Reconstitution of nuclear protein transport with semi-intact yeast cells

We have developed an in vitro nuclear protein import reaction from semi- intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS- containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.     

A rapid method for determination of acid phosphatase activity of whole yeast cells

A simple and rapid procedure for determination of intracellular acid phosphatase activity without the need for disruption of cells is described. Candida lipolytica cell suspension was treated with 0.1% Triton X-100 for 30 min at room temperature and with intermittent shaking. The enzyme assay is carried out directly with the permeabilized cell suspension. Permeabilization of the yeast cells to p -nitrophenylphosphate by Triton X-100 provides almost 100% efficiency in determining the total acid phosphatase activity compared to results obtained with disrupted yeast cells.     

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.     

An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B" spliceosome proteins

Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B" is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B" in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-beta family of nucleocytoplasmic transport receptors, U1A/U2B" import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B" import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B" import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B" import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.     

In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells

DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM. The initial incorporation rate at 10 microM TTP concentration was about 12% of the in vivo synthesis and declined very strongly within 1 h. A similar decline of the incorporation rate was found at 0.12 microM TTP concentration. DNAase I digestion of DNA-matrix complexes obtained from isolated nuclei in 2 M NaCl revealed that newly replicated DNA was preferentially bound to the nuclear matrix. A similar digestion with S1 nuclease caused a selective release of short duplexes of Okazaki fragments with the complementary parental strand. The results show that in vivo replication continues in permeabilized cells in an almost unchanged way, except for a gradual decline of its rate which is mainly due to inactivation of one or more essential components.     

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.     

Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.     

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis

Calmodulin (CaM) potentiates Ca(2+)-dependent signaling pathways in both the cytoplasm and nucleus. We have investigated the mechanism of CaM nuclear transport using tissue culture cell microinjection and a permeabilized cell import assay. The inhibition of CaM import by the translocation inhibitor wheat germ agglutinin (WGA) and by chilling, indicates that CaM import is facilitated, but because ATP depletion does not affect CaM import, the mechanism does not appear to be active. Chilling and WGA arrest persist in ATP-depleted cells, indicating that CaM is not retained in the cytoplasm by an ATP-dependent mechanism. In permeabilized cells, both Ca(2+)-CaM and Ca(2+)-free CaM are sensitive to extract-dependent WGA and chilling import inhibition. Titration experiments in microinjected and permeabilized cells indicate that a saturable cytosolic factor(s) mediates chilling and WGA arrest.     

Human anti-DNA secretory immunglobulins A possess endonuclease activity and they are able to cause the destruction of nuclear chromatin in vitro

sIgA possessing ability to hydrolyse plasmid DNA to linear forms was purified from human milk by sequential chromatography on protein A-sepharose, DEAE-Fractogel and DNA-cellulose. It was discovered that incubation of sIgA with nuclei of porcine embryo kidney cells permeabilized by Triton X-100 causes formation of electrophoretically mobile forms of nuclear nucleic acids and inhibition of phosphorylation of nuclear proteins. We suppose that sIgA possessing affinity to DNA and endonuclease activity can cause degradation of cell nuclear chromatin.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.     

On the association of centrioles with the interphase nucleus.

Preparations of nuclei from rat liver and bovine spleen purified by centrifugation through dense sucrose solutions are shown to contain centrioles. These centrioles retain their in situ ultrastructure and are surrounded by a network of filaments adjacent to the nucleus and probably attached to it. The number of centrioles in isolated nuclei depends on the conditions of cell homogenization. Under certain conditions of homogenization, the fraction of purified nuclei contains almost all centrioles of the original tissue. The number of centrioles in isolated nuclei sharply decreases if the nuclei are rehomogenized under conditions that do not cause damage to nuclei. The number of nucleus-associated centrioles does not decrease after solubilization of nuclear membranes by Triton X-100. Nuclei retain the associated centrioles after treatmentwith RNase-free DNase I. It is concluded that in interphase the centrioles are associated with the nucleus and that this association which is probably mediated by filaments involves nuclear structures other than nuclear membranes or whole chromatin.     

The influence of Triton X-100 on the nuclear envelope of the isolated liver cell nuclei.

In order to obtain more precise information on an eventual presence of extra-membranous lipids in the interior of the nucleus, the effects of Triton X-100 on the lipid content and ultrastructure of isolated rat liver nuclei was investigated. Enzyme markers (a.o. glucose-6-phosphatase) were used to control impurities of the nuclear fractions biochemically along with transmission electron microscopy and qualitative and quantitative light microscopy to check the condition of the nuclei obtained. Treatment of the nuclear fraction with increasing concentrations of Triton X-100 resulted in a decrease of the phospholipid content down to 25% at a Triton X-100/protein ratio of 0.4. A further decrease to 8% was measured at a ratio of 1.5. Electron microscopy of nuclei of the latter group showed nuclei containing outer membrane fragments in 2.5% of their surfaces. The composition of lipids extracted from a nuclear fraction appeared to be markedly changed after treatment with Triton X-100 with an increase of the percentage of neutral lipids and the phospholipids diphosphatidyl-glycerol and spingomyelin. From the chemical and morphological data obtained, the conclusion was drawn that a substantial part of the lipids remaining in the isolated nuclei after treatment with Triton X-100 is localized in both membranes of the nuclear envelope. It cannot however, be excluded that a small portion would be present in the interior of the nuclei.     

Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models

The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X-100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X-100 demembranated sperm while 0.01% and 0.05% Triton X-100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1% Triton X-100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X-100 permeabilized sperm models. Reactivated motility of 0.05% Triton X-100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.     

On the attachment of the nuclear pore complex

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin. Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes. The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

BACKGROUND: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals. METHODS: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230. RESULTS: PI4K230 gives a prominent signal in the nucleolus of ethanol fixed rat brain cryosections and of several cell types in addition to its presence in the nucleus and cytoplasm. The PI4K230 immunoreactivity of the nucleolus is masked in PFA fixed cells, but it can be restored by treatment of PFA fixed cells with hot wet citrate buffer or by washing the cryosections with PBS prior to PFA fixation. Nucleolar PI4K230 occurs in a Triton X-100 resistant complex. Treatment of COS-7 cells with siRNA targeting PI4K230 and permeabilized B50 cells with DNase or RNase results in the loss of PI4K230 signal from the nucleolus. CONCLUSION: These experiments suggest the participation of PI4K230 in a DNase and RNase sensitive complex with a unique localization and function in the nucleolus.     

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.     

The major polypeptides of the nuclear pore complex

Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [³H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.