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.     

Targeting and function in mRNA export of nuclear pore complex protein Nup153

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH- terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2- terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.     

The RNA binding domain within the nucleoporin Nup153 associates preferentially with single-stranded RNA

The nuclear pore protein Nup153 is important for the transport of protein and RNA between the nucleus and cytoplasm. Recently, a novel RNA binding domain (RBD) was mapped within the N-terminal region of Nup153; however, the determinants of RNA association were not characterized. Here we have tested a range of RNAs with different general features to better understand targets recognized by this domain. We have found that the RBD associates with single-stranded RNA with little sequence preference. These results provide new information about a novel RNA binding domain and suggest new models to consider for the contribution of Nup153 to nucleocytoplasmic transport.     

The nucleoporin nup153 plays a critical role in multiple types of nuclear export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.     

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.     

Molecular characterization of the Ran-binding zinc finger domain of Nup153

The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.     

In vitro analysis of nuclear transport mediated by the C-terminal shuttle domain of Tap

The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitro assay to study nuclear export mediated by the C-terminal shuttle domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin beta-related export receptor CRM1 competes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.     

Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Background: Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin β, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it.Results: Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin α/β, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin β binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim.Conclusions: Two nuclear import pathways, mediated by importin β and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

The interaction between importin-α and Nup153 promotes importin-α/β-mediated nuclear import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.     

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.     

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import.     

The crystal structure of the Ran-Nup153ZnF2 complex: a general Ran docking site at the nuclear pore complex

Nucleoporin (Nup) 153 is a highly mobile, multifunctional, and essential nuclear pore protein. It contains four zinc finger motifs that are thought to be crucial for the regulation of transport-receptor/cargo interactions via their binding to the small guanine nucleotide binding protein, Ran. We found this interaction to be independent of the phoshorylation state of the nucleotide. Ran binds with the highest affinity to the second zinc finger motif of Nup153 (Nup153ZnF2). Here we present the crystal structure of this complex, revealing a new type of Ran-Ran interaction partner interface together with the solution structure of Nup153ZnF2. According to our complex structure, Nup153ZnF2 binding to Ran excludes the formation of a Ran-importin-beta complex. This finding suggests a local Nup153-mediated Ran reservoir at the nucleoplasmic distal ring of the nuclear pore, where nucleotide exchange may take place in a ternary Nup153-Ran-RCC1 complex, so that import complexes are efficiently terminated.     

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.