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PubMedDate not supplied

Identification of protein complexes by comparative analysis of yeast and bacterial protein interaction data.

Mounting evidence shows that many protein complexes are conserved in evolution. Here we use conservation to find complexes that are common to the yeast S. cerevisiae and the bacteria H. pylori. Our analysis combines protein interaction data that are available for each of the two species and orthology information based on protein sequence comparison. We develop a detailed probabilistic model for protein complexes in a single species and a model for the conservation of complexes between two species. Using these models, one can recast the question of finding conserved complexes as a problem of searching for heavy subgraphs in an edge- and node-weighted graph, whose nodes are orthologous protein pairs. We tested this approach on the data currently available for yeast and bacteria and detected 11 significantly conserved complexes. Several of these complexes match very well with prior experimental knowledge on complexes in yeast only and serve for validation of our methodology. The complexes suggest new functions for a variety of uncharacterized proteins. By identifying a conserved complex whose yeast proteins function predominantly in the nuclear pore complex, we propose that the corresponding bacterial proteins function as a coherent cellular membrane transport system. We also compare our results to two alternative methods for detecting complexes and demonstrate that our methodology obtains a much higher specificity.

Bacterial Proteins↗
PubMed1998

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

3T3 Cells↗
PubMed1982

[Localization of high-molecular alkali-insoluble polypeptides of rat liver cell nuclear matrix revealed by immunological methods].

By means of chicken immunization, antibodies were obtained to two high molecular weight polypeptides of an alkali-insoluble highly dispersed fraction of rat liver cell nuclear matrix. Using methods of indirect immunofluorescence these antibodies were seen fixed selectively on the periphery of the cell nuclei of rat liver, cultured human fibroblasts and of cultured human fibrosarcoma 8387 cells. The same pattern of antibody fixation, with non-specific net-like staining in the cytoplasm, was observed in the light microscope after the immunoperoxidase staining. Using the similar peroxidase staining with electron microscopy, antibody fixation was recorded with nuclear pore complexes, ribosomes, fibrous lamina and intranuclear granular structures in isolated rat liver cell nuclei and in the nuclear matrix. In mitotic cells the cytoplasm displayed a bright fluorescence, whereas the condensed chromosomes showed a fainter fluorescence. Thus, the examined high molecular weight antigens revealed no organ or species specificity. They appear to be constituents of nonmembraneous structures of the nuclear envelope, more likely of the pore complexes.

Animals↗
PubMed2003

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages.

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

Active Transport, Cell Nucleus↗
PubMed1995

Histone-poly(A) hybrid molecules as tools to block nuclear pores.

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

Animals↗
PubMed2005

Structural basis for nuclear import complex dissociation by RanGTP.

Nuclear protein import is mediated mainly by the transport factor importin-beta that binds cytoplasmic cargo, most often via the importin-alpha adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-beta consists of 19 HEAT repeats that are based on a pair of antiparallel alpha-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-beta (Kap95p or karyopherin-beta) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-alpha or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.

Active Transport, Cell Nucleus↗
PubMed2006

Nuclear export of simian immunodeficiency virus Vpx protein.

Lentiviruses, human immunodeficiency viruses (HIVs), and simian immunodeficiency viruses (SIVs) are distinguished from oncoretroviruses by their ability to infect nondividing cells such as macrophages. Retroviruses must gain access to the host cell nucleus for replication and propagation. HIV and SIV preintegration complexes (PIC) enter nuclei after traversing the central aqueous channel of the limiting nuclear pore complex without membrane breakdown. Among the nucleophilic proteins, namely, matrix, integrase, Vpx, and Vpr, present in HIV type 2/SIV PIC, Vpx is implicated in nuclear targeting and is also available for incorporation into budding virions at the plasma membrane. The mechanisms of these two opposite functions are not known. We demonstrate that Vpx is a nucleocytoplasmic shuttling protein and contains two novel noncanonical nuclear import signals and a leptomycin B-sensitive nuclear export signal. In addition, Vpx interacts with the cellular tyrosine kinase Fyn through its C-terminal proline-rich motif. Furthermore, our data indicate that Fyn kinase phosphorylates Vpx and regulates its export from nucleus. Replacement of conserved tryptophan residues within domain 41 to 63 and tyrosine residues at positions 66, 69, and 71 in Vpx impairs its nuclear export, virion incorporation, and SIV replication in macrophages. Nuclear export is essential to ensure the availability of Vpx in the cytoplasm for incorporation into virions, leading to efficient viral replication within nondividing cells.

Active Transport, Cell Nucleus↗
PubMed2004

Import and export of nuclear proteins: focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor.

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor alpha from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER alpha appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg2+ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.

Active Transport, Cell Nucleus↗
PubMed2000

Monitoring biomolecular interactions by time-lapse atomic force microscopy.

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.

Actins↗
PubMed2000

New steps toward the nucleocytoplasmic traffic of macromolecules.

The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.

Active Transport, Cell Nucleus↗
PubMed1999

Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei.

A fraction from human milk containing spf-multimer alpha-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER-Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca2+ was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca2+. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.

Animals↗
PubMed2005

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim.

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.

Active Transport, Cell Nucleus↗
PubMed1998

Nuclear import of polypeptides, polynucleotides and supramolecular complexes.

Nuclear import of macromolecules or particles is a strictly regulated phenomenon that is now understood in some depth at the molecular level. Transport across nuclear membranes is controlled by nuclear pores, with the involvement of cytoplasmic receptors and accessory molecules. Each nuclear pore complex is an assembly of multiple copies of at least 30 distinct proteins, with a total mass of approximately 10(7) kDa. Passive transport of small molecules through nuclear pores is unrestricted, but efficient uptake of macromolecules requires nuclear localization signals, which facilitate the interaction with cytoplasmic receptor proteins. The basic architecture of the nuclear pore and the mechanisms that regulate nuclear import are summarized, in relation to import of endogenous molecules and viruses. Subsequently, the significance of these mechanisms in controlling gene delivery is discussed. Access of DNA to the nucleus is a major barrier to the success of gene therapy, although viruses have evolved mechanisms to exploit the active transport machinery within the host cell. A key step in the future development of non-viral gene therapy will be the design and development of synthetic systems for active delivery of DNA to the nucleus.

Journal Article↗
PubMed1996

Ran binding domains promote the interaction of Ran with p97/beta-karyopherin, linking the docking and translocation steps of nuclear import.

Nuclear protein import is accomplished by two sequential events; docking at the nuclear pore complex followed by ATP-dependent translocation across the nuclear envelope. Docking of nuclear targeted proteins requires a 56-kDa nuclear localization signal receptor (alpha-karyopherin, importin-alpha, SRP1 alpha) and a 97-kDa protein (beta-karyopherin, importin-beta). Components necessary for translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of these factors at a molecular level remain unclear. We have now found that a complex of Ran, in the GTP-bound state, with either the Ran binding protein, RanBP1, or an isolated Ran binding domain binds with high affinity and specificity to beta-karyopherin to form a ternary complex. We find that a C-terminal truncation mutant of Ran, delta-DE Ran, also binds to beta-karyopherin and that delta-DE Ran can associate with a cytosolic, multiprotein complex that contains beta-karyopherin and another delta-DE Ran binding protein of 115/120 kDa. These data suggest a physical link between docking and translocation mediated by a Ran GTPase-Ran binding protein complex.

Amino Acid Sequence↗
PubMed2002

Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes.

The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood. However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats. We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording. NTF2, the transport receptor of RanGDP, was exported approximately 30 times faster than green fluorescent protein, an inert molecule of approximately the same size. The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗
PubMed1999

Nuclear import and export: transport factors, mechanisms and regulation.

Molecules enter and exit the nucleus of eukaryotic cells through aqueous channels formed in the nuclear envelope by nuclear pore complexes (NPC). Proteins entering or leaving the nucleus use nuclear localization signals (NLSs), or nuclear export signals (NESs), respectively. Different types of NLSs and NESs are specifically recognized directly, or indirectly via adapters, by transport receptors. In recent years, many transport receptors, adapters, and the specific cargoes they carry into or out of the nucleus have been identified, revealing an increasing level of complexity. All the transport receptors identified are members of the same family and have in common the ability to shuttle between the nucleus and cytoplasm, and to interact with RanGTP and with nucleoporins at the NPC. The GTPase Ran regulates the interaction between receptors and cargoes, or adapters, and is crucial in providing directionality to nuclear import and export. One of the challenges for the future will be establishing the mechanisms of translocation through the NPC of receptors together with their cargoes. Nuclear import and export of many cargoes is regulated. The molecular mechanisms of regulation of nucleocytoplasmic transport during cell growth, development, viral infections, and different diseases constitute important areas of investigation.

Biological Transport↗
PubMed2001

Harnessing nuclear localization pathways for transgene delivery.

Inefficient transport of DNA from the cytoplasm into the nucleus remains a limiting step in the development of non-viral gene delivery systems. This is particularly acute in non-dividing cells, where entry to the nucleus is thought to occur only through the nuclear pore complex. Active import of physiological proteins is mediated by nuclear localization sequences (NLSs) within cargo proteins such as transcription factors. Here we review current knowledge of this import machinery and consider its exploitation by mammalian viruses. Significant research effort has been directed at incorporating NLSs into synthetic gene delivery systems to take advantage of this physiological pathway. Both non-covalent and covalent methods of conjugation are evaluated, with NLS linkage to both DNA and carrier, and compared with activities of simple cationic polymers. Finally, progress in the field of DNA sequence-specific nuclear import is examined and the current state of the technology assessed.

Active Transport, Cell Nucleus↗
PubMed1989

Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins.

Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.

Amino Acid Sequence↗