The effects of variations in the number and sequence of targeting signals on nuclear uptake

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.     

The permeability of the nuclear envelope in dividing and nondividing cell cultures

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.     

Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.     

Foreign protein can be carried into the nucleus of mammalian cell by conjugation with nucleoplasmin

In studies on the specific migration of macromolecules across the nuclear envelope, a karyophilic protein was injected into the cytoplasm of cultured cells and its subsequent location in the cell was examined. Nucleoplasmin of frog nuclear protein was used for this experiment. When [125I]nucleoplasmin was introduced into the cytoplasm of mammalian cells (human and mouse) by red blood cell-mediated microinjection, it rapidly accumulated in the nucleus. When nucleoplasmin conjugated with [125I]IgG against chromosomal protein was introduced similarly, it also accumulated rapidly in the nucleus, and reacted with its antigen inside the nucleus. On the contrary, when IgG alone or IgG conjugated with BSA were introduced, they did not migrate from the cytoplasm into the nucleus. These findings imply that the migration of macromolecules from the cytoplasm to the nucleus does not depend only on their molecular size but also on a specific transport mechanism, and that karyophilic proteins may act as useful carriers in the transfer of exogenous proteins into the nucleus.     

The function of the nuclear envelope in nuclear protein accumulation

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.     

Translocation of RNA-coated gold particles through the nuclear pores of oocytes

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.     

Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells

The importance of glycoproteins located in the nuclear envelope in nuclear transport was tested by microinjection of karyophilic proteins into the cytoplasm of cultured human cells together with various lectins. Wheat germ agglutinin (WGA) blocked the nuclear transport of nucleoplasmin, a nuclear protein of Xenopus laevis oocytes, and of nonnuclear proteins conjugated with a synthetic peptide containing the nuclear localization signal sequence for simian virus 40 (SV40) large T antigen. Its inhibitory activity persisted for about 1 h after its injection into the cells and then gradually decreased. Export of at least some kinds of RNA from the nucleus seemed not to be affected by WGA even when import of the proteins into the nucleus was completely blocked (within 1 h after WGA injection). Moreover, WGA did not inhibit the passive diffusion of fluorescein isothiocyanate (FITC)-dextran (average Mr 17,900) into the nucleus. Wistaria floribunda agglutinin (WFA), concanavalin A (Con A), and lentil lectin did not block nuclear transport. These results indicate that WGA specifically blocks active protein import, but not passive diffusion of materials into the nucleus.     

Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis

Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not interfere with normal nuclear accumulation. Upon injection into the cytoplasm of various mammalian cell types nucleoplasmin was rapidly taken up by the nucleus. In rat hepatoma cells the half-time of nuclear uptake was approx. 5 min at 37 degrees C; the nucleocytoplasmic equilibrium concentration ratio had a maximum of 6.5 +/- 1.4 and depended on the injected amount. Upon co-injection of ATPases or reduction of temperature to 10 degrees C a nucleocytoplasmic equilization but no nuclear accumulation was observed. Equilization was fast (time constant 65 s at 23 degrees C), similar to that of 10-kDa dextran permeating the nuclear envelope by simple diffusion through functional pores. Nucleoplasmin (160 kDa), however, is too large to permeate passively the nuclear envelope, which is apparent from the fact that its tryptic 'core' fragment (100 kDa) could not permeate the nuclear envelope. On the other hand, a large fluorescent protein, phycoerythrin (240 kDa), was targeted to the nucleus by conjugation with nucleoplasmin. In the nucleus-to-cytoplasm direction the nuclear envelope was completely impermeable to nucleoplasmin, independently of temperature or ATP depletion. Nucleoplasmin, its core fragment, phycoerythrin and the phycoerythrin-nucleoplasmin conjugate were mobile in both cytoplasm and nucleus.     

THE NUCLEAR ANNULI AS PATHWAYS FOR NUCLEOCYTOPLASMIC EXCHANGES

Colloidal gold particles, 25 to 55 A in diameter, which had been coated with polyvinylpyrrolidone, were microinjected into the ground cytoplasm of amebas (Chaos chaos). At time intervals of 1 minute, 2 minutes, 10 minutes, and 24 hours after injection the cells were fixed for electron microscopy. After 24 hours, gold particles were found in both the nuclei and the ground cytoplasm, the concentration being higher in the nuclei. Colloidal particles were also present in the nuclei after 10 minutes, but at this time interval the concentration did not appear to be greater than that in the ground cytoplasm. One and 2 minutes after injection, the gold particles were located almost exclusively in the ground cytoplasm; however, individual particles were often found within the annuli of the nuclear envelope, and were located specifically in the centers of these structures. The results suggest that at least some of the gold particles which enter the nuclei pass through the annuli, and that passage through these structures may be restricted to a central channel.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.     

THE EFFECT OF THE ELECTRON-OPAQUE PORE MATERIAL ON EXCHANGES THROUGH THE NUCLEAR ANNULI

To investigate the extent to which the electron-opaque pore material can regulate nucleocytoplasmic exchanges which occur through the nuclear annuli, experiments were performed in which polyvinylpyrrolidone (PVP)-coated colloidal gold particles (25 to 170 A in diameter) were microinjected into the cytoplasm of amebas (Amoeba proteus). The cells were fixed at various times after injection and examined with the electron microscope in order to determine the location of the gold particles. High concentrations of gold were found associated with the pore material at specific points adjacent to and within the pores. It is tentatively suggested that such specific accumulations could be a means of selecting substances from the cytoplasm for transport through the pores. Particles were also scattered throughout the ground cytoplasm and nucleoplasm. A comparison of the diameters of particles located in these two regions showed that the ability of materials to penetrate the nuclear envelope is a function of their size. It was estimated that the maximum size of the particles able to enter the nucleus is approximately 125 to 145 A indiameter. The regulation of exchanges with regard to particle size is thought to be dependent on the specific organization of the electron-opaque pore material.     

Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium.

We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein. Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes. In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific. Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells. The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes. These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway.     

Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.     

Nucleoplasmin: the archetypal molecular chaperone

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.     

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.     

Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin

The lectin wheat germ agglutinin (WGA), which has been reported to inhibit nuclear protein uptake in vitro by isolated nuclei (Finlay et al. (1987) J. Cell Biol. 104, 189), also blocks, on microinjection into living cells, the migration of proteins into the cell nucleus. Radioactively labeled nuclear proteins were injected into the cytoplasm of Xenopus oocytes and their reentry into the nucleus was analyzed in the presence or absence of WGA by two-dimensional gel electrophoresis. In another set of experiments, fluorescently labeled nucleoplasmin was injected, alone or together with WGA, into the cytoplasm of rat hepatoma cells, and its nucleocytoplasmic distribution was studied by quantitative laser fluorescence microscopy. The results indicate that WGA inhibits the uptake of karyophilic proteins in general, independent of their sizes. Since the nucleocytoplasmic flux of a dextran with Mr 10,000 was not affected it can be excluded that WGA acts by a general blockade or constriction of the functional pore channel. At reduced WGA concentrations, the rate but not the final extent of nuclear protein accumulation was decreased. These findings support the concept that the O-glycosidically bound carbohydrates of certain nuclear pore complex proteins are exposed to the pore interior and that these regions are probably involved in nucleocytoplasmic translocation processes.     

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

We analysed the soluble form in which the nuclear pore complex protein p68 is stored in Xenopus laevis eggs and its involvement in pore complex assembly processes. We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein of nuclear pore complexes from Xenopus oocytes, is located in the pore channel and participates in mediated transport of karyophilic proteins. Using a monoclonal antibody directed against p68 (PI1) we removed this protein from Xenopus egg extract by immunoadsorption. On addition of lambda DNA the immunodepleted extract supported reconstitution of nuclei which were surrounded by a continuous double-membrane envelope but lacked pore complexes and were unable to import karyophilic proteins such as nucleoplasmin or lamin L_III. Essentially identical results were obtained with extract depleted of WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extract necessary for pore complex assembly but did not interfere with nuclear membrane formation demonstrates that these processes are independent of each other. Analysis of the immunoprecipitate on silver-stained SDS-polyacrylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel filtration we showed that p68 together with associated protein(s) forms a stable, approximately globular complex plex with an M_r of 254,000, a Stokes radius of 5.2 nm and a sedimentation coefficient of 11.3 S. Our finding that p68 occurs in the form of larger macromolecular assemblies offers an explanation for the distinctly punctate immunofluorescence pattern observed in the cytoplasm of mitotic cells after staining with antibodies to p68.W. Hennig