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.     

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co- purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.     

In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.     

Structural basis of the nic96 subcomplex organization in the nuclear pore channel

Nic96 is a conserved nucleoporin that recruits the Nsp1-Nup49-Nup57 complex, a module with Phe-Gly (FG) repeats, to the central transport channel of the nuclear pore complex (NPC). Nic96 binds the Nsp1 complex via its N domain and assembles into the NPC framework via its central and C domain. Here, we report the crystal structure of a large structural nucleoporin, Nic96 without its N domain (Nic96DeltaN). Nic96DeltaN is composed of three domains and is a straight molecule that--although almost entirely helical--exhibits strong deviations from the predicted alpha-solenoid fold. The missing N domain projects midway from the Nic96 molecule, indicating how the Nsp1 complex might be located with respect to the rod-like Nic96. Notably, Nic96DeltaN binds in vitro to FG repeats of the Nsp1 complex. These data suggest a model of how Nic96 could organize a transport module with coiled-coil domains and FG repeats in the central pore channel.     

Crystal structure of nucleoporin Nic96 reveals a novel, intricate helical domain architecture

The nuclear pore complex (NPC) is an elaborate protein machine that mediates macromolecular transport across the nuclear envelope in all eukaryotes. The NPC is formed by nucleoporins that assemble in multiple copies around an 8-fold symmetry axis. Homology modeling suggests that most architectural nucleoporins are composed of simple beta-propeller and alpha-helical repeat domains. Here we present the crystal structure of Nic96, the Nup93 homolog in Saccharomyces cerevisiae, one of the major components of the NPC. This is the first structure of an alpha-helical nucleoporin domain. The protein folds into an elongated, mostly alpha-helical structure. Characteristically, non-canonical architectural features define the Nic96 structure. Sequence conservation among Nup93 homologs across all eukaryotes strongly suggests that the distinct topology is evolutionarily well maintained. We propose that the unique Nic96/Nup93 fold has a conserved function in all eukaryotes.     

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57p^E17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.     

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.     

The Nsp1p Carboxy-Terminal Domain Is Organized into Functionally Distinct Coiled-Coil Regions Required for Assembly of Nucleoporin Subcomplexes and Nucleocytoplasmic Transport

Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1 coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640S temperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.     

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.     

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.     

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.     

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.     

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.     

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.     

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.     

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.     

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.     

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.     

Nup192p is a conserved nucleoporin with a preferential location at the inner site of the nuclear membrane.

Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown. We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin. Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p. Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p. NUP192 is essential for cell growth. A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature. By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 +/- 15 nm from the central plane of the pore. This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane.