Identification and analysis of LNO1-Like and AtGLE1-Like Nucleoporins in plants

Nucleoporins (Nups) are building blocks of the nuclear pore complex (NPC) that mediate cargo trafficking between the nucleus and the cytoplasm. Although the physical structure of the NPC is well studied in yeast and vertebrates, little is known about the structure of NPCs or the function of most Nups in plants. Recently we demonstrated two Nups in Arabidopsis: LONO1 (LNO1), homolog of human NUP214 and yeast Nup159, and AtGLE1, homolog of yeast Gle1, are required for early embryogenesis and seed development. To identify LNO1 and AtGLE1 homologs in other plant species, we searched the protein databases and identified 30 LNO1-like and 35 AtGLE1-like proteins from lower plant species to higher plants. Furthermore, phylogenetic analyses indicate that the evolutionary trees of these proteins follow expected plant phylogenies. High sequence homology and conserved domain structure of these nucleoporins suggest important functions of these proteins in nucleocytoplasmic transport, growth and development in plants.     

Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.     

Proteomic analysis of nucleoporin interacting proteins

The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex.     

Deciphering networks of protein interactions at the nuclear pore complex

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.     

Analysis of the yeast nucleoporin Nup188 reveals a conserved S-like structure with similarity to karyopherins

Nuclear pore complexes (NPCs) embedded in the double nuclear membrane mediate the entire nucleocytoplasmic transport between the nucleus and cytoplasm. Each NPC is composed of about 30 different proteins (nucleoporins or Nups), which exist in multiple (8, 16 or 32) copies within the NPC scaffold. Recently, we have identified and characterized the large structural Nups, Nup188 and Nup192, from the thermophilic eukaryote Chaetomium thermophilum, which exhibited superior properties for biochemical and structural studies, when compared to their mesophilic orthologs. Here, we study the large structural Nups from the model organism yeast Saccharomyces cerevisiae. Our data show that yeast Nup188 like its thermophilic orthologue ctNup188 exhibits a twisted S-like structure, which flexibly binds the linker nucleoporin Nic96 via a short conserved α-helix motif. Using bioinformatic methods, we have generated a pseudo-atomic structural model of Nup188 and its related Nup192, which further strengthens the view that the large α-solenoid structural Nups are related to karyopherins.     

Reconstitution of Nup157 and Nup145N into the Nup84 complex

About 30 different nucleoporins (Nups) constitute the nuclear pore complex. We have affinity-purified 28 of these nuclear pore proteins and identified new nucleoporin interactions by this analysis. We found that Nup157 and Nup170, two members of the large structural Nups, and the Gly-Leu-Phe-Gly nucleoporin Nup145N specifically co-purified with members of the Nup84 complex. In addition, Nup145N co-enriched during Nup157 purification. By in vitro reconstitution, we demonstrate that Nup157 and Nup145N form a nucleoporin subcomplex. Moreover, we show that Nup157 and Nup145N bind to the heptameric Nup84 complex. This assembly thus represents approximately one-third of all nucleoporins. To characterize Nup157 structurally, we purified and analyzed it by electron microscopy. Nup157 is a hollow sphere that resembles a clamp or a gripping hand. Thus, we could reconstitute an interaction between a large structural Nup, an FG repeat Nup, and a major structural module of the nuclear pore complex.     

Identification and characterization of nuclear pore complex components in Arabidopsis thaliana

The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities. The NPC comprises ~30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.     

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.     

Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae

The eukaryotic nuclear permeability barrier and selective nucleocytoplasmic transport are maintained by nuclear pore complexes (NPCs), large structures composed of ∼30 proteins (nucleoporins [Nups]). NPC structure and function are disrupted in aged nondividing metazoan cells, although it is unclear whether these changes are a cause or consequence of aging. Using the replicative life span (RLS) of Saccharomyces cerevisiae as a model, we find that specific Nups and transport events regulate longevity independent of changes in NPC permeability. Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100-null mutants. We show that Nup116 mediates nuclear import of the karyopherin Kap121, and each protein is required for mitochondrial function. Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast. Overexpression of GSP1, the small GTPase that powers karyopherin-mediated transport, rescued mitochondrial and RLS defects in nup116 mutants and increased longevity in wild-type cells. Together, these studies reveal that specific NPC nuclear transport events directly influence aging.     

Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.     

Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes

The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.     

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.     

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.     

Nuclear accumulation of the small GTPase Gsp1p depends on nucleoporins Nup133p,Rat2p/Nup120p,Nup85p,Nic96p,and the acetyl-CoA carboxylase Acc1p

The small GTPase Ran/Gsp1p plays an essential role in nuclear trafficking of macromolecules, as Ran/Gsp1p regulates many transport processes across the nuclear pore complex (NPC). To determine the role of nucleoporins in the generation of the nucleocytoplasmic Gsp1p concentration gradient, mutations in various nucleoporin genes were analyzed in the yeast Saccharomyces cerevisiae. We show that the nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the enzyme acetyl-CoA carboxylase (MTR7) control the distribution and cellular concentration of Gsp1p. At the restrictive temperature the reporter protein GFP-Gsp1p, which is too large to diffuse across the nuclear envelope, fails to concentrate in nuclei of nup133delta, rat2-1, nup85delta, nic96deltaC, and mtr7-1 cells, demonstrating that GFP-Gsp1p nuclear import is deficient. In addition, the concentration of Gsp1p is severely reduced in mutants nup133Delta and mtr7-1 under these conditions. We have now identified the molecular mechanisms that contribute to the dissipation of the Gsp1p concentration gradient in these mutants. Loss of the Gsp1p gradient in nup133delta and rat2-1 can be explained by reduced binding of the Gsp1p nuclear carrier Ntf2p to NPCs. Likewise, nup85delta cells that mislocalize GFP-Gsp1p at the permissive as well as non-permissive temperature have a diminished association of Ntf2p-GFP with nuclear envelopes under both conditions. Moreover, under restrictive conditions Prp20p, the guanine nucleotide exchange factor for Gsp1p, mislocalizes to the cytoplasm in nup85delta, nic96deltaC, and mtr7-1 cells, thereby contributing to a collapse of the Gsp1p gradient. Taken together, components of the NPC subcomplex containing Rat2p/Nup120p, Nup133p, and Nup85p, in addition to proteins Nic96p and Mtr7p, are shown to be crucial for the formation of a nucleocytoplasmic Gsp1p gradient.     

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.     

Functional Characterization of a Nup159p-containing Nuclear Pore Subcomplex

Nup159p/Rat7p is an essential FG repeat–containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)⁺ RNA export and NPC distribution. A detailed structural–functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Δ108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Δ108 cells grown at 37°C, a temperature at which the Nup82Δ108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Δ108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)⁺ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.     

Leader-Induced Phosphorylation of Nucleoporins Correlates with Nuclear Trafficking Inhibition by Cardioviruses

Picornaviruses disrupt nucleocytoplasmic trafficking pathways during infection. Poliovirus and rhinovirus inhibit nuclear protein import/export through a series of 2A protease-dependent cleavages within nuclear pore proteins (nucleoporins [Nups]), including Nup62, Nup98, and Nup153. Cardioviruses lack the same protease and instead affect trafficking inhibition through an activity mapped to their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide with no known enzymatic activity. We have shown that L from encephalomyocarditis virus (EMCV) binds and inhibits the activity of Ran-GTPase, a key regulator of nucleocytoplasmic transport. We now report that recombinant EMCV L triggers the unregulated efflux of protein cargo from preloaded HeLa cell nuclei in cell-free reactions dependent upon Xenopus egg cytosol or HeLa cell-derived cytosol. Recombinant L was the only viral protein necessary for this activity or for nuclear protein import inhibition. Mutational disruption of the L protein zinc finger domain (C₁₉A) abrogated the inhibitory activity for both import and efflux in cell extracts, but mutations in the C-terminal acidic domain of L (aa 37 to 61) did not. Notably, HeLa cell nuclei treated with L, or those from EMCV-infected cells, showed reproducibly altered patterns of nucleoporin phosphorylation. Nup62, Nup153, and Nup214 each became hyperphosphorylated in an L-dependent manner. Staurosporine, a broad-spectrum kinase inhibitor, blocked this phosphorylation and rescued nuclear import/export activity from L-dependent inhibition. Therefore, cardioviruses target the same group of nucleoporins as enteroviruses, but the effector mechanism triggered by L (or L-Ran complexes) involves a unique cytosol-dependent phosphorylation cascade rather than proteolysis.     

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.     

Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96.

The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.