Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.     

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1(-) yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1(-) yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.     

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.     

Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.     

Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.     

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.     

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.     

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.     

Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export.

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

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> Ran-binding protein,AtRanBP1c,is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.     

Induction of Ran GTP drives ciliogenesis

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin-Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.     

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.     

Characterization of membrane-bound small GTP-binding proteins from Nicotiana tabacum.

We have cloned nine cDNAs encoding small GTP-binding proteins from leaf cDNA libraries of tobacco (Nicotiana tabacum). These cDNAs encode distinct proteins (22-25 kD) that display different levels of identity with members of the mammalian Rab family: Nt-Rab6 with Rab6 (83%), Nt-Rab7a-c with Rab7 (63-70%), and Nt-Rab11a-e with Rab11 (53-69%). Functionally important regions of these proteins, including the "effector binding" domain, the C-terminal Cys residues for membrane attachment, and the four regions involved in GTP-binding and hydrolysis, are highly conserved. Northern and western blot analyses show that these genes are expressed, although at slightly different levels, in all plant tissues examined. We demonstrate that the plant Rab5, Rab6, and Rab11 proteins, similar to their mammalian and yeast counterparts, are tightly bound to membranes and that they exhibit different solubilization characteristics. Furthermore, we show that the yeast GTPase-activating protein Gyp6, shown to be specifically required to control the GTP hydrolysis of the yeast Ypt6 protein, could interact with tobacco GTP-binding proteins. It increases in vitro the GTP hydrolysis rate of the wild-type Nt-Rab7 protein. In addition, it also increases, at different levels, the GTP hydrolysis rates of a Nt-Rab7m protein with a Rab6 effector domain and of two other chimaeric Nt-Rab6/Nt-Rab7 proteins. However, it does not interact with the wild-type Nt-Rab6 protein, which is most similar to the yeast Ypt6 protein.     

RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.     

Anchorage of plant RanGAP to the nuclear envelope involves novel nuclear-pore-associated proteins

The Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1-4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5-8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9-14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15-18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms.     

Phosphorylation of Ran-binding protein-1 by Polo-like kinase-1 is required for interaction with Ran and early mitotic progression

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.     

Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta

The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin. cargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might function like RanBP1. We used biophysical assays based on fluorescence-labeled probes and on surface plasmon resonance to investigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and with importin-beta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities for Ran.GTP (10(8)-10(9) M(-1)). Deletion of Ran's C-terminal (211)DEDDDL(216) sequence weakens the interaction of Ran.GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran.GTP with importin-beta 10-fold. Importin-beta binds Ran.GTP with a moderate rate, but attains a high affinity for Ran (K(D) = 140 pM) via an extremely low dissociation rate of 10(-5) s(-)(1). Association with Ran is accelerated 3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hydrolysis in Ran and relieve the GTPase block in a Ran.GTP.transportin complex. Our data suggest that the C-terminus of Ran functions like a loose tether in Ran.GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by the multiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran.GTP complexes.     

Solution structure of the Ran-binding domain 2 of RanBP2 and its interaction with the C terminus of Ran

The termination of export processes from the nucleus to the cytoplasm in higher eukaryotes is mediated by binding of the small GTPase Ran as part of the export complexes to the Ran-binding domains (RanBD) of Ran-binding protein 2 (RanBP2) of the nuclear pore complex. So far, the structures of the first RanBD of RanBP2 and of RanBP1 in complexes with Ran have been known from X-ray crystallographic studies. Here we report the NMR solution structure of the uncomplexed second RanBD of RanBP2. The structure shows a pleckstrin homology (PH) fold featuring two almost orthogonal beta-sheets consisting of three and four strands and an alpha-helix sitting on top. This is in contrast to the RanBD in the crystal structure complexes in which one beta-strand is missing. That is probably due to the binding of the C-terminal alpha-helix of Ran to the RanBD in these complexes. To analyze the interaction between RanBD2 and the C terminus of Ran, NMR-titration studies with peptides comprising the six or 28 C-terminal residues of Ran were performed. While the six-residue peptide alone does not bind to RanBD2 in a specific manner, the 28-residue peptide, including the entire C-terminal helix of Ran, binds to RanBD2 in a manner analogous to the crystal structures. By solving the solution structure of the 28mer peptide alone, we confirmed that it adopts a stable alpha-helical structure like in native Ran and therefore serves as a valid model of the Ran C terminus. These results support current models that assume recognition of the transport complexes by the RanBDs through the Ran C terminus that is exposed in these complexes.