Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.     

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.     

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.     

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.     

Nop53p is required for late 60S ribosome subunit maturation and nuclear export in yeast

We report that Ypl146cp/Nop53p is associated with pre-60S ribosomal complexes and localized to the nucleolus and nucleoplasm. In cells depleted of Nop53p synthesis of the rRNA components of the 60S ribosomal subunit is severely inhibited, with strikingly strong accumulation of the 7S pre-rRNA and a 5' extended form of the 25S rRNA. In cells depleted of Nop53p pre-60S subunits accumulate in the nucleus. However, a heterokaryon assay demonstrated that Nop53p is not transferred between nuclei, indicating that it is not released into the cytoplasm. We conclude that Nop53p is a late-acting factor in the nuclear maturation of 60S ribosomal subunits, which is required for normal acquisition of export competence. The strong accumulation of preribosomes in the Nop53p-depleted strain further suggests that it may participate in targeting aberrant preribosomes to surveillance and degradation pathways.     

Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex

Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.     

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.     

A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.     

The yeast nucleoporin Nup2p is involved in nuclear export of importin alpha/Srp1p.

The importin alpha.beta heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin alpha subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in Deltanup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in Deltanup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an N-terminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export. Formation of the Srp1p.Cse1p. RanGTP export complex releases Srp1p from its binding site in Nup2p. We propose that Nup2p may act as a scaffold that facilitates formation of the Srp1p export complex.     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.     

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.     

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.     

The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm,respectively

We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.     

Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes

A number of chemicals, mostly adenosine analogues, cause the nucleolus of the chick embryo fibroblast to lose material and unravel over a period of several hours into beaded strands termed nucleolar necklaces (NN). The results of analyses of the fibroblasts, treated with the NN-forming chemical dichlororibobenzimidazole (DRB), suggests that the following biochemical alterations occur: DRB almost completely prevents the increase in both messenger RNA (mRNA) and heterogeneous nuclear RNA. It interferes with ribosome synthesis by decreasing the rate of 45S ribosomal RNA (rRNA) accumulation by 50%, slowing the rate of 18S rRNA appearance by 50%, and causing an extensive degradation (80%) of the 32S and 28S rRNA-containing preribisomes. Most of this preribosome degration probably occurs at or before the 32S rRNA preribosome stage. The degradation of these preribosomes appears to be due to the formation of defective 45S rRNA preribosomes rather than to a direct DRB interference with preribosome processing enzyme action. DRB inhibits total cellular RNA synthesis in less than 15 min, suggesting a direct interference with RNA synthesis. DRB also inhibits the uptake of nucleosides into the cell. DRB in the concentrations used does not appear to directly interfere with the translation of mRNA (i.e., protein synthesis). Other NN-forming adenoside analogues and high concentrations of adenosine (2 mM) cause biochemical alterations similar to those produced by DRB. To explain the preribosome degradation, we propose the hypothesis that DRB inhibits the synthesis of mRNA; as a consequence, some of the preribosomal proteins that normally coat the 32S rRNA portion of the 45S precursor RNA become limiting, and this defective portion is then subject to degradation by nucleases.     

Molecular basis for the anchoring of proto-oncoprotein Nup98 to the cytoplasmic face of the nuclear pore complex

The cytoplasmic filament nucleoporins of the nuclear pore complex (NPC) are critically involved in nuclear export and remodeling of mRNA ribonucleoprotein particles and are associated with various human malignancies. Here, we report the crystal structure of the Nup98 C-terminal autoproteolytic domain, frequently missing from leukemogenic forms of the protein, in complex with the N-terminal domain of Nup82 and the C-terminal tail fragment of Nup159. The Nup82 β propeller serves as a noncooperative binding platform for both binding partners. Interaction of Nup98 with Nup82 occurs through a reciprocal exchange of loop structures. Strikingly, the same Nup98 groove promiscuously interacts with Nup82 and Nup96 in a mutually excusive fashion. Simultaneous disruption of both Nup82 interactions in yeast causes severe defects in mRNA export, while the severing of a single interaction is tolerated. Thus, the cytoplasmic filament network of the NPC is robust, consistent with its essential function in nucleocytoplasmic transport.     

Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we discovered an unexpected role for yeast dynein light chain (Dyn2) in the NPC. Dyn2 is a previously undescribed nucleoporin that functions as molecular glue to dimerize and stabilize the Nup82-Nsp1-Nup159 complex, a module of the cytoplasmic pore filaments. Biochemical analyses showed that Dyn2 binds to a linear motif (termed DID(Nup159)) inserted between the Phe-Gly repeat and coiled-coil domain of Nup159. Electron microscopy revealed that the reconstituted Dyn2-DID(Nup159) complex forms a rigid rod-like structure, in which five Dyn2 homodimers align like 'pearls on a string' between two extented DID(Nup159) strands. These findings imply that the rigid 20 nm long Dyn2-DID(Nup159) filament projects the Nup159 Phe-Gly repeats from the Nup82 module. Thus, it is possible that dynein light chain plays a role in organizing natively unfolded Phe-Gly repeats within the NPC scaffold to facilitate nucleocytoplasmic transport.     

Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.     

The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37 degrees C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37 degrees C, the mutant Rat7-1p/Nup159-1p is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rss1p did not result in retention of the mutant Rat7-1p/Nup159-1p in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rss1p by placing the RSS1 open reading frame (ORF) under control of the GAL1 promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rss1p are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rss1p overexpression suppresses the growth defect of rat7-1 cells at 37 degrees C by acting to maintain mRNA export.