Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

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.     

Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A(2) (PLA(2)) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA(2) (10(-5) approximately 10(-2)/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA(2), regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA(2)-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA(2)-treated group. Messenger RNA export rate in PLA(2) (10(-3) unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA(2) plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.     

The effect of chick-liver ribonucleic acid in amino acid-incorporation systems from rat liver

1. Rat-liver microsomes, ribonucleoprotein particles and a fraction mainly consisting of microsomal membranes were tested for their ability to incorporate amino acids into protein in the presence of ATP, GTP, phosphoenolpyruvate and pyruvate kinase. Addition of polyuridylic acid or of ribonucleic acid from rat-liver nuclei stimulated the incorporating activities. 2. These protein-synthesizing systems were found to be susceptible to ribonucleic acid from chick-liver nuclei as well. The biological activity of the ribonucleic acid from chick liver was measured by its capacity to stimulate amino acid incorporation. 3. In the presence of chick-liver ribonucleic acid, the ribonucleoprotein particles from rat liver showed an increased radioactivity in ribosomal units with a sedimentation constant higher than 70s. 4. This was investigated by sucrose-gradient centrifugation or by column chromatography on agarose suspensions.     

An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.     

Thermal diminution and augmentation of the retention of transportable rRNA in nuclear envelope-free nuclei

We have examined the effect of temperature on the rRNA transport from nuclei isolated from Tetrahymena after removal of both nuclear membranes and pore complexes by 1% Triton X-100. These nuclei export rRNA as precursor ribosomal ribonucleoprotein particles at both 28 degrees C and 8 degrees C which are qualitatively the same in terms of rRNA pattern, sedimentation coefficients and buoyant densities. At 8 degrees C, however, significantly fewer ribosomal ribonucleoprotein particles can be maximally exported than at 28 degrees C, though nuclei contain enough potentially transportable particles. These are increasingly released with increasing temperatures. Under conditions non-permissive for export, temperature elevation decreases the number of the potentially transportable ribosomal ribonucleoprotein particles in nuclei. Our data show: transportable ribosomal ribonucleoprotein particles inside nuclei are not 'free', but rather are subject to a complex temperature-sensitive retention: this retention is gradually diminished under export conditions and augmented under non-permissive export conditions with increasing temperatures. These retention mechanisms operate at an intranuclear level preceding the ribosomal ribonucleoprotein passage through the nuclear envelope pore complexes, i.e., the nuclear envelope regulates neither the number of potentially transportable ribosomal ribonucleoprotein particles in nuclei nor the number of those particles which can be maximally exported from nuclei at a given temperature. We suggest that these retention mechanisms involve temperature-sensitive domains of the nuclear matrix.     

肿瘤坏死因子诱导B16细胞凋亡的核蛋白质组分析(英文)

为深入了解细胞凋亡的机制,研究了细胞凋亡过程中细胞核蛋白的变化.通过分离肿瘤坏死因子TNFα诱导的小鼠黑色素瘤B16细胞的细胞核并进行二维电泳分析,获得了分辨率和重复性较好的双向电泳图谱.图像分析比较对照细胞和凋亡细胞的核蛋白发现,在凋亡细胞中有11个蛋白发生了明显的变化,6个蛋白下调,5个蛋白上调,对这11个差异蛋白质点分别进行肽质指纹分析.经数据库查询,初步鉴定出这些蛋白.其中4个含量增加的蛋白(HSP84,calreticulin,vimentin,GAPDH)均直接或间接地参与诱导细胞凋亡,另外1个含量增加的蛋白(plasminogen)尚未见其与细胞凋亡有关的报道.而6个含量降低的蛋白分别属于信号转导相关蛋白(guaninenucleotidebindingprotein,lamininreceptor1)、转录调控蛋白、mRNA转运蛋白(heterochromatinprotein1alpha,heterogeneousnuclearribonucleoproteinA3,heterogeneousnuclearribonucleoproteinA2B1)和未知功能蛋白(nucleolarproteinNO38).细胞凋亡过程中这些变化的核蛋白质的发现将有助于深入认识细胞凋亡的分子机制.     

Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process

To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.     

Ionic Permeability on Isolated Mouse Liver Nuclei: Influence of ATP and Ca2+

Patch-clamp experiments on isolated nuclei revealed the existence of ionic channels on the nuclear envelope, but their exact localization and function are still unknown. Recent studies have demonstrated that ATP and calcium ions play an important role in nucleocytoplasmic protein traffic. ATP is essential to allow big molecules in and out of the nucleus. However, a cytoplasmic rise of calcium ions above 300 nm decreases both ATP-dependent transport and passive diffusion through the nuclear envelope. The use of isolated nuclei placed in a saline solution provides the possibility for testing only the compounds added in the bath or in the recording pipette. In the present study, we show that ATP is responsible for an increase of nuclear ionic permeability on isolated nuclei. This result not only confirms data previously reported in in situ nuclei, but also suggests that ATP is directly involved in the modulation of passive ionic permeability. In these particular experimental conditions, calcium ions decrease the channel current starting from a concentration of 1 μm. The parallelism in the modulation action of ATP and Ca⁺⁺ between nuclear pores and ionic channels present on the nuclear envelope contributes to the support of the idea that an ionic pathway is associated with the pore complex. Received: 5 September 1996/Revised: 13 January 1997     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events

Messenger RNA (mRNA) export involves the unidirectional passage of ribonucleoprotein particles (RNPs) through nuclear pore complexes (NPCs), presumably driven by the ATP-dependent activity of the DEAD-box protein Dbp5. Here we report that Dbp5 functions as an RNP remodeling protein to displace the RNA-binding protein Nab2 from RNA. Strikingly, the ADP-bound form of Dbp5 and not ATP hydrolysis is required for RNP remodeling. In vivo studies with nab2 and dbp5 mutants show that a Nab2-bound mRNP is a physiological Dbp5 target. We propose that Dbp5 functions as a nucleotide-dependent switch to control mRNA export efficiency and release the mRNP from the NPC.     

Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei.

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.