Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.     

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.     

Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi- intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.     

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy- dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Burglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non- nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.     

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.     

Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0°C and is Ca²⁺-independent. By contrast, translocation requires ATP hydrolysis, Ca²⁺, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells. © 1996 Wiley-Liss, Inc.     

Characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-T antigens.

Simian virus 40 small-t and large-T antigen were synthesized in vitro and labeled with methionine donated by initiator tRNA. Tryptic peptide fingerprinting was used to identify the amino-terminal peptide of the two proteins. Similar fingerprint analysis of small-t and large-T made in vitro in the absence of acetyl coenzyme A showed that the mobility of the amino-terminal peptide was changed under these conditions and suggested that it is acetylated. These data establish that the amino-terminal methionine residue of simian virus 40 small-t and large-T results from an initiation event, not post-translational cleavage, and provides additional evidence that the amino terminus of both proteins is acetylated. The identification of the amino-terminal peptide provides a useful marker for further studies on different forms of T-antigen from cells infected with and transformed by simian virus 40 and related viruses.     

Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen.

Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.     

A plant in vitro system for the nuclear import of proteins

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-γ-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-γ-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.     

Biochemical properties of the 145,000-dalton super-T antigen from simian virus 40-transformed BALB/c 3T3 clone 20 cells

SV3T3 C120 cells contain a 145,000-dalton form of simian virus 40 (SV40) super-T antigen but little if any normal-sized large-T. The subcellular location of super-T, its DNA binding properties, and its interaction with nonviral tumor antigen (NVT) were examined. Immunofluorescence microscopy and subcellular fractionation indicated that super-T is almost exclusively nuclear. Chromatography on double-stranded DNA-cellulose showed that super-T binds to double-stranded DNA and has an elution profile indistinguishable from normal-sized large-T. Super-T also binds specifically to a fragment of SV40 DNA which contains the origin of DNA replication. However, immunoprecipitation of super-T or large-T either with anti-tumor cell serum or with anti-NVT serum from fractions obtained by sucrose density centrifugation of 32P-labeled or [35S]methionine-labeled extracts revealed clear differences in the sedimentation characteristics of these proteins. The bulk of labeled 145,000-dalton super-T sedimented between 4S and 10S, whereas the bulk of 32P-labeled large-T from normal SV40-transformed cells sedimented as two peaks at 23S to 25S and 16S to 18S. By contrast, the sedimentation properties of NVT from the SV3T3 C120 cells were similar to those normally observed with other SV3T3 cell lines. The reason for this apparent difference in complex formation between super-T and NVT and that normally observed with large-T is unclear, but it probably has no deleterious effect on the ability of super-T to maintain transformation.     

Nuclear envelope permeability measured by fluorescence microphotolysis of single liver cell nuclei

Fluorescence microphotolysis ("photobleaching") has been widely used to measure translational diffusion coefficients of lipids and proteins in cell membranes. This communication shows that fluorescence microphotolysis can be also employed for measurement of membrane transport in single cells and organelles. The influx of fluorescently labeled dextrans of graded molecular size into leaky human erythrocyte ghosts and isolated rat liver cell nuclei has been measured. For the nuclear envelope, a functional pore radius of 56-59 A is derived.     

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0°C, are present in both the nucleus and cytoplasm. © 1996 Wiley-Liss, Inc.     

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.     

The nuclear matrix continues DNA synthesis at in vivo replicational forks

Alkaline cesium chloride gradient analysis of in vivo [3H]bromodeoxyuridine-labeled and in vitro [alpha-32P]dCTP-labeled DNA was used to determine whether in vitro DNA synthesis in regenerating rat liver nuclei and nuclear matrices continued from sites of replication initiated in vivo. At least 70 and 50% of the products of total nuclear and matrix-bound in vitro DNA synthesis, respectively, were continuations of in vivo initiated replicational forks. The relationship of the in vitro DNA synthetic sites in total nuclei versus the nuclear matrix was examined by using [3H]bromodeoxyuridine triphosphate to density label in vitro synthesized DNA in isolated nuclei and [alpha-32P]dCTP to label DNA synthesized in isolated nuclear matrix. A minimum of about 40% of matrix-bound DNA synthesis continued from sites being used in vitro by isolated nuclei. Furthermore, nuclear matrices prepared from in vitro labeled nuclei were 5-fold enriched in DNA synthesized by the nuclei and were several-fold enriched, compared to total nuclear DNA, in a particularly high density labeled population of DNA molecules.     

A 40 kDa nuclear membrane protein changes its concentration and localization within the nuclear envelope of the yeast Saccharomyces cerevisiae in a cell-cycle dependent manner

In order to identify and characterize structural components in the nuclear membrane of Saccharomyces cerevisiae which show a cell-cycle dependent regulation, we have undertaken a combined biochemical/immunofluorescence microscopy approach. Antisera raised against nuclear membrane proteins from yeast lead to the identification of a 40 kDa membrane protein which cofractionated with nuclei upon cell fractionation. This 40 kDa membrane protein partitioned into the Triton X-114 phase and was not extracted from purified nuclei at alkaline pH. Using affinity-purified antibodies against this protein, the antigen was localized at the nuclear periphery suggesting that it is an integral constituent of the nuclear envelope. However, the 40 kDa antigen revealed a heterogenous distribution within the nuclear membrane: in indirect immunofluorescence microscopy, nuclei isolated from an asynchronously growing yeast culture showed either no immunodetectable antigen or contained it in a cap-, dot- or ring-like conformation. Using synchronized yeast cultures, we could demonstrate cell-cycle dependent changes of concentration and localization of the 40 kDa protein within the nuclear envelope.     

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.