The Ran GTPase cycle is required for yeast nuclear pore complex assembly

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.     

Concentration of Ran on chromatin induces decondensation,nuclear envelope formation and nuclear pore complex assembly

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.     

Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system

The molecular mechanism of nuclear envelope (NE) assembly is poorly understood, but in a cell-free system made from Xenopus eggs NE assembly is controlled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1]. Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the roles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei that actively transport proteins across the NE. Both RCC1 and the GTPase-activating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and RanGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precursor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by RanGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to form an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the recruitment of vesicles and their fusion to form NEs.     

Toward a more complete 3-D structure of the nuclear pore complex

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.     

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.     

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.     

Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.     

Cell cycle regulated transport controlled by alterations in the nuclear pore complex

Eukaryotic cells have developed mechanisms for regulating the nuclear transport of macromolecules that control various cellular events including movement through defined stages of the cell cycle. In yeast cells, where the nuclear envelope remains intact throughout the cell cycle, these transport regulatory mechanisms must also function during mitosis. We have uncovered a mechanism for regulating transport that is controlled by M phase specific molecular rearrangements in the nuclear pore complex (NPC). These changes allow a transport inhibitory nucleoporin, Nup53p, to bind the karyopherin Kap121p specifically during mitosis, slowing its movement through the NPC and inducing cargo release. Yeast strains that possess defects in the function of Kap121p or the fidelity of the inhibitory pathway are delayed in mitosis. We propose that fluctuations in Kap121p transport mediated by the NPC contribute to controlling the subcellular distribution of molecules that direct progression through mitosis.     

A Role for the Karyopherin Kap123p in Microtubule Stability

Several components of the nuclear transport machinery play a role in mitotic spindle assembly in higher eukaryotes. To further investigate the role of this family of proteins in microtubule function, we screened for mutations in Saccharomyces cerevisiae that confer sensitivity to microtubule-destabilizing drugs. One mutant exhibiting this phenotype lacked the gene encoding the karyopherin Kap123p. Analysis of kap123 Δ cells revealed that the drug sensitivity was caused by a defect in microtubule stability and/or assembly. In support of this idea, we demonstrated genetic interactions between the kap123 Δ mutation and mutated alleles of genes encoding α-tubulins and factors controlling microtubule dynamics. Moreover, kap123 Δ cells exhibit defects in spindle structure and dynamics as well as nuclear positioning defects during mitosis. Cultures of kap123 Δ strains are enriched for mononucleated large-budded cells often containing short spindles and nuclei positioned away from the budneck, phenotypes indicative of defects in both cytoplasmic and nuclear microtubules. Finally, we identified a gene, CAJ1 , which when deleted in combination with KAP123 exacerbated the microtubule-related defects of the kap123 Δ mutants. We propose that Kap123p and Caj1p, a member of the Hsp40 family of proteins, together play an essential role in normal microtubule function.     

A Nuclear Export Signal in Kap95p Is Required for Both Recycling the Import Factor and Interaction with the Nucleoporin GLFG Repeat Regions of Nup116p and Nup100p

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A–tagged Nup116p or protein A–tagged Nup100p complexes. The protein A–tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.     

Kap120 functions as a nuclear import receptor for ribosome assembly factor Rpf1 in yeast

The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.     

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.     

High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores

The nuclear envelope (NE) of amphibian oocytes can be readily isolated in relatively structurally intact and pure form and has been used extensively for structural studies. Using high resolution scanning electron microscopy (HRSEM), both surfaces of the NE can be visualized in detail. Here, we demonstrate the use of HRSEM to obtain high resolution information of NE structure, confirming previous data and providing some new information. NEs, manually isolated from Triturus cristatus oocytes, have been mounted on conductive silicon chips, fixed, critical point dried and coated with a thin, continuous film of chromium or tantalum and viewed at relatively high accelerating voltage in a field emission scanning electron microscope with the sample within the objective lens. Both nucleoplasmic and cytoplasmic surfaces of the nuclear pore complexes (NPC) have been visualized, revealing the cytoplasmic coaxial ring, associated particles, central plug/transporter and spokes. The nucleoplasmic face is dominated by the previously described basketlike structure attached to the nucleoplasmic coaxial ring. In Triturus, a novel, highly regular flat sheet of fibers, termed the NE lattice (NEL) has been observed attached to the distal ring of the NPC basket. The NEL appears to be distinct from the nuclear lamina. Evidence for the NEL is also presented in thin TEM sections from Triturus oocytes and GVs and in spread NEs from Xenopus. A model is presented for NEL structure and its interaction with the NPCs is discussed.     

A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex

Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.     

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

Yeast nucleoporins involved in passive nuclear envelope permeability

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.     

Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p,Nup59p,and Nup170p

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.     

Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae

In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.     

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C‐terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.     

Interactions between a Nuclear Transporter and a Subset of Nuclear Pore Complex Proteins Depend on Ran GTPase

Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-α and -β (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.