Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.     

Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function

PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68,000 pore complex glycoprotein) or of wheat germ agglutinin (WGA) complete cytokinesis. However, their nuclei remain stably arrested in a telophase-like organization characterized by highly condensed chromatin and the absence of nucleoli, indicating a requirement for pore-mediated transport for the reassembly of interphase nuclei. We have now examined this requirement more closely by monitoring the behavior of individual nuclear macromolecules in microinjected cells using immunofluorescence microscopy and have investigated the effect of microinjecting the antibody or WGA on cellular ultrastructure. The absence of nuclear transport did not affect the sequestration into daughter nuclei of components such as DNA, DNA topoisomerase I and the nucleolar protein fibrillarin that are carried through mitosis on chromosomes. On the other hand, lamins, snRNAs and the p68 pore complex glycoprotein, all cytoplasmic during mitosis, remained largely cytoplasmic in the telophase-arrested cells. Electron microscopy showed the nuclei to be surrounded by a double-layered membrane with some inserted pore complexes. In addition, however, a variety of membranous structures with associated pore complexes was regularly noted in the cytoplasm, suggesting that chromatin may not be essential for the postmitotic formation of pore complexes. We propose that cellular compartmentalization at telophase is a two-step process. First, a nuclear envelope tightly encloses the condensed chromosomes, excluding non-selectively all macromolecules not associated with the chromosomes. Interphase nuclear organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm.     

Nuclear transport as an ultimate step of multidrug resistance

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.     

Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication

Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport. Such cell- free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were raised and affinity purified. Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116. An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family. A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted. The p97-depleted nuclei remained largely competent for nuclear protein import. However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ssDNA replication in such extracts remains unaffected. Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects. The possible role(s) of p97 in these nuclear functions is discussed.     

Estradiol-stimulated nuclear ribonucleoprotein transport in the rat uterus: a molecular basis

The present investigation probes the intranuclear molecular changes that serve to link the nuclear binding of estradiol with the hormone-stimulated ribonucleoprotein (RNP) transport in the rat uterus. Within 2 min of in vitro exposure of isolated uterine nuclei to 10 nM 17 beta-estradiol a Mg2+-dependent nuclear ATPase becomes activated and reaches its peak activity. This is immediately followed by a phase of ATP resynthesis. This newly synthesized ATP serves as the substrate for the nuclear protein kinases. Cyclic AMP inhibits this ATP resynthesis and, as a consequence, prevents the estradiol-stimulated nuclear protein kinase activity and the exit of the RNP-estradiol complex from the nuclei. cGMP is stimulatory to the estradiol-mediated nuclear ribonucleoprotein transport.     

Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei.

Isolated yeast nuclei were able to incorporate 3H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg2+. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated [3H]pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.     

Isolation and initial characterization of residual nuclear structures from yeast

Residual nuclear structures have previously been isolated from a wide range of eukaryotic organisms. When nuclei are isolated from Saccharomyces cerevisiae and then treated with 1.95 M NaCl and DNase I, sedimentable residual structures are obtained similar in several respects to structures isolated from organisms previously studied. These yeast residual nuclear structures retain less than 7% of nuclear DNA, less than 17% of nuclear RNA and less than 50% of nuclear proteins. Electron microscopy suggests that these structures are derived from the nuclear interior and are composed of a sparse fibrogranular network. Replicating DNA is preferentially bound to these yeast residual nuclear structures, just as it is to residual nuclear structures from other organisms.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 degrees C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 degrees C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca(2+)ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.     

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II.

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193. The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer. The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities. Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA. These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable. We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks. The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.     

Preparation and characterization of nuclear-envelope vesicles from rat liver nuclei.

We describe a procedure for the preparation of sealed nuclear-envelope vesicles from rat liver nuclei. These vesicles are strikingly similar in their polypeptide composition when compared with those of nuclear envelopes prepared conventionally using deoxyribonuclease I. Subfractionation analysis by means of extraction with high salt and urea show that the components of the nuclear envelope, e.g. the pore-complex/lamina fraction, are present. The residual DNA content is only 1.5%, and typical preparations consist of about 80% vesicles, with the vesicular character of these envelopes shown by microscopic and biochemical studies. The vesicles can be obtained in high yield, are tight and stable for at least two days and are enriched in a nucleoside triphosphatase thought to be involved in nucleocytoplasmic transport processes. Because the vesicles are largely free of components of the nuclear interior, but retain properties of intact nuclei, we believe that they are a valuable model system to study nucleocytoplasmic transport. Although in transport studies with isolated nuclei interference from intranuclear events has to be considered, the nuclear-envelope vesicles provide the possibility of studying translocation alone. Furthermore, the less complex nature of these vesicles compared with whole nuclei should facilitate investigation of the components involved in the regulation of nuclear transport processes.     

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.     

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.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

The fluidity of the nuclear envelope lipid does not affect the rate of nucleocytoplasmic RNA transport in mammalian liver

The effects of in vitro and in vivo modifications of nuclear envelope lipid on DNA leakage and on ATP-stimulated RNA release from isolated rat liver nuclei were investigated. The modifications included corn-oil feeding of the animals to alter the fatty acid composition of the lipids, phospholipase treatment of the isolated nuclei, and extraction of the total lipid with Triton X-100. Significant changes in lipid composition and approximate order parameter values of the spin-label 5-doxylstearate resulted, but there was no significant effect on RNA transport rate. It was concluded that the nuclear envelope lipid does not play any important part in nucleocytoplasmic RNA transport in mammalian liver.     

Mutation of the yeast epsilon-COP gene ANU2 causes abnormal nuclear morphology and defects in intracellular vesicular transport.

Previously we reported an original method of visualizing the shape of yeast nuclei by the expression of green fluorescent protein (GFP)-tagged Xenopus nucleoplasmin in Saccharomyces cerevisiae. To identify components that determine nuclear structure, we searched for mutants exhibiting abnormal nuclear morphology from a collection of temperature-sensitive yeast strains expressing GFP-tagged nucleoplasmin. Four anu mutant strains (anu1-1, 2-1, 3-1 and 4-1; ANU=abnormal nuclear morphology) that exhibited strikingly different nuclear morphologies at the restrictive temperature as compared to the wild-type were isolated. The nuclei of these mutants were irregularly shaped and often consisted of multiple lobes. ANU1, 3 and 4 were found to encode known factors Sec24p, Sec13p and Sec18p, respectively, all of which are involved in the formation or fusion of intracellular membrane vesicles of protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus. On the other hand, ANU2 was not well characterized. Disruption of ANU2 (delta anu2) was not lethal but conferred temperature-sensitivity for growth. Electron microscopic analysis of anu2-1 cells revealed not only the abnormal nuclear morphology but also excessive accumulation of ER membranes. In addition, both anu2-1 and delta anu2 cells were defective in protein transport between the ER and the Golgi, suggesting that Anu2p has an important role in vesicular transport in the early secretory pathway. Here we show that ANU2 encodes a 34 kDa polypeptide, which shares a 20% sequence identity with the mammalian epsilon-COP. Our results suggest that Anu2p is the yeast homologue of mammalian epsilon-COP and the abrupt accumulation of the ER membrane caused by a blockage of the early protein transport pathway leads to alteration of nuclear morphology of the budding yeast cells.     

A mitochondrial presequence can transport a chloroplast-encoded protein into yeast mitochondria

Ribulose-1,5-bisphosphate carboxylase/oxygenase of chloroplasts contains eight large and eight small subunits. The small subunit is encoded by nuclear DNA, synthesized in the cytoplasm, and imported into chloroplasts. The large subunit is encoded by chloroplast DNA and synthesized within chloroplasts. We show in this communication that the large subunit of Chlamydomonas chloroplasts could be efficiently imported into isolated yeast mitochondria if it was attached to the presequence of a protein transported into the yeast mitochondrial matrix. Thus, synthesis of the large subunit within chloroplasts does not reflect the inability of this subunit to cross membranes. The same mitochondrial presequence could also transport the nuclear-encoded small subunit into yeast mitochondria. However, when the two types of subunits were coimported into mitochondria, they did not assemble with each other inside the heterologous organelle.