Perinuclear and nuclear envelope localizations of <Emphasis Type="Italic">Arabidopsis</Emphasis> Ran proteins

Using phragmoplastin-interacting protein 1 (PhrIP1) as bait, we isolated an Arabidopsis cDNA encoding Ran2, a small Ras-like GTP-binding protein. The interaction between PhrIP1 and Ran2 was confirmed by an in vitro protein–protein interaction assay with purified Ran2 and PhrIP1. The plant Ran2 shares high sequence homology, 78 and 86% at the amino acid level, with human Ran/TC4 and C. elegans Ran, respectively. Our results obtained from enzyme assays and Western blot analysis show that Ran2 has intrinsic GTPase activity and is present in the soluble fraction of Arabidopsis seedling extract. Fluorescent microscopy using anti-Ran2 antibody revealed that the Ran protein is localized in the perinuclear region with the highest concentration at the nuclear envelope. In contrast to its animal counterparts that are present in the nucleoplasm, the Ran protein is absent inside the nucleus. These results suggest that plant Ran proteins may be involved in mediation of nucleocytoplasmic transport and assembly of the nuclear envelope after karyokinesis in plant cells.     

ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope

Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mm) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-γ-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore ``contraction.' Received: 10 February 1997/Revised: 10 February 1998     

Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.     

Composition of the plant nuclear envelope: theme and variations

The nuclear envelope is the hallmark of all eukaryotic cells, separating the nucleoplasm from the cytoplasm. At the same time, the nuclear envelope allows for the controlled exchange of macromolecules between the two compartments through nuclear pores and presents a surface for anchoring and organizing cytoskeletal components and chromatin. Although our molecular understanding of the nuclear envelope in higher plants is only just beginning, fundamental differences from the animal nuclear envelope have already been found. This review provides an updated investigation of these differences with respect to nuclear pore complexes, targeting of Ran signalling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis.     

The ultrastructure of the nuclear envelope of amphibian oocytes

Summary In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10^–16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10^–15 g/³) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.Additionally, the results of the chemical analyses as well as of the³H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.The author thanks Miss Ulrika Lempert, Miss Marianne Winter, and Miss Sigrid Krien for skilful technical help as well as Dr. W. W. Franke for many helpful discussions. The work has been supported by a Deutsche Forschungsgemeinschaft grant given to Dr. W. W. Franke (SFB Molgrudent, 46).     

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport‐competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well‐established function in nuclear transport. RNAi‐mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/β, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N‐terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild‐type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

The karyopherin Kap95 regulates nuclear pore complex assembly into intact nuclear envelopes in vivo

Nuclear pore complex (NPC) assembly in interphase cells requires that new NPCs insert into an intact nuclear envelope (NE). Our previous work identified the Ran GTPase as an essential component in this process. We proposed that Ran is required for targeting assembly factors to the cytoplasmic NE face via a novel, vesicular intermediate. Although the molecular target was not identified, Ran is known to function by modulating protein interactions for karyopherin (Kap) beta family members. Here we characterize loss-of-function Saccharomyces cerevisiae mutants in KAP95 with blocks in NPC assembly. Similar to defects in Ran cycle mutants, nuclear pore proteins are no longer localized properly to the NE in kap95 mutants. Also like Ran cycle mutants, the kap95-E126K mutant displayed enhanced lethality with nic96 and nup170 mutants. Thus, Kap95 and Ran are likely functioning at the same stage in assembly. However, although Ran cycle mutants accumulate small cytoplasmic vesicles, cells depleted of Kap95 accumulated long stretches of cytoplasmic membranes and had highly distorted NEs. We conclude that Kap95 serves as a key regulator of NPC assembly into intact NEs. Furthermore, both Kap95 and Ran may provide spatial cues necessary for targeting of vesicular intermediates in de novo NPC assembly.     

Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran.     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca²⁺-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.     

The molecular mechanism of transport of macromolecules through nuclear pore complexes

Trafficking of macromolecules between nuclear and cytoplasmic compartments takes place through the nuclear pore complexes (NPCs) of the nuclear envelope. Nuclear trafficking involves a complex series of interactions between cargo, soluble transport factors (carriers) and nuclear pore proteins (nucleoporins) that are orchestrated by the Ras-family GTPase Ran. The primary role of Ran is probably to establish directionality and to sort molecules to be transported by controlling the interaction between carriers and cargoes, so that they bind in one compartment but dissociate in the other. Translocation of carriers and cargo-carrier complexes through NPCs requires interactions between the carriers and nucleoporins that contain distinctive tandem sequence repeats based on cores rich in glycine and phenylalanine residues that are separated by hydrophilic linkers. Much recent work has focused on these interactions and, in particular, their specificity, regulation and function. Evidence is accumulating that carriers move through the NPC by distinct but overlapping routes using specific subsets of nucleoporins.     

Spatial Distribution and Mobility of the Ran GTPase in Live Interphase Cells

The GTPase Ran is a key regulator of molecular transport through nuclear pore complex (NPC) channels. To analyze the role of Ran in its nuclear transport function, we used several quantitative fluorescence techniques to follow the distribution and dynamics of an enhanced yellow fluorescent protein (EYFP)-Ran in HeLa cells. The diffusion coefficient of the majority of EYFP-Ran molecules throughout the cells corresponded to an unbound state, revealing the remarkably dynamic Ran regulation. Although we observed no significant immobile Ran populations in cells, ∼10% of the cytoplasmic EYFP-Ran and 30% of the nuclear EYFP-Ran exhibited low mobility indicative of molecular interactions. The high fraction of slow nuclear EYFP-Ran reflects the expected numerous interactions of nuclear RanGTP with nuclear transport receptors. However, it is not high enough to support retention mechanisms as the main cause for the observed nuclear accumulation of Ran. The highest cellular concentration of EYFP-Ran was detected at the nuclear envelope, corresponding to ∼200 endogenous Ran molecules for each NPC, and exceeding the currently estimated NPC channel transport capacity. Together with the relatively long residence time of EYFP-Ran at the nuclear envelope (33 ± 14 ms), these observations suggest that only a fraction of the Ran concentrated at NPCs transits through NPC channels.     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

Structural Characterisation of the Nuclear Import Receptor Importin Alpha in Complex with the Bipartite NLS of Prp20

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Ų of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters ¹⁷RAKKMSK²³ and ³KR⁴, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala¹⁸ between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C‐terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.     

On the universality of nuclear pore complex structure

Summary Substructural details of the nuclear pore complex were studied in diverse plant and animal cells with both section technique and negative staining of isolated nuclear envelope pieces. The structures observed after the different techniques, including a variety of fixation procedures, are compared and their significance is discussed. It is shown that, down to the 15–20 Å level, the architecture of the nuclear pore complex is universal among such diverse cell types as from, e. g., onion root tips, bean leaves, mammalian liver parenchyma, HeLa cell cultures, and amphibian germ material. The fundamental substructures of the pore complex such as (1) the annular granules, (2) the fibrils attached to the annuli, (3) the central granules, (4) the fibrils in the pore interior including those which make up the inner ring and/or those which connect the central granule to the pore margin, are recognized in all cell types studied. The dynamic variability of the central granule morphology is emphasized and observations are presented which suggest that the view of such centrally located material as representing ribonucleoproteins in a transitory state of nucleocytoplasmic migration can be extended to generality. General concepts of the nuclear pore complex structure are presented as alternative model views revealing either a more compact, predominantly granular, or a more fibrillar aspect.The author gratefully acknowledges the frequent discussions and cooperation with his team-colleagues Drs. H. Falk (in the work on leaf material) and U. Scheer (in working with amphibian oocytes) as well as the skillful technical assistance of Miss Marianne Winter and Miss Sigrid Krien. The work was supported by the Deutsche Forschungsgemeinschaft.