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.     

Amino-terminal sequences that direct nucleoporin Nup153 to the inner surface of the nuclear envelope

Nup153 is a large O-linked glycoprotein that is a component of the basket-like structure that forms the nucleoplasmic face of nuclear pore complexes (NPCs). The Nup153 molecule has a tripartite structure consisting of N- and C-terminal domains flanking a central zinc finger domain. All of the targeting and assembly information contained within Nup153 is contributed by the N-domain. In fact this region of the molecule can target a cytosolic protein, pyruvate kinase, to the nucleoplasmic face of the NPC. The zinc finger and C-terminal domains appear to have no role in these targeting and assembly activities. Deletion analysis reveals that there are two distinct regions within the Nup153 N-domain that contain different targeting functions. One of these is directly involved in assembly into the NPC while a second overlapping region may target Nup153, as well as other reporter molecules, to the inner face of the nuclear envelope. Received: 2 March 1998; in revised form: 4 June 1998 / Accepted: 24 June 1998     

The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155

The protein Gle1 is required for export of mRNAs from the nucleus to the cytoplasm in both lower and higher eukaryotic cells. In human (h) cells, shuttling of hGle1 between the nucleus and cytoplasm is essential for bulk mRNA export. To date, no hGle1-interacting proteins have been reported and the mechanism by which hGle1 interacts with the nuclear pore complex (NPC) and mediates export is unknown. To identify proteins that can interact with hGle1, a genome-wide yeast two-hybrid screen was performed. Three potential hGle1-interacting partners were isolated, including clones encoding the C-terminal region of the NPC protein hNup155. This interaction between hGle1 and full-length hNup155 was confirmed in vitro, and deletion analysis identified the N-terminal 29 residues of hGle1 as the hNup155-binding domain. Experiments in HeLa cells confirmed that the nuclear rim localization of the major hGle1 protein variant (hGle1B) was dependent on the presence of these 29 N-terminal residues. This suggests that this domain of hGle1 is necessary for targeting to the NPC. This work also characterizes the first domain in hNup155, a 177 C-terminal amino acid span that binds to hGle1. The mutual interaction between hGle1 and the symmetrically distributed nuclear pore protein Nup155 suggests a model in which hGle1's association with hNup155 may represent a step in the Gle1-mediated mRNA export pathway.     

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.     

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.     

Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.     

Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.     

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.     

Structural constraints on autoprocessing of the human nucleoporin Nup98

Nucleoporin Nup98, a 98-kDa protein component of the nuclear pore complex, plays an important role in both protein and RNA transport. During its maturation process, Nup98 undergoes post-translational autoproteolysis, which is critical for targeting to the NPC. Here we present high-resolution crystal structures of the C-terminal autoproteolytic domains of Nup98 (2.3 A for the wild type and 1.9 A for the S864A precursor), and propose a detailed autoproteolysis mechanism through an N-O acyl shift. Structural constraints are found at the autocleavage site, and could thus provide a driving force for autocleavage at the scissile peptide bond. Such structural constraints appear to be generated, at least in part, by anchoring a conserved phenylalanine side chain into a highly conserved hydrophobic pocket at the catalytic site. Our high-resolution crystal structures also reveal that three highly conserved residues, Tyr866, Gly867, and Leu868, provide most of the interactions between the autoproteolytic domain and the C-terminal tail. These results suggest that Nup98 may represent a new subtype of protein that utilizes autoprocessing to control biogenesis pathways and intracellular translocation.     

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.     

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.     

Intranuclear dynamics of the Nup107-160 complex

Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat–containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup–containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies.     

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.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

RNA association defines a functionally conserved domain in the nuclear pore protein Nup153

Traffic between the nucleus and cytoplasm takes place through a macromolecular structure termed the nuclear pore complex. To understand how the vital process of nucleocytoplasmic transport occurs, the contribution of individual pore proteins must be elucidated. One such protein, the nucleoporin Nup153, is localized to the nuclear basket of the pore complex and has been shown to be a central component of the nuclear transport machinery. Perturbation of Nup153 function was demonstrated previously to block the export of several classes of RNA cargo. Moreover, these studies also showed that Nup153 can stably associate with RNA in vitro. In this study, we have mapped a domain within Nup153, encompassing amino acids 250-400 in human Nup153, that is responsible for RNA association. After cloning this region of Xenopus Nup153, we performed a cross-species analysis. Despite variation in sequence conservation between Drosophila, Xenopus, and human, this domain of Nup153 displayed robust RNA binding activity in each case, indicating that this property is a hallmark feature of Nup153 and pointing toward a subset of amino acid residues that are key to conferring this ability. We have further determined that a recombinant fragment of Nup153 can bind directly to RNA and that this fragment can interact with endogenous RNA targets. Our findings identify a functionally conserved domain in Nup153 and suggest a role for RNA binding in Nup153 function at the nuclear pore.     

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.     

Molecular determinants of binding between Gly-Leu-Phe-Gly nucleoporins and the nuclear pore complex

The vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N.