP2''s RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2''s RBDs, which is prevented by the transport factor NTF2.  相似文献   

17.
The small GTPase Ran functions in several critical processes in eukaryotic cells including nuclear transport, nuclear envelope formation, and spindle formation. A RanGDP-binding protein, NTF2, facilitates translocation of RanGDP through the nuclear pore complex and also acts to stabilize RanGDP against nucleotide exchange. Here, we identify a novel activity that stimulates release of GDP from Ran in the presence of NTF2. Hydrolyzable ATP enhances the GDP dissociation activity, and this enhancement is inhibited by nonhydrolyzable ATP analogues. In contrast, neither hydrolyzable ATP nor nonhydrolyzable ATP analogues affect GDP dissociation from Ran catalyzed by recombinant RCC1 or inhibition of GDP dissociation from Ran by recombinant NTF2. The ATP-dependent RanGDP dissociation activity therefore has the properties of a RanGDP dissociation inhibitor (GDI) displacement factor (RanGDF) where the GDI is NTF2. A protein phosphatase inhibitor mixture stimulates the RanGDF activity, suggesting the activity is regulated by phosphorylation. We propose that the ATP-dependent NTF2 releasing factor may have a role in the RanGDP/GTP cycle.  相似文献   

18.
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.  相似文献   

19.
Anne-Christine Ström  Karsten Weis 《Genome biology》2001,2(6):reviews3008.1-reviews30089
In recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been identified. Most of these receptors belong to one large family of proteins, all of which share homology with the protein import receptor importin β (also named karyopherin β). Members of this family have been classified as importins or exportins on the basis of the direction they carry their cargo. To date, the family includes 14 members in the yeast Saccharomyces cerevisiae and at least 22 members in humans. Importins and exportins are regulated by the small GTPase Ran, which is thought to be highly enriched in the nucleus in its GTP-bound form. Importins recognize their substrates in the cytoplasm and transport them through nuclear pores into the nucleus. In the nucleoplasm, RanGTP binds to importins, inducing the release of import cargoes. In contrast, exportins interact with their substrates only in the nucleus in the presence of RanGTP and release them after GTP hydrolysis in the cytoplasm, causing disassembly of the export complex. Thus, common features of all importin-β-like transport factors are their ability to shuttle between the nucleus and the cytoplasm, their interaction with RanGTP as well as their ability to recognize specific transport substrates.  相似文献   

20.
Transport between the cytoplasm and the nucleus   总被引:1,自引:0,他引:1  
Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC.  相似文献   

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