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At least 937 records · Page 52Linked to original sources
PubMed1999

Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin alpha2beta1 heterodimers.

L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor-mediated manner into the nuclei of digitonin-permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via karyopherin alpha2beta1 heterodimers. Anti-karyopherin-beta1 and anti-karyopherin alpha2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using karyopherin alpha2, beta1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to karyopherin alpha2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the karyopherin alpha2beta1 pathway.

Amino Acid Sequence↗
PubMed2006

The nuclear barrier is structurally and functionally highly responsive to glucocorticoids.

Nuclear pore complexes mediate and control transport between the cytosol and the nucleus. They form a highly selective and, thus, tight nuclear barrier between these compartments. The nuclear barrier provides the cell with the opportunity to control access to its DNA, a defining feature of eukaryotes. The tightness of the nuclear barrier is therefore physiologically pivotal and any remarkable change in its structure and permeability can prove pathophysiological, e.g. as a result of viral attack. However, there is accumulating evidence that nuclear barrier structure and permeability are highly responsive to hydrophobic cargos of crucial physiological and therapeutic relevance, glucocorticoids (steroid hormones). The present review highlights the glucocorticoid-induced effects on the nuclear barrier structure and permeability concluding that they are physiologically essential to mediate glucocorticoid action.

Animals↗
PubMed2004

Identification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion.

The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu44 and Phe45 in the (Lxx)4 domain or deletion of the whole (Lxx)4 domain (amino acid #35-52 of the Gag p6 domain) did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1-23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1-23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.

Alanine↗
PubMed2004

CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm.

Messenger RNA is formed from precursors known as pre-mRNA. These precursors associate with proteins to form pre-mRNA-protein (pre-mRNP) complexes. Processing machines cap, splice and polyadenylate the pre-mRNP and in this way build the mRNP. These processing machines also affect the export of the mRNP complexes from the nucleus to the cytoplasm. Export to the cytoplasm takes place through a structure in the nuclear membrane called the nuclear pore complex (NPC). Export involves adapter proteins in the mRNP and receptor proteins that bind to the adapter proteins and to components of the NPC. We show that the export receptor chromosomal region maintenance protein 1 (CRM1), belonging to a family of proteins known as importin-beta-like proteins, binds to gene-specific Balbiani ring (BR) pre-mRNP while transcription takes place. We also show that the GTPase known as Ran binds to BR pre-mRNP, and that it binds mainly in the interchromatin. However, we also show using leptomycin B treatment that a NES-CRM1-RanGTP complex is not essential for export, even though both CRM1 and Ran accompany the BR mRNP through the NPC. Our results therefore suggest that several export receptors associate with BR mRNP and that these receptors have redundant functions in the nuclear export of BR mRNP.

Animals↗
PubMed2000

Distinct ion channel classes are expressed on the outer nuclear envelope of T- and B-lymphocyte cell lines.

The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system.

Animals↗
PubMed2001

Adenovirus hexon protein enhances nuclear delivery and increases transgene expression of polyethylenimine/plasmid DNA vectors.

Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon-PEI/DNA complexes and NLS-albumin-PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon-PEI/DNA>NLS-albumin-PEI/DNA>PEI/DNA>DNA alone>albumin-PEI/DNA. The activities of both NLS-albumin-PEI and hexon-PEI were abolished by co-injection of wheat germ agglutinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS-albumin abolished transgene expression with NLS-albumin-PEI/DNA, but only partially inhibited hexon-PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependent on DNA concentration for all materials, although hexon conjugates showed much better activity than NLS-albumin at low DNA doses (500-1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear import enables substantially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.

Active Transport, Cell Nucleus↗
PubMed2002

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus: II. Isolation and characterization of three small nuclear ribonucleoprotein proteins which bind to nER-II.

Three proteins of a goat uterine small nuclear ribonucleoprotein (snRNP) fraction, which bind to nuclear estrogen receptor-II (nER-II) have been isolated and purified. These are the p32, p55, and p60 of which p32 is the major nER-II binding protein. Indirect evidence reveals that p32 binds to the nuclear export signal (NES) on the nER-II. nER-II is a snRNA binding protein while p32 does not bind to the RNA. nER-II along with p32 and p55 form an effective Mg(++)ATPase complex, the activation of which appears to be the immediate reason behind the RNP exit from the nuclei following estradiol exposure. The three nER-II binding proteins bind to the nuclear pore complex; nER-II does not possess this property.

Adenosine Triphosphatases↗
PubMed1978

Rat liver nuclear skeleton and ribonucleoprotein complexes containing HnRNA.

Rat liver nuclei deprived of chromatin and nucleoplasm show a spongelike network which preserves its connection with nucleoli, the inner membrane of the nuclear envelope, and nuclear pore complexes. It contains all of the HnRNA, provided the endogenous proteolytic activity is inhibited by a proteolytic inhibitor such as phenylmethyl sulfonyl chloride (PMSC) or the fluoride form (PMSF). In the absence of these proteolytic inhibitors, HnRNA is dissociated from the spongelike network and sediments in a sucrose gradient as polydispersed ribonucleoprotein complexes. Furthermore, purified HnRNA as well as rRNA do not bind to the spongelike network when added to these nuclei. These observations demonstrate that the association of HnRNA to the nuclear skeleton is not an artifact. RNase treatment of the spongelike network digests the majority of the rapidly labeled RNA but does not alter the morphological aspect nor the architecture of this network. EDTA and heparin treatments affect neither the attachment of HnRNA nor the structural organization of this network. Electron microscope studies of the network reveal a characteristic flexuous configuration. Its relationship with diffused and condensed chromatin is discussed.

Animals↗
PubMed1998

Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy.

At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.

Animals↗
PubMed2003

The Mtr2-Mex67 NTF2-like domain complex. Structural insights into a dual role of Mtr2 for yeast nuclear export.

The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.

Active Transport, Cell Nucleus↗
PubMed1996

Atomic force microscopy visualizes ATP-dependent dissociation of multimeric TATA-binding protein before translocation into the cell nucleus.

The TATA-binding protein (TBP) is a universal transcription factor which plays an essential role in eukaryotic gene expression. As a karyophilic molecule, this cytosolic protein reaches its DNA-binding site through the transport channel of the nuclear pore complex. As occurs with other major cellular proteins, TBP forms multimers in solution, which is a limiting factor for nuclear translocation. While studying the nuclear translocation of TBP, we detected ATP-dependent multimerization of TBP with atomic force microscopy. In physiological solutions containing ATP, 14-molecule multimers dissociated into four-molecule multimers with a half-maximum dissociation constant of 10 microM. Electrophysiological experiments using isolated cell nuclei of cultured kidney cells revealed that TBP translocates into the cell nucleus only in the presence of ATP. When ATP was replaced with its slowly hydrolysing analogue, ATP[gamma-S] [i.e. adenosine 5'-o-(3-thiotriphosphate)], the aggregates remained intact and nuclear translocation was not possible. Taken together, our investigations suggest that TBP exhibits ATPase activity similar to that observed in relation to molecular chaperons. This activity secures physiological translocation of the transcription factor into the nucleus.

Adenosine Triphosphate↗
PubMed1988

Association of Autographa californica nuclear polyhedrosis virus (AcMNPV) with the nuclear matrix.

Nuclear matrices from uninfected Spodoptera frugiperda cells and those infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) were isolated and their protein constituents were compared. Proteins were characterized according to size and several different antibodies to Drosophila nuclear proteins were employed in an attempt to identify the proteins comprising this nuclear substructure. Three species of lamins were identified as major constituents of the nuclear matrix of Spodoptera cells. Two DNA-binding proteins having molecular weights of 54 and 36 kDa were also identified as components of the nuclear matrix of uninfected cells. Infection resulted in a superimposition of viral proteins upon the nuclear matrix of the host cell. Polyhedrin, the basic viral DNA-binding protein (p6.9), and the major capsid protein of AcMNPV were identified immunologically as components of the nuclear matrix fraction of infected cells. Infection also resulted in the increased association of cellular histones with the nuclear matrix. DNA-binding assays demonstrated histones and p6.9 were the predominant DNA-binding proteins associated with the nuclear matrix of infected cells. Nuclear matrices from uninfected cells and cells infected with AcMNPV for 10 and 24 hr were examined using transmission electron microscopy. Morphologically, the nuclear matrix of the uninfected cell consists of the outer nuclear lamina (including nuclear pore complexes), an internal fibrogranular protein constituent, and a residual nucleolar structure. Numerous viral capsids were observed associated with the nuclear matrix in cells infected with either wild-type AcMNPV or a polyhedrin-deletion mutant by 10 hr p.i. The capsids appeared to be attached in an end-on association with the internal fibrogranular protein network of the nuclear matrix. The matrix-associated capsids were similar in width and length to those packaged within the polyhedra. In addition to the capsids, polyhedra in various stages of maturation were seen at 24 hr following infection of the cells with the wild-type virus. The nuclear matrix of the infected cell appears to play an important role in baculovirus assembly.

Animals↗
PubMed1997

Oxidation of nuclear membrane cholesterol inhibits nucleoside triphosphatase activity.

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Acid Anhydride Hydrolases↗
PubMed2006

Structural basis for the nuclear protein import cycle.

Transport of macromolecules between the nuclear and cytoplasmic compartments through NPCs (nuclear pore complexes) is mediated by soluble transport factors that are commonly members of the importin-beta superfamily. In the nuclear protein import cycle, importin-beta binds cargo in the cytoplasm (usually via the importin-alpha adaptor) and transports it through NPCs with which it interacts transiently by way of NPC proteins ('nucleoporins') that contain distinctive FG (Phe-Gly) sequence repeats. In the nucleus, Ran-GTP binds to importin-beta, dissociating the import complex. The importin-beta-Ran-GTP complex recycles to the cytoplasm, whereas importin-alpha is recycled by the importin-beta family member CAS (cellular apoptosis susceptibility protein) complexed with Ran-GTP. Cytoplasmic RanGAP (Ran GTPase-activating protein) dissociates these complexes, freeing the importins for another import cycle. Crystallography and biochemical and cellular studies have enabled a molecular description of the transport cycle to be developed and tested using protein engineering and computer modelling. Importin-beta family members are elongated flexible molecules that adapt their shape to encircle their cargoes. Ran-GTP binds at three sites along importin-beta and CAS, locking the molecules into a rigid conformation that is unable to bind cargoes effectively. Interactions between transport factors and key nucleoporins (such as Nup1p, Nup2p and Nup50) accelerate the formation and dissolution of many of these complexes.

Animals↗
PubMed2003

The strategy for coupling the RanGTP gradient to nuclear protein export.

The Ran GTPase plays critical roles in both providing energy for and determining the directionality of nucleocytoplasmic transport. The mechanism that couples the RanGTP gradient to nuclear protein export will determine the rate of and limits to accumulation of export cargoes in the cytoplasm, but is presently unknown. We reasoned that plausible coupling mechanisms could be distinguished by comparing the rates of reverse motion of export cargoes through the nuclear pore complex (NPC) with the predictions of a mathematical model. Measurement of reverse export rates in Xenopus oocytes revealed that nuclear export signals can facilitate RanGTP-dependent cargo movement into the nucleus against the RanGTP gradient at rates comparable to export rates. Although export cargoes with high affinity for their receptor are exported faster than those with low affinity, their reverse transport is also greater. The ratio of the rates of reverse and forward export of a cargo is proportional to its rate of diffusion through the NPC, i.e., to the ability of the cargo to penetrate the NPC permeability barrier. The data substantiate a diffusional mechanism of coupling and suggest the existence of a high concentration of RanGTP-receptor complexes within the NPC that decreases sharply at the cytoplasmic boundary of the NPC permeability barrier.

Models, Biological↗
PubMed2000

Differential regulation of sentrinized proteins by a novel sentrin-specific protease.

Sentrin-1, also called SUMO-1, is a protein of 101 residues that is distantly related to ubiquitin and another ubiquitin-like protein, NEDD8. Here we report the cloning of a novel sentrin-specific protease, SENP1, which has no homology to the known de-ubiquitinating enzymes or ubiquitin C-terminal hydrolases. However, SENP1 is distantly related to the yeast Smt3-specific protease, Ulp1. A COS cell expression system was used to demonstrate the activity of SENP1 in vivo. When HA-tagged sentrin-1 was co-expressed with SENP1, the higher molecular weight sentrin-1 conjugates were completely removed. Surprisingly, the major sentrinized band at 90 kDa remained intact. The disappearance of the high molecular weight sentrin-1 conjugates also coincided with an increase in free sentrin-1 monomers. SENP1 is also active against proteins modified by sentrin-2, but not those modified by ubiquitin or NEDD8. In addition, sentrinized PML, a tumor suppressor protein that resides in the nucleus, was selectively affected by SENP1, whereas sentrinized RanGAP1, which is associated with the cytoplasmic fibrils of the nuclear pore complex, remained intact. The inability of SENP1 to process sentrinized RanGAP1 in vivo is most likely due to its nuclear localization because SENP1 is active against sentrinized RanGAP1 in vitro. The identification of a nuclear-localized, sentrin-specific protease will provide a unique tool to study the role of sentrinization in the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.

Amino Acid Sequence↗
PubMed2002

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast.

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.

Active Transport, Cell Nucleus↗
PubMed1995

Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport.

Transport of RNAs within nuclei and through nuclear pore complexes (NPCs) are essential, but poorly understood, steps in gene expression. In experiments with mammalian cells, RCC1, the abundant nuclear guanine nucleotide exchange factor for the guanosine triphosphatase Ran/TC4, was shown to be required for nucleocytoplasmic transport of precursors of spliceosomal small nuclear RNAs (snRNAs), intranuclear transport of U3 snRNA, and processing of ribosomal RNAs, but not for export of transfer RNAs. It is proposed that guanosine triphosphate (GTP)-bound Ran/TC4 associates with ribonucleoprotein particles (RNPs) during intranuclear movement, and that GTP hydrolysis promotes deposition of RNPs at targeted sites such as NPCs or nucleoli.

Biological Transport↗