The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88.

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.     

Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca²⁺ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca²⁺ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M₁ receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca²⁺ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.     

Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore,Nuclear Import and Integration

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.     

G2 arrest and impaired nucleocytoplasmic transport in mouse embryos lacking the proto-oncogene CAN/Nup214.

The vertebrate nucleopore complex (NPC) is a 125 MDa multiprotein assembly that mediates nucleocytoplasmic transport. One of its components, CAN/Nup214, is an FXFG repeat-containing protein known to be involved in myeloid leukemia in humans. We have devised a powerful genetic approach, using maternally derived protein in murine null embryos, to show that CAN/ Nup214 is essential for NPC function in vivo. We demonstrate that CAN-/- mouse embryonic stem (ES) cells are not viable and that CAN-/- embryos die in utero between 4.0 and 4.5 days postcoitum, following the depletion of their CAN from maternal sources. In 3.5-day-old mutant embryos, cultured in vitro, progressive depletion of CAN leads to cell cycle arrest in G2 phase, and eventually to blastocoel collapse, impaired NLS-mediated protein uptake and nuclear accumulation of polyadenylated RNA. Remarkably, these defective CAN-depleted embryos do not display any gross morphological abnormalities in their nuclear envelopes or NPCs. Our data suggest that CAN is critical to cell cycle progression and required for both nuclear protein import and mRNA export.     

Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis

Background: The transport of macromolecules between the nucleus and cytoplasm is an energy-dependent process. Substrates are translocated across the nuclear envelope through nuclear pore complexes (NPCs). Translocation requires nucleocytoplasmic transport receptors of the importin β family, which interact both with the NPC and, either directly or via an adaptor, with the transport substrate. Although certain receptors have recently been shown to cross the NPC in an energy-independent manner, translocation of substrate–receptor complexes through the NPC has generally been regarded as an energy-requiring step.Results: We describe an in vitro system that is based on permeabilised cells and supports nuclear export mediated by leucine-rich nuclear export signals. In this system, export is dependent on exogenous CRM1/Exportin1 – a nuclear export receptor – the GTPase Ran and nucleotide triphosphates (NTPs), and is further stimulated by Ran-binding protein 1 (RanBP1) and nuclear transport factor 2 (NTF2). Unexpectedly, non-hydrolysable NTP analogues completely satisfy the NTP requirements for a single-round of CRM1-mediated translocation of protein substrates across the NPC. Similarly, single transportin-mediated nuclear protein import events are shown not to require hydrolysable NTPs and to occur in the absence of the Ran GTPase.Conclusions: Our data show that, contrary to expectation and prior conclusions, the translocation of substrate–receptor complexes across the NPC in either direction occurs in the absence of NTP hydrolysis and is thus energy independent. The energy needed to drive substrate transport against a concentration gradient is supplied at the step of receptor recycling in the cytoplasm.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.     

Kindlin-2 is required for myocyte elongation and is essential for myogenesis

Background

Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.

Results

Separation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G₀/G₁ cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G₀/G₁ growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.

Conclusion

The findings demonstrated that monocytic differentiation and G₀/G₁ growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.     

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

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

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t_1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G₀ are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t_1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1⁻ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.     

Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153_C). NUP153_C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153_C) potently restricted HIV-1, providing an intracellular readout for the NUP153_C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153_C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153_C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153_C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153_C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153_C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153_C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.     

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.     

Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p,Nup59p,and Nup170p

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.     

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP₆) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP₆ are also required for proper translation termination. However, the specificity of the IP₆-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP₆ and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP₆ binding and show that these residues are needed for IP₆-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP₆ for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP₆-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP₆. We conclude that Gle1 specifically binds IP₆ and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.     

Nuclear pore complex function in Saccharomyces cerevisiae is influenced by glycosylation of the transmembrane nucleoporin Pom152p

The regulated transport of proteins across the nuclear envelope occurs through nuclear pore complexes (NPCs), which are composed of >30 different protein subunits termed nucleoporins. While some nucleoporins are glycosylated, little about the role of glycosylation in NPC activity is understood. We have identified loss-of-function alleles of ALG12, encoding a mannosyltransferase, as suppressors of a temperature-sensitive mutation in the gene encoding the FXFG-nucleoporin NUP1. We observe that nup1Delta cells import nucleophilic proteins more efficiently when ALG12 is absent, suggesting that glycosylation may influence nuclear transport. Conditional nup1 and nup82 mutations are partially suppressed by the glycosylation inhibitor tunicamycin, while nic96 and nup116 alleles are hypersensitive to tunicamycin treatment, further implicating glycosylation in NPC function. Because Pom152p is a glycosylated, transmembrane nucleoporin, we examined genetic interactions between pom152 mutants and nup1Delta. A nup1 deletion is lethal in combination with pom152Delta, as well as with truncations of the N-terminal and transmembrane regions of Pom152p. However, truncations of the N-glycosylated, lumenal domain of Pom152p and pom152 mutants lacking N-linked glycosylation sites are viable in combination with nup1Delta, suppress nup1Delta temperature sensitivity, and partially suppress the nuclear protein import defects associated with the deletion of NUP1. These data provide compelling evidence for a role for glycosylation in influencing NPC function.     

A Nuclear Export Signal in Kap95p Is Required for Both Recycling the Import Factor and Interaction with the Nucleoporin GLFG Repeat Regions of Nup116p and Nup100p

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A–tagged Nup116p or protein A–tagged Nup100p complexes. The protein A–tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37°?C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.     

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.     

RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclear pore complex and functions in nuclear mRNA export

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.