The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.     

The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function

The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus.     

Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores

How individual nucleoporins (Nups) perform their role in nuclear pore structure and function is largely unknown. In this study, we examined the structure of purified Nup170 to obtain clues about its function. We show that Nup170 adopts a crescent moon shape with two structurally distinct and separable domains, a β-propeller N terminus and an α-solenoid C terminus. To address the individual roles of each domain, we expressed these domains separately in yeast. Notably, overexpression of the Nup170 C domain was toxic in nup170Δ cells and caused accumulation of several Nups in cytoplasmic foci. Further experiments indicated that the C-terminal domain anchors Nup170 to nuclear pores, whereas the N-terminal domain functions to recruit or retain a subset of Nups, including Nup159, Nup188, and Pom34, at nuclear pores. We conclude that Nup170 performs its role as a structural adapter between cytoplasmically oriented Nups and the nuclear pore membrane.     

Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.     

The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.     

Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.     

The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88.

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.     

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.     

Nup155 is a novel nuclear pore complex protein that contains neither repetitive sequence motifs nor reacts with WGA

We have molecularly cloned and sequenced a rat liver nuclear pore complex (NPC) protein of calculated molecular mass of 155 kD. Consistent with recently proposed nomenclature this protein is termed nucleoporin 155, or nup155. Unlike other nups that have so far been molecularly cloned and sequenced, nup155 does not contain repetitive sequence domains. It does not show similarity to the sequences of other proteins, including any nups, so far compiled in the data bases. Like other vertebrate nups which have been characterized nup155 possesses abundant (46 in total) consensus sites for various kinases. By immunoelectron microscopy, nup155 is associated with both the nucleoplasmic and the cytoplasmic aspect of the NPC and is therefore possibly a component of the symmetrically arranged NPC substructures. In mitotic cells, nup155 assumes a diffuse cytoplasmic distribution. Nup155 is among the integral of 30 proteins that were extracted from rat liver nuclear envelopes by 2.0 M urea/1.0 mM EDTA, separated from WGA-reactive proteins by WGA-Sepharose and further subfractionated by SDS-hydroxylapatite. These proteins are potential candidates for being nups.     

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.     

Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export

Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.     

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.     

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.     

Upf1p, a highly conserved protein required for nonsense-mediated mRNA decay, interacts with the nuclear pore proteins Nup100p and Nup116p

Saccharomyces cerevisiae Upf1p is a 971-amino-acid protein that is required for the nonsense-mediated mRNA decay (NMD) pathway, a pathway that degrades mRNAs with premature translational termination codons. We have identified a two-hybrid interaction between Upf1p and the nuclear pore (Nup) proteins, Nup100p and Nup116p. Both nucleoporins predominantly localize to the cytoplasmic side of the nuclear pore and participate in mRNA transport. The two-hybrid interaction between Upf1p and the nuclear pore proteins, Nup100p and Nup116p, is dependent on the presence of the C-terminal 158 amino acids of Upf1p. Nup100p and Nup116p can be co-immunoprecipitated from whole-cell extracts with Upf1p, confirming in vitro the interaction identified by the two-hybrid analysis. Finally, we see a genetic interaction between UPF1 and NUP100. The growth of upf1Delta, can1-100 cells is inhibited by canavanine. The deletion of NUP100 allows upf1Delta, can1-100 cells to grow in the presence of canavanine. Physiologically, the interaction between Upf1p and the nuclear pore proteins, Nup100p and Nup116p, is significant because it suggests a mechanism to ensure that Upf1p associates with newly synthesized mRNA as it is transported from the nucleus to the cytoplasm prior to the pioneer round of translation.     

The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex

We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.     

A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96

The mammalian nuclear pore complex (NPC) is comprised of approximately 50 unique proteins, collectively known as nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.     

Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.

Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide. In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p). Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half. The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles. Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.     

Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.     

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.     

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.